Latest Articles Include:
- Keeping up with the times
- Nat Struct Mol Biol 17(12):1399 (2010)
Nature Structural & Molecular Biology | Editorial Keeping up with the times Journal name:Nature Structural & Molecular BiologyVolume: 17,Page:1399Year published:(2010)DOI:doi:10.1038/nsmb1210-1399Published online03 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. As the season changes, daylight shifts and a new year looms, we consider cycles and the intrinsic clock that drives us. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Understanding of the Hsp90 molecular chaperone reaches new heights
- Nat Struct Mol Biol 17(12):1400-1404 (2010)
Nature Structural & Molecular Biology | Meeting Report Understanding of the Hsp90 molecular chaperone reaches new heights * Cara K Vaughan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Len Neckers2 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter W Piper3 Contact Peter W Piper Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1400–1404Year published:(2010)DOI:doi:10.1038/nsmb1210-1400Published online03 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Heat shock protein 90 (Hsp90) was the focus of a recent meeting in the Swiss Alps, where the Hsp90 community met to discuss the operation and functions of this ubiquitous and essential molecular chaperone. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Cara K. Vaughan is at the Institute of Structural Molecular Biology, Birkbeck College, London, UK. * Len Neckers is at the National Cancer Institute, Bethesda, Maryland, USA. * Peter W. Piper is at the University of Sheffield, Sheffield, UK. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Peter W Piper Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Research highlights
- Nat Struct Mol Biol 17(12):1405 (2010)
Nature Structural & Molecular Biology | Research Highlights Research highlights Journal name:Nature Structural & Molecular BiologyVolume: 17,Page:1405Year published:(2010)DOI:doi:10.1038/nsmb1210-1405Published online03 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Muscle talk Ryanodine receptors (RyR) are large, tetrameric channels that regulate the release of calcium from the endoplasmic reticulum (ER). RyR1 is found in skeletal muscle and is required for excitation-contraction coupling. Mutations of this receptor can result in severe diseases and disorders, which can now be explained by the crystal structure of the three N-terminal domains of RyR1 obtained by Van Petegem and colleagues. Domain A has a β-trefoil motif, in line with previous reports, as does domain B, and domain C forms a bundle of five α-helices. The three N-terminal domains interact with each other through hydrophilic interfaces, and docking of the RyR1 crystal structure into electron microscopy maps show that the domains are located in the cytoplasmic part of the channel. Together, they form a vestibule that surrounds the fourfold symmetry axis. Thirty-three disease-associated mutations could be mapped to the RyR1 structure, resulting in three groups of effects: the first gr! oup destabilizes the interface between each of the three N-terminal domains, the second affects the folding of each domain, and the third alters the interface between the domains and the rest of the receptor. From this information, a model is proposed whereby the opening of RyR1 changes the position of the three N-terminal domains, and the mutations act to destabilize the closed state and promote the open state, producing either 'leaky' channels or ones that are more easily activated. (Nature doi: 10.1038/nature09471, published online 3 Nov 2010) MH Islets in the stream Comprehensive genome-wide mapping of epigenomic modifications has the potential to increase understanding of a multitude of diseases, including type 2 diabetes (T2D). However, to date few tissues have been thoroughly examined with regard to epigenomic status. Collins and colleagues have now undertaken extensive profiling of human pancreatic islets to gain insight into gene regulation in the endocrine pancreas. Using DNase-seq and ChIP-seq, the authors characterized open chromatin, several histone methylation marks and CTCF binding sites across the genome. Over 18,000 potential transcription start sites were identified (of which over 30% were not previously annotated), along with at least 34,000 distal regulatory elements. CTCF binding sites, which often function as insulators, comprised 22% of these distal elements. Many putative distal regulatory elements were unique to the islet and were clustered together in the genome, indicating that they may function together to regula! te islet gene expression. Surprisingly, several highly expressed islet hormone genes lacked the usual chromatin conformation and histone methylation patterns found at active genes. Four predicted regulatory elements unique to the islet functioned as transcriptional enhancers. Two of those elements, found within the genes TCF7L2 and WFS1 (Wolfram syndrome 1), contained known T2D-associated single-nucleotide polymorphisms (SNPs) that conferred allele-specific differences in enhancer activity, indicating they may have altered function in T2D. The abundance of regulatory elements unique to the islet raises intriguing questions about how gene expression is regulated in the pancreas and what role it may play in the etiology of diabetes. This extensive characterization will be foundational for studies probing gene regulation in normal and diseased human pancreatic islet tissue. (Cell Metab.12, 443–455, 2010) SM View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Transcriptional regulation of BRCA1 expression by a metabolic switch
- Nat Struct Mol Biol 17(12):1406-1413 (2010)
Nature Structural & Molecular Biology | Article Transcriptional regulation of BRCA1 expression by a metabolic switch * Li-Jun Di1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alfonso G Fernandez1 Search for this author in: * NPG journals * PubMed * Google Scholar * Adriana De Siervi2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dan L Longo3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Gardner1 Contact Kevin Gardner Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1406–1413Year published:(2010)DOI:doi:10.1038/nsmb.1941Received02 March 2010Accepted16 September 2010Published online21 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Though the linkages between germline mutations of BRCA1 and hereditary breast cancer are well known, recent evidence suggests that altered BRCA1 transcription may also contribute to sporadic forms of breast cancer. Here we show that BRCA1 expression is controlled by a dynamic equilibrium between transcriptional coactivators and co-repressors that govern histone acetylation and DNA accessibility at the BRCA1 promoter. Eviction of the transcriptional co-repressor and metabolic sensor, C terminal–binding protein (CtBP), has a central role in this regulation. Loss of CtBP from the BRCA1 promoter through estrogen induction, depletion by RNA interference or increased NAD+/NADH ratio leads to HDAC1 dismissal, elevated histone acetylation and increased BRCA1 transcription. The active control of chromatin marks, DNA accessibility and gene expression at the BRCA1 promoter by this 'metabolic switch' provides an important molecular link between caloric intake and tumor suppressor expr! ession in mammary cells. View full text Figures at a glance * Figure 1: Estrogen induction increases histone acetylation at the BRCA1 promoter. Top, schematic of bidirectional promoter of BRCA1-NBR2 gene locus with positions of ChIP amplicons. () BRCA1 nascent and mature RNA expression in control or MCF-7 cell treated 24 h with 10 nM estradiol (E2). Error bars, s.e.m. of n = 3 biological replicates. Ctrl, control. (–) ChIP profiles of resting and E2-stimulated MCF-7 cells using antibodies against Pol II (), p300 (), CREB (), acetylated histone H4 (AcH4, ) and acetylated histone H3 (AcH3, ) at the BRCA1 promoter. Error bars, s.e.m. for n = 3 (Pol II), n = 2 (p300), n = 2 (CREB), n = 3 (AcH4) and n = 3 (AcH3) biological replicates. NS, nonspecific. * Figure 2: A multicomponent co-repressor complex containing CtBP is dismissed and elongation factors are recruited to the BRCA1 promoter after estrogen induction. (–) ChIP profiles of MCF-7 cells stimulated 24 h with estradiol (E2) using antibodies against HDAC1, p130, BRCA1 and CtIP as indicated. Error bars, s.e.m. for n = 2 biological replicates. Ctrl, control. NS, nonspecific. () Top, schematic of location of ChIP primer pairs (–) across the BRCA1 locus. ChIP profiles of CtBP, E2F1 and E2F4 enrichment across the 85-kb BRCA1 locus before (blue) and after (red) estrogen induction. Values are mean of n = 2 biological replicates; average s.e.m., 24.6% of mean. () ChIP profiles of NELF, Cdk9 and ELL enrichment across the BRCA1 locus before (blue); and after (red); estrogen induction. Values are mean of n = 2 biological replicates; average s.e.m., 19.5% of mean. * Figure 3: CtBP regulates BRCA1 expression by influencing histone acetylation at the BRCA1 promoter. Top, schematic illustration of the bidirectional promoter of the BRCA1-NBR2 gene locus showing the positions of the amplicons. () Left, immunoblot of CtBP and BRCA1 expression in control and MCF-7 cells depleted of CtBP by RNAi. Actin, endogenous control. Right, nascent and mature BRCA1 RNA levels in control and CtBP-depleted MCF-7 cells. Error bars, s.e.m. for n = 3 biological replicates. Ctrl, control. NS, nonspecific. (–) Estrogen-stimulated enrichment of HDAC1 (), acetylated histone H3 (AcH3, ), acetylated histone H4 (AcH4, ), Pol II () and E2F1 () at the BRCA1 promoter in control and CtBP-depleted MCF-7 cells. KD, knockdown. Error bars, s.e.m. for n = 2 independent biological replicates. * Figure 4: CtBP control of BRCA1 is gene specific, functionally influences cell cycle progression and is chromatin dependent. () mRNA levels of BLM, H2AZ, MAD3L, TFF1 and NBR2 in control and CtBP-depleted MCF-7 cells. Error bars, s.e.m. for n = 3 biological replicates. () Cell cycle profiles (percent distribution in G1, S and G2/M phases) of MCF-7 cells depleted of CtBP for 48 h and 72 h, or overexpressing BRCA1 for 48 h and 72 h. () mRNA profiles of TFF1, BRCA1 and NBR2 in control MCF-7 or cells 48 h after transfection with empty vector or CtBP1-expressing plasmids. Error bars, s.e.m. for n = 3 independent biological replicates. () Top, schematic of dual NBR2-BRCA1 promoter reporter. Bottom, firefly and R. reniformis luciferase activity profiles of MCF-7 cells cotransfected with dual NBR2-BRCA1 luciferase reporter and either control or vectors expressing CtBP1, BRCA1 or p300. () Firefly and R. reniformis luciferase mRNA levels in MCF-7 cells expressing a transiently (left) or stably integrated (right) BRCA1 bidirectional firefly or R. reniformis luciferase reporter after 24 h stimulation with estr! adiol (E2). Error bars, s.e.m. for n = 2 biological replicates. * Figure 5: TSA mimics estrogen-induced activation of BRCA1 by increasing p300-dependent histone acetylation at the BRCA1 promoter. () Time course of TFF1, NBR2 and BRCA1 expression in MCF-7 cells treated 0–24 h with either estradiol (E2), E2 plus cycloheximide (E2+CH, 10 μg ml−1), TSA (500 ng ml−1), or TSA plus cycloheximide (TSA+CH). Error bars, s.e.m. of n = 2 independent biological replicates. () DNase I hypersensitivity profile of BRCA1 promoter and an HBB locus control from MCF-7 cells treated with either estrogen or TSA. Error bars, s.e.m. for n = 3 biological replicates. () Acetylated histone H3 (AcH3), acetylated histone H4 (AcH4), HDAC1, BRCA1, p130 and CtBP ChIP profiles at the BRCA1 promoter in control or MCF-7 cells treated 1 h with 500 ng ml−1 TSA. Error bars, s.e.m. of n = 2 biological replicates. Ctrl, control. NS, nonspecific. () Top, TSA-stimulated expression of BRCA1 nascent and mature RNA levels in either control or p300-depleted MCF-7. Error bars, s.e.m. of n = 2 biological replicates. Bottom, ChIP enrichment for H3 and H4 histone acetylation at the BRCA1 locus in control ve! rsus p300-depleted MCF-7 cells with or without TSA stimulation. Values are mean of n = 2 independent biological replicates. * Figure 6: CtBP functions as a metabolic switch to control BRCA1 expression. () Left, relative change in NAD+/NADH ratio in lysates from MCF-7 cells treated with vehicle or estradiol (E2) for 24 h, or TSA for 1 h. Unstim, unstimulated. Right, time course of relative change in NAD+/NADH ratio in MCF-7 cells treated 0–24 h with 10 mM 2-deoxyglucose (2-DG). () Relative enrichment of TFF1, NBR2 and nascent and mature BRCA1 RNA in MCF-7 cells treated 0–24 h with 2-DG. Error bars, s.e.m. for n = 2 independent biological replicates. (–) ChIP enrichment for CtBP (), acetylated histone H3 (AcH3, ) and acetylated histone H4 (AcH4, ) at the BRCA1 promoter in MCF-7 cells treated 3 h with 2-DG. Error bars, s.e.m. for n = 2 independent biological replicates. * Figure 7: Hypoxia inhibits estrogen-induced changes in the NAD+/NADH ratio and selectively represses estrogen induction of BRCA1 transcription. () Assay of relative change in NAD+/NADH ratio, and BRCA1, TFF1, NBR2 and CtBP1 expression in control (Ctrl) versus hypoxic cells with without estradiol (E2) stimulation. () Schematic hypothetical model for mechanism of CtBP control of BRCA1 transcription. The nucleosome positioning is as described39 by the genome-wide sequencing of micrococcal nuclease–generated fragments (MNase-seq). E2, changing NAD+/NADH ratio, CtBP knockdown or TSA treatment induces removal or inactivation of a repressive complex composed of CtBP, BRCA1, HDAC1 and HDAC2 at the dual BRCA1 promoter. Acetylation-associated destabilization of the centrally positioned nucleosome (Nuc), in combination with the asymmetric nucleosome distribution at the BRCA1 locus, biases expression more toward BRCA1 compared to NBR2 in response to the activating signals. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, Maryland, USA. * Li-Jun Di, * Alfonso G Fernandez & * Kevin Gardner * Department of Biological Chemistry, School of Sciences, University of Buenos Aires, Buenos Aires, Argentina. * Adriana De Siervi * Laboratory of Immunology, National Institute on Aging, Baltimore, Maryland, USA. * Dan L Longo Contributions L.-J.D. and A.G.F. carried out the experiments. A.D. and D.L.L. helped write the paper and contributed valuable reagents. L.-J.D. and K.G. designed experiments and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kevin Gardner Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (720K) Supplementary Figures 1–8 and Supplementary Methods Additional data - The histone methyltransferase MLL1 permits the oscillation of circadian gene expression
- Nat Struct Mol Biol 17(12):1414-1421 (2010)
Nature Structural & Molecular Biology | Article The histone methyltransferase MLL1 permits the oscillation of circadian gene expression * Sayako Katada1 Search for this author in: * NPG journals * PubMed * Google Scholar * Paolo Sassone-Corsi1 Contact Paolo Sassone-Corsi Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1414–1421Year published:(2010)DOI:doi:10.1038/nsmb.1961Received17 July 2010Accepted25 October 2010Published online28 November 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The classical view of the molecular clock is based on interlocked transcriptional-translational feedback loops. Because a substantial fraction of the mammalian genome is expressed in a circadian manner, chromatin remodeling has been proposed to be crucial in clock function. Here we show that Lys4 (K4) trimethylation of histone H3 is rhythmic and follows the same profile as previously described H3 acetylation on circadian promoters. MLL1, a mammalian homolog of Drosophila trithorax, is an H3K4-specific methyltransferase implicated in transcriptional control. We demonstrate that MLL1 is essential for circadian transcription and cyclic H3K4 trimethylation. MLL1 is in a complex with CLOCK–BMAL1 and contributes to its rhythmic recruitment to circadian promoters and to H3 acetylation. Yet MLL1 fails to interact with CLOCKΔ19, providing an explanation for this mutation's dominant negative phenotype. Our results favor a scenario in which H3K4 trimethylation by MLL1 is required to! establish a permissive chromatin state for circadian transcription. View full text Figures at a glance * Figure 1: Histone H3K4 methyltransferase MLL1 synergistically activate CLOCK–BMAL1-mediated gene transcription. () H3K4me3, but not me1 or me2, levels change between two time points on the Dbp promoter E-box region. ChIP analyses were done in MEFs at 18 or 30 h after dexamethasone synchronization using antibodies against acetylated H3K9 or H3K14 (white) and me1, me2 or me3 H3K4 (black). (Means ± s.e.m. of three independent samples; *P < 0.05, ***P < 0.0001.) The amount at CT 18 was set to 1. () H3K4me3 follows the same circadian rhythmicity as acetylation of H3K9 or H3K14 on the Dbp promoter E-box region. ChIP analyses were done in MEFs after dexamethasone synchronization. (Means ± s.e.m. of three independent samples from a representative experiment. Analogous results were observed in three independent experiments.) () Histone H3K4-specific methyltransferase MLL1 and SET1A were transiently transfected with or without CLOCK–BMAL1 in 293 cells. MLL1, but not SET1A, showed substantial increases in dbp-luc transcription. On the other hand, a methyltransferase-dead mutant of MLL1 (MLL1! Δ)) abolished transcriptional activity. (Means ± s.e.m. of eight samples in two independent experiments.) () Synergistic transcriptional activation on E-box motif by MLL1 together with CLOCK–BMAL1. E-box motif–containing sequences (E-box), NF-κB-responsive element (NF-κB), estrogen responsive element (ERE) or PPAR-responsive element (PPRE) luciferase reporter genes were transfected in presence or absence of CLOCK–BMAL1 in 293 cells. (Means ± s.e.m. of eight samples from two independent experiments.) * Figure 2: MLL1 governs circadian oscillation of histone modifications and gene expression. () MLL1-deficient MEFs have impaired CLOCK–BMAL1-mediated transcriptional activities. MLL1 was expressed with or without CLOCK–BMAL1 in MEFs derived from wild-type (black) or MLL1-KO (gray) mice. In MLL1-KO MEFs, CLOCK–BMAL1-mediated Dbp and Per2 promoter transactivation was basically abolished; however, ectopic expression of MLL1 rescued the expression. (Means ± s.e.m. of seven samples from two independent experiments; *P < 0.001.) () Circadian H3K4me3 is completely impaired in MLL1-KO MEFs. Dexamethasone-treated wild-type (square) and MLL1-KO (triangle) MEFs were analyzed by ChIP using antibodies again H3K4me3 (left) or against acetylated H3K9 and H3K14 (right). (Means ± s.e.m. of three independent samples; ANOVA P < 0.001, and *P < 0.05, **P < 0.001 at t-test.) () Circadian rhythmic expression of Dbp and Per2 were greatly decreased in MLL1-KO MEFs. mRNA was extracted from wild-type (black) or MLL1-KO (gray) MEFs after dexamethasone shock and analyzed by quantitati! ve real-time PCR. The amount of the highest expression time point was set to 1. (Means ± s.e.m. of three samples; ANOVA P < 0.001, and *P < 0.05 at t-test.) * Figure 3: Global analysis of gene expression profile in WT and MLL1-KO MEFs. mRNA was extracted from wild-type or MLL1-KO MEFs after 18 h or 30 h after serum shock, and microarray analyses were done using GeneChip Mouse Gene 1.0 ST Array. Out of 35,557 probes, 41 genes were identified as having expression levels that changed significantly (>1.6-fold) between two time points in WT MEFs. The heat map represents the fold changes of these 41 genes in WT and MLL1-KO MEFs. The pie chart represents functional categories of genes that changed in wild-type MEFs. Eight out of the 41 genes are listed as typical circadian-regulated genes in Table 1. * Figure 4: MLL1 interacts with CLOCK and BMAL1 and is recruited in circadian fashion. () CLOCK and BMAL1, but not CLOCKΔ19, were co-immunoprecipitated with MLL1. 293 cells were transfected with indicated expression vectors together with or without Flag-MLL1-Myc. Flag-tagged MLL1 N-terminal fragments (320 kDa) were immunoprecipitated by Flag-Agar, and co-immunoprecipitated proteins were determined by western analysis using anti-Myc and anti-V5 antibodies. Together with Myc-tagged 180-kDa MLL1 C-terminal fragments (arrow), Myc-BMAL1 (white asterisk) and Myc-CLOCK (black asterisk) proteins were co-immunoprecipitated with MLL1 N-terminal fragments. On the other hand, CLOCKΔ19, CRY1, CRY2, PER1 and PER2 were not co-immunoprecipitated with MLL1. Lower panel shows the results of total cell lysates as an input. () Endogenous MLL1 interacts with CLOCK in a circadian manner. MEFs were entrained by serum shock and harvested at various circadian times, and then cellular extracts were immunoprecipitated using anti-CLOCK antibody. Co-immunoprecipitated proteins were dete! rmined using anti-BMAL1 and anti-MLL1 antibodies. The lower panel shows the results of total cell lysates as an input. () MLL1 recruited to E-box proximal region of the Dbp promoter in a circadian manner. ChIP analyses were done on dual cross-linked MEFs at 18 or 30 h after dexamethasone synchronization using antibodies against BMAL1 (gray) and MLL1 (black), and quantitative PCR was done using 5′ UTR, E-box or 3′ UTR primers. (Means ± s.e.m. of six independent samples; *P < 0.05, **P < 0.01) () ChIP analyses on the Dbp promoter E-box region in wild-type or MLL1-KO MEFs. Analyses were done on dual cross-linked MEFs at 18 or 30 h after dexamethasone treatment using antibodies against BMAL1, CLOCK and MLL1. The amount of the highest binding time point (18 h) in wild-type MEFs was set to 1. (Means ± s.e.m. of six independent samples; *P < 0.01.) * Figure 5: Impaired H3K4me3 and less recruitment of BMAL1 and MLL1 proteins in c/c mutant MEFs. () Rhythmic H3K4me3 was completely abolished in c/c mutant MEFs. ChIP analyses were done on the Dbp promoter E-box region in wild-type (square) or c/c mutant (triangle) MEFs at each time point after dexamethasone shock. () Luciferase reporter gene assay using the Dbp promoter in c/c mutant MEFs. MLL1 was transiently transfected with or without CLOCK–BMAL1 in wild-type (black) or c/c mutant (gray) MEFs, and luciferase activity was measured after 48 h. Relative light units obtained from each CLOCK–BMAL1 and MLL1-transfected MEFs were set to 100 (%). (Means ± s.e.m. of four independent samples; *P < 0.001.) () Circadian recruitment of BMAL1 and MLL1 on the Dbp promoter E-box regions was abolished in c/c mutant MEFs. Dexamethasone-shocked and dual cross-linked wild-type and c/c mutant MEFs were analyzed by ChIP using anti-BMAL1 (left) or anti-MLL1 (right) antibodies. (Means ± s.e.m. of three independent samples; *P < 0.05, **P = 0.0512.) () MLL1 protein levels of MLL1 do n! ot change in the liver from c/c mice. Proteins were extracted from each time point of liver from WT or c/c mutant mice, and MLL1 expression levels were detected using anti-MLL antibodies. () Rescue of circadian mRNA expression of clock genes by stably expressing MLL1 in MLL1-KO MEFs. mRNA was extracted from wild-type (square), Flag-MLL1 stably expressed MLL1-KO (diamond) or mock transfected MLL1-KO (triangle) MEFs after dexamethasone shock, and Dbp and Per2 expression levels were analyzed by quantitative real-time PCR. (Means ± s.e.m. of four independent samples; *P < 0.05, **P < 0.001.) * Figure 6: CLOCK and MLL1 physically interact at the level of clock-controlled promoters (with E-boxes). Together they induce PTMs on histone N-terminal tails associated with circadian transcriptional activation. The mutant protein CLOCKΔ19 fails to associate with MLL1 (bottom), providing a molecular explanation of its impaired activation potential. As MLL1 and CLOCKΔ19 do not interact, there is also a lack of PTMs on the H3 tail, leading to a lack of circadian transcription. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24964 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Unite 904 INSERM–Epigenetic Control and Neuronal Plasticity, Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, California, USA. * Sayako Katada & * Paolo Sassone-Corsi Contributions S.K. conceived the project, designed and conducted the experiments, and wrote the manuscript. P.S.-C. conceived the project, provided conceptual support and contributed to writing the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paolo Sassone-Corsi Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (216K) Supplementary Figures 1–8 and Supplementary Table 1 Additional data - Optical trapping with high forces reveals unexpected behaviors of prion fibrils
- Nat Struct Mol Biol 17(12):1422-1430 (2010)
Nature Structural & Molecular Biology | Article Optical trapping with high forces reveals unexpected behaviors of prion fibrils * Jijun Dong1 Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos E Castro2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mary C Boyce2 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew J Lang2, 3, 5 Contact Matthew J Lang Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Lindquist1, 4 Contact Susan Lindquist Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1422–1430Year published:(2010)DOI:doi:10.1038/nsmb.1954Received04 June 2010Accepted18 October 2010Published online28 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we describe methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A crucial advance was to use the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces before fibril rupture. Experiments with selective amyloid-disrupting agents and mutations demonstrated that such discontinuities were caused by the unfolding of individual subdomains. Thus, our results reveal unusually strong noncovalent intermolecular contacts that maintain fibril integrity even when individual monomers partially unfold and e! xtend fibril length. View full text Figures at a glance * Figure 1: The biological homogeneity of NM fibrils was confirmed by protein-only transformation. () Diagram of wild-type Sup35. The black segments in the N-terminal region of Sup35 (N) represent oligopeptide repeats. The highly charged nature of the middle region (M) is indicated. The C-terminal region (C) functions in translation termination. In –, biologically distinct prion states were induced when prion-minus ([psi−]) cells were transformed with fluorescently labeled fibrils formed under different conditions. () Colonies transformed with buffer alone. () Colonies transformed with spontaneously assembled NM fibrils. A mixture of prion phenotypes is highlighted in the blue box. () Colonies after protein-only transformation at 4 °C with NM fibrils seeded by lysates of yeast cells that carried a strong prion element. The uniform strong prion phenotypes ([PSI+]) are indicated by the light pink-colored colonies in the blue box. () Colonies after protein-only transformation at 37 °C with NM fibrils seeded by lysates from cells carrying a weak prion element. The unifo! rm weak prion phenotypes are indicated by the dark salmon-colored colonies in the blue box. () Quantification of transformation efficiency and specificity of distinct prion fibrils. 'Strong fibrils' refers to the fibrils used in ; 'weak fibrils' refers to the fibrils used in . * Figure 2: NM monomers effectively and specifically bind individual NM fibrils to a glass surface on one end and a polystyrene bead on the other. () Schematic diagram of the method for the attachment of NM fibrils. Monomers (blue) are adhered to the glass surface, and the remaining surface is coated with casein (light green). Green circles, fluorescently labeled NM monomers; white circles, wild-type NM monomers. Polystyrene beads (light blue circles) labeled with Alexa 488 (green triangle) and Alexa 555–NM (blue) are introduced and captured by the other free ends of the fibrils. () Top, the molecular architectures and amino acid compositions of Saccharomyces NM (blue) and Candida NM (orange). Bottom, schematic illustration of the experiment showing the specificity between prebound monomers and fibrils. From left to right, deposition of Candida NM on the glass surface; incubation of Saccharomyces NM fibrils in the flow channel; imaging after washing. () Schematic diagram of the detachment of NM fibrils from the glass surface with a treatment of TEV protease. The TEV recognition site is shown in red and highlighted in! the enlarged segments. The M domain (blue line) binds with the glass surface, and the N domain (blue circle) interacts with the ends of NM fibrils. The bead was held in position by the optical trapping (light pink). * Figure 3: Stretching NM fibrils by optical trapping at high forces with simultaneous fluorescent imaging. () Schematic of the trapping instrument with dual trapping capabilities used for high-force experiments (see Online Methods for details). 1,024 nm, trapping laser; 975 nm, position-detection laser; 532 nm, fluorescence imaging. () Schematic representation and fluorescence snapshots of the experiments. From left to right, the bead was first centered on top of the attachment point, and then the stage was moved along the x axis until the fibril was fully extended. () Representative pulling and relaxation traces for NM fibrils in the presence of normal assembly buffer. The force and the position of the stage were plotted as a function of time. Black line, stretching and holding phase; red line, relaxation phase; blue line, position of the bead relative to the attachment point of the tether on the glass surface. () Left, force applied versus fibril extension length (force-extension curve) for the tethered fibril experiencing one stretching-relaxation cycle. Black line, stretching! and holding phase; red line, relaxation phase. The x axis is expanded to illustrate the behavior in the fully extended position. Right, the force-extension curves for a single tethered fibril that experienced four sequential stretching-relaxation cycles, whose curves are superimposed upon each other. For clarity, the holding phase is not shown. Stretching and relaxation curves from the same cycle are shown in the same color, with subsequent curves in a different color. * Figure 4: Representative pulling traces (force versus time) for NM fibrils under different experimental conditions. () Force-extension curves of NM fibrils in the presence of 1.2 M GdHCl. Black arrows mark sudden force changes in the stretching and holding traces. Red arrows mark rupture of the tethered fibril with a concomitant drop to zero force. () Representative force-extension curves for two NM fibrils that experienced multiple stretching-holding-relaxation cycles in the presence of 1.2 M GdHCl. Black, light blue, blue, light pink and green correspond to cycles 1–5, respectively, for each fibril. For clarity, only stretching phases are shown. () Force-extension curves in the presence of 50 μM DAPH. () The structures of DAPH and SA. * Figure 5: Expansion or deletion of the repeat region markedly changed mechanical behavior of prion fibrils. () A previously proposed β-helix model for NM fibrils. The M domain is not involved in the amyloid core and is not shown. Top left, color scheme for the N domain. Inset at top right, enlarged view of two monomers. (,) Top, schematic illustrations of R2E2 and RΔ2–5. The blue segments in the N-terminal region represent oligopeptide repeats. Middle, R2E2 () and RΔ2–5 () fibrils imaged by transmission EM. Scale bar, 100 nm. The R2E2 fibrils had an average diameter of 16 ± 2 nm, slightly wider than that of wild-type 'strong' NM fibrils, 12 ± 2 nm. The RΔ2–5 fibrils had a diameter comparable to that of wild-type fibrils, but many showed a distinct wavy morphology. Bottom, protein-only transformation of R2E2 () and RΔ2–5 () fibrils. () Representative force-extension curves for R2E2 (top) and RΔ2–5 (bottom) fibrils. Black traces, stretching phase; red traces, relaxation phase; black arrows, force-dropping events in the stretching curves; green arrows, force plateau! s in the relaxation curves at low forces. Inset in bottom graph shows expansion under the low-force regime. The stretching and relaxation curves were fit to modified WLC models. The deviation of the relaxation curves from the WLC fitting (red line in the inset) is indicated by the green arrow. * Figure 6: The distribution of the length of extension is broad. () Left, an example of WLC fits used to determine the length added to the fibril at each force-dropping event that occurred during the stretching phase. Right, a linear correlation was obtained between the length of extension determined from WLC fits and the instantaneous bead displacement that occurred upon unfolding. Black points, extension lengths from WLC fits to wild-type NM fibrils; gray points, extension lengths from WLC fits to RΔ2–5 fibrils. () Histogram of extension lengths for NM fibrils at each force-dropping event in the presence of 1.2 M GdHCl (left) or 50 μM DAPH (right). () Histogram of extension lengths obtained with RΔ2–5 fibrils. A 12-nm and a 24-nm bin width were used for and , respectively (Supplementary Methods). () Rupture time versus force for the RΔ2–5 fibrils. The rupture events that occurred during the holding phase were fit to a Bell model as described in the Online Methods. * Figure 7: Cartoon representation of the mechanical unfolding of NM amyloid fibrils with different structures. () A possible mechanism with the β-pleated-sheet model. () A possible mechanism with the β-helix model. See Discussion for details. Author information * Abstract * Author information * Supplementary information Affiliations * Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. * Jijun Dong & * Susan Lindquist * Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Carlos E Castro, * Mary C Boyce & * Matthew J Lang * Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Matthew J Lang * Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Susan Lindquist * Present address: Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, Tennessee, USA. * Matthew J Lang Contributions J.D. initiated the project and carried out sample preparation and yeast biology; J.D. and C.E.C. designed and carried out the optical-trapping experiments and data analysis; M.C.B., M.J.L. and S.L. supervised the projects and interpreted the results; J.D. and S.L. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Matthew J Lang or * Susan Lindquist Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (594K) Movie S1. The NM fibrils were tethered to the glass surface. The movie was taken from a standard inverted fluorescence microscope with a 100× objective lens (field of observation 60 μm × 60 μm). The main bodies of the tethered fibrils were often out of focus as seen at the beginning of the movie. In the middle of the experiment, flow was introduced to straighten the fibrils for better visualization. The direction of the flow was changed during the experiments to demonstrate that these fibrils were indeed attached to the surface at one end while the main bodies were mobile. * Supplementary Movie 2 (12M) Movie S2. Fluorescence image of tethered NM fibrils on the optical trapping instrument (field of observation 15 μm × 15 μm). The distribution of tethered fibrils in the movie was illustrated below. Fibrils with one end attached to the glass surface are highlighted by green arrows, while fibrils with both ends attached to the glass surface are highlighted by red arrows. We optimized the procedure for each experiment to reduce the number of fibrils double-tethered to the glass surface. * Supplementary Movie 3 (9M) Movie S3. The NM prion domain from Candida albicans Sup35 homolog did not capture S. cerevisiae NM fibrils. When CaNM monomers were pre-deposited to the glass surface followed by casein blocking, ScNM fibrils were rarely recruited and tethered to the surface. Typically no more than three ScNM fibrils were observed in the field of observation (60 μm × 60 μm) and the movie showed the extreme. * Supplementary Movie 4 (1M) Movie S4 – 6. Cleavage of NM monomers at the junction between the fibril and the glass surface released tethered NM fibrils. S4. Before the TEV treatment to release tethered NM fibrils. The fluorescent channel was opened transiently for imaging to minimize fluorescence bleaching. * Supplementary Movie 5 (1M) Movie S4 – 6. Cleavage of NM monomers at the junction between the fibril and the glass surface released tethered NM fibrils. S5. After the TEV treatment to release tethered NM fibrils. 20 μl of 20 unit μl−1 TEV protease was introduced into the chamber by flow. This fluorescent image was recorded after 10-minute incubation of TEV protease. The bead that was previously attached to one end of the fibril and was trapped with the optical laser is visible in the image. However, the previously tethered fibril shown in movie S4 was no longer detected after the TEV protease treatment. We suspected that, after release from the glass surface, the flexible fibril might collapse and be hidden from view by the bead. * Supplementary Movie 6 (1M) Movie S4 – 6. Cleavage of NM monomers at the junction between the fibril and the glass surface released tethered NM fibrils. S6. After the TEV treatment to release tethered NM fibrils, imaged with a constant flow. To test if the released fibril was hidden from view by the bead, we asked if we could extend the hidden fibril with a constant flow of 1xCRBB buffer through the flow channel. The flow is readily detectable in this movie by the free objects in the flow channel flowing from the left lower corner to the upper right corner in the movie. Indeed, in the presence of a constant flow, the fibril that had been released from the glass surface by the TEV cleavage was now visible, aligning itself with the flow. * Supplementary Movie 7 (2M) Movie S7 – 8. Typical pulling behavior of tethered NM fibrils. Prior to each pulling experiment, the tethered fibril and the attached bead were fluorescently imaged to confirm the nature of the tethers. The instrument we employed to achieve low trapping forces allowed an interlaced optical force and fluorescence (IOFF). In short, fluorescence and excitation lasers were cycled out of phase at 50 kHz with a 10% duty cycle lag time in between to allow excited electrons to return to their ground state. This method prolonged the fluorescence signal from the bead and the fibril and enabled imaging of the fibril morphology throughout the force-extension experiment. S7. Fluorescent image of the tethered NM fibril when the trapping laser was not turned on. * Supplementary Movie 8 (8M) Movie S7 – 8. Typical pulling behavior of tethered NM fibrils. Prior to each pulling experiment, the tethered fibril and the attached bead were fluorescently imaged to confirm the nature of the tethers. The instrument we employed to achieve low trapping forces allowed an interlaced optical force and fluorescence (IOFF). In short, fluorescence and excitation lasers were cycled out of phase at 50 kHz with a 10% duty cycle lag time in between to allow excited electrons to return to their ground state. This method prolonged the fluorescence signal from the bead and the fibril and enabled imaging of the fibril morphology throughout the force-extension experiment. S8. The trapping laser was turned on to perform the pulling experiments. * Supplementary Movie 9 (2M) Movie S9– 14. Direct observation of the rupture of tethered NM fibrils by fluorescent imaging. We trapped the bead at the extended position and manually aligned the fibril with the x-axis of the piezo stage without centering. Without the prolonged process of centering, trap-accelerated photo bleaching was minimized and fluorescent images could be recorded stably before rupture. Note however that the fragment of the fibril closest to the bead still became photobleached during this process. Constant high force was then applied to the bead in the absence of fluorescent imaging until the tether was ruptured. To establish the position of rupture, fluorescent imaging was re-initiated. In movies recorded after rupture, fragments of the previously tethered fibrils could be seen still attached to the surface, and these represented varying fractions of the initial fibril length. Other objects in the field of view provide a point of reference for the ruptured fibril. S9 – 10. Ruptu! re occurred in the middle of this fibril. S9, before rupture; S10, after rupture. The portion of the fibril left on the glass surface moved freely relative to the trapped bead after rupture. The trapped bead was able to move freely with pulling force, confirming that the connection to the fibril had been ruptured. * Supplementary Movie 10 (26M) Movie S9 – 14. Direct observation of the rupture of tethered NM fibrils by fluorescent imaging. We trapped the bead at the extended position and manually aligned the fibril with the x-axis of the piezo stage without centering. Without the prolonged process of centering, trap-accelerated photo bleaching was minimized and fluorescent images could be recorded stably before rupture. Note however that the fragment of the fibril closest to the bead still became photobleached during this process. Constant high force was then applied to the bead in the absence of fluorescent imaging until the tether was ruptured. To establish the position of rupture, fluorescent imaging was re-initiated. In movies recorded after rupture, fragments of the previously tethered fibrils could be seen still attached to the surface, and these represented varying fractions of the initial fibril length. Other objects in the field of view provide a point of reference for the ruptured fibril. S9 – 10.Ruptu! re occurred in the middle of this fibril. S9, before rupture; S10, after rupture. The portion of the fibril left on the glass surface moved freely relative to the trapped bead after rupture. The trapped bead was able to move freely with pulling force, confirming that the connection to the fibril had been ruptured. * Supplementary Movie 11 (3M) Movie S9 – 14. Direct observation of the rupture of tethered NM fibrils by fluorescent imaging. We trapped the bead at the extended position and manually aligned the fibril with the x-axis of the piezo stage without centering. Without the prolonged process of centering, trap-accelerated photo bleaching was minimized and fluorescent images could be recorded stably before rupture. Note however that the fragment of the fibril closest to the bead still became photobleached during this process. Constant high force was then applied to the bead in the absence of fluorescent imaging until the tether was ruptured. To establish the position of rupture, fluorescent imaging was re-initiated. In movies recorded after rupture, fragments of the previously tethered fibrils could be seen still attached to the surface, and these represented varying fractions of the initial fibril length. Other objects in the field of view provide a point of reference for the ruptured fibril. S11 – 12.Rupt! ure occurred close to the bead. S11, before rupture; S12, after rupture. * Supplementary Movie 12 (13M) Movie S9 – 14. Direct observation of the rupture of tethered NM fibrils by fluorescent imaging. We trapped the bead at the extended position and manually aligned the fibril with the x-axis of the piezo stage without centering. Without the prolonged process of centering, trap-accelerated photo bleaching was minimized and fluorescent images could be recorded stably before rupture. Note however that the fragment of the fibril closest to the bead still became photobleached during this process. Constant high force was then applied to the bead in the absence of fluorescent imaging until the tether was ruptured. To establish the position of rupture, fluorescent imaging was re-initiated. In movies recorded after rupture, fragments of the previously tethered fibrils could be seen still attached to the surface, and these represented varying fractions of the initial fibril length. Other objects in the field of view provide a point of reference for the ruptured fibril. S11 – 12.Rupt! ure occurred close to the bead. S11, before rupture; S12, after rupture. * Supplementary Movie 13 (3M) Movie S9 – 14. Direct observation of the rupture of tethered NM fibrils by fluorescent imaging. We trapped the bead at the extended position and manually aligned the fibril with the x-axis of the piezo stage without centering. Without the prolonged process of centering, trap-accelerated photo bleaching was minimized and fluorescent images could be recorded stably before rupture. Note however that the fragment of the fibril closest to the bead still became photobleached during this process. Constant high force was then applied to the bead in the absence of fluorescent imaging until the tether was ruptured. To establish the position of rupture, fluorescent imaging was re-initiated. In movies recorded after rupture, fragments of the previously tethered fibrils could be seen still attached to the surface, and these represented varying fractions of the initial fibril length. Other objects in the field of view provide a point of reference for the ruptured fibril. S13 – 14.Rupt! ure occurred close to the glass surface. S13, before rupture; S14, after rupture. The small fragment remaining on the surface couldn't be identified but the fibril, still attached to the bead, could be visualized. To ensure that rupture of the tether had occurred we then flowed buffer through the chamber. This reoriented the fibril – now tethered only to the bead – relative to its original orientation. * Supplementary Movie 14 (22M) Movie S9 – 14. Direct observation of the rupture of tethered NM fibrils by fluorescent imaging. We trapped the bead at the extended position and manually aligned the fibril with the x-axis of the piezo stage without centering. Without the prolonged process of centering, trap-accelerated photo bleaching was minimized and fluorescent images could be recorded stably before rupture. Note however that the fragment of the fibril closest to the bead still became photobleached during this process. Constant high force was then applied to the bead in the absence of fluorescent imaging until the tether was ruptured. To establish the position of rupture, fluorescent imaging was re-initiated. In movies recorded after rupture, fragments of the previously tethered fibrils could be seen still attached to the surface, and these represented varying fractions of the initial fibril length. Other objects in the field of view provide a point of reference for the ruptured fibril. S13 – 14. Rup! ture occurred close to the glass surface. S13, before rupture; S14, after rupture. The small fragment remaining on the surface couldn't be identified but the fibril, still attached to the bead, could be visualized. To ensure that rupture of the tether had occurred we then flowed buffer through the chamber. This reoriented the fibril – now tethered only to the bead – relative to its original orientation. PDF files * Supplementary Text and Figures (604K) Supplementary Figures 1–4 and Supplementary Methods Additional data - Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain
- Nat Struct Mol Biol 17(12):1431-1437 (2010)
Nature Structural & Molecular Biology | Article Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain * Stefano Gianni1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ylva Ivarsson1, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Alfonso De Simone2 Search for this author in: * NPG journals * PubMed * Google Scholar * Carlo Travaglini-Allocatelli1 Search for this author in: * NPG journals * PubMed * Google Scholar * Maurizio Brunori1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michele Vendruscolo2 Contact Michele Vendruscolo Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1431–1437Year published:(2010)DOI:doi:10.1038/nsmb.1956Received31 March 2010Accepted08 October 2010Published online14 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Incorrectly folded states transiently populated during the protein folding process are potentially prone to aggregation and have been implicated in a range of misfolding disorders that include Alzheimer's and Parkinson's diseases. Despite their importance, however, the structures of these states and the mechanism of their formation have largely escaped detailed characterization because of their short-lived nature. Here we present the structures of all the major states involved in the folding process of a PDZ domain, which include an off-pathway misfolded intermediate. By using a combination of kinetic, protein engineering, biophysical and computational techniques, we show that the misfolded intermediate is characterized by an alternative packing of the N-terminal β-hairpin onto an otherwise native-like scaffold. Our results suggest a mechanism of formation of incorrectly folded transient compact states by which misfolded structural elements are assembled together with more ! extended native-like regions. View full text Figures at a glance * Figure 1: Folding kinetics of D1pPDZ. () Representative refolding trace of D1pPDZ. Gray line is the best fit to a double exponential. () Trace of a mutant (L245A) that populates almost exclusively the misfolded intermediate. The gray line is the best fit to a single exponential. Although refolding of D1pPDZ is biphasic, the mutant shows only the fast phase, reflecting the transition from the denatured state to the misfolded intermediate. * Figure 2: Comparison between the chevron plots of D1pPDZ and of six representative mutants (Y169A, A183G, V198A, L210A, S211G and L245A). Empty circles refer to the kinetic data of intermediate (un)folding, and filled circles to native (un)folding. The folding of D1pPDZ is indicated by gray symbols and lines. Mutant proteins are indicated by black symbols and lines. Data for all variants (except L245A) are fitted to a three-state equation with an off-pathway intermediate. Because of the complexity of the kinetic model, the m values for the microscopic rate constants were assumed to be the same as those determined for wild-type D1pPDZ. The kinetic data of L245A (un)folding were fitted to a two-state equation. * Figure 3: Structural distribution of the ΦI values in the misfolding reaction of D1pPDZ. () Mapping of the experimentally measured ΦI values on the native structure of D1pPDZ. The ΦI values are divided into four categories and color coded accordingly: white, 0 < ΦI < 0.33; magenta, 0.33 < ΦI < 0.66; deep blue, 0.66 < ΦI < 1; and cyan, ΦI > 1. () Mutations at ten positions (shown in yellow or red spheres) destabilize the native state but do not substantially destabilize the intermediate, thereby shifting the equilibrium toward the intermediate (Supplementary Table 1). The V177A and L212A mutations (at the positions marked by red spheres) stabilize the intermediate while destabilizing the native state, indicating that the two residues have a negative ΦI value. * Figure 4: Comparison of the structures of the intermediate and the native states. () Intermediate state. () Native state. The upper panels illustrate the packing between the N-terminal β1-β2 hairpin and the rest of the protein. The β1-β2 hairpin is drawn as cyan ribbons, with side chains explicitly shown; the remaining part of the protein is drawn as red ribbons and gray surfaces. The structures of the intermediate and native states are shown in the lower panels. The β1-β2 hairpin is drawn as red ribbons and the rest of the protein as cyan ribbons. The single tryptophan residue in position 178 is partly solvent exposed in the native state yet is completely buried in the intermediate state. * Figure 5: Comparison of the spectroscopic and functional properties of D1pPDZ (gray) and three mutants that populate the intermediate state, V163A (blue), A193G (red) and L231A (black). () Fluorescence emission spectra of D1pPDZ. () Near-UV circular dichroism spectra. () Equilibrium binding titration to the peptide EAPSVNA monitored by the change in intrinsic fluorescence at 350 nm. The experiments were done in 50 mM sodium phosphate, pH 7.2, at a constant protein concentration of 5 μM. Whereas D1pPDZ yields a simple hyperbolic transition, no detectable change in fluorescence could be observed with the mutants. * Figure 6: Comparison of the TSN and TSI structures of D1pPDZ. () Conformational ensemble of TSI. () Conformational ensemble of TSN. The color codes are gray (loops), purple (helices), yellow (β-strands) and red (β1 and β2). () Contact map of TSI. () Contact map of TSN. The contact maps are averaged over the structural ensembles; for each member of the ensemble, a contact is assigned if at least an inter-residue distance (heavy atoms only) is below 6.5 Å. Probabilities are normalized to range from 0 (for not contacting residues) to 1 (for tightly contacting residues). Red circles indicate contacts between strands β1 and β2. Orange circles indicate contacts between the β1-β2 hairpin and the rest of the protein. * Figure 7: Comparison of the solvent exposure of the aggregation-prone regions of D1pPDZ in the native and in the misfolded intermediate case. () Native structure, which has been color coded according to the sequence-dependent aggregation propensity45, which is shown in ; blue indicates highly aggregation-prone regions, and red indicates aggregation-resistant regions. () Sequence-dependent aggregation propensity of D1pPDZ; green shaded areas indicate regions that are highly aggregation prone. () Misfolded intermediate structure, color coded in the same manner as for the native state. () Comparison of the solvent-exposed surface area (SASA) between the native state (black line) and the misfolded intermediate (blue line); the aggregation-prone regions in the N-terminal part of the protein, which corresponds to the β1-β2 hairpin, are substantially more solvent exposed in the misfolded intermediate than in the native state. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Stefano Gianni & * Ylva Ivarsson Affiliations * Istituto Pasteur–Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Università di Roma 'La Sapienza', Rome, Italy. * Stefano Gianni, * Ylva Ivarsson, * Carlo Travaglini-Allocatelli & * Maurizio Brunori * Department of Chemistry, University of Cambridge, Cambridge, UK. * Alfonso De Simone & * Michele Vendruscolo * Present address: Department of Human Genetics, Katholieke Universiteit Leuven, Herestraat, Leuven, Belgium. * Ylva Ivarsson Contributions S.G., Y.I., C.T.-A. and M.B. conceived and designed the experimental work; A.D.S. and M.V. conceived and designed the computational work; Y.I. expressed and purified the protein samples; S.G. and Y.I. conducted the experiments; A.D.S. did the simulations; S.G., Y.I., A.D.S., C.T.-A., M.B. and M.V. analyzed the data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michele Vendruscolo Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (480K) Supplementary Figure 1 and Supplementary Table 1 Additional data - Reduced Rif2 and lack of Mec1 target short telomeres for elongation rather than double-strand break repair
- Nat Struct Mol Biol 17(12):1438-1445 (2010)
Nature Structural & Molecular Biology | Article Reduced Rif2 and lack of Mec1 target short telomeres for elongation rather than double-strand break repair * Jean S McGee1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jane A Phillips1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Angela Chan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michelle Sabourin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Katrin Paeschke1 Search for this author in: * NPG journals * PubMed * Google Scholar * Virginia A Zakian1 Contact Virginia A Zakian Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1438–1445Year published:(2010)DOI:doi:10.1038/nsmb.1947Received04 May 2010Accepted07 September 2010Published online07 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Telomerase in Saccharomyces cerevisiae binds and preferentially elongates short telomeres, and this process requires the checkpoint kinase Tel1. Here we show that the Mre11 complex bound preferentially to short telomeres, which could explain the preferential binding of Tel1 to these ends. Compared to wild-type length telomeres, short telomeres generated by incomplete replication had low levels of the telomerase inhibitory protein Rif2. Moreover, in the absence of Rif2, Tel1 bound equally well to short and wild-type length telomeres, suggesting that low Rif2 content marks short telomeres for preferential elongation. In congenic strains, a double-strand break bound at least 140 times as much Mec1 in the first cell cycle after breakage as did a short telomere in the same time frame. Binding of replication protein A was also much lower at short telomeres. The absence of Mec1 at short telomeres could explain why they do not trigger a checkpoint-mediated cell-cycle arrest. View full text Figures at a glance * Figure 1: MRX binds preferentially to short telomeres. () Schematic short telomere assay: structures of VII-L end before (parental) and after FLP recombination in experimental (left) and control (right) strains. Recombination generates a VII-L telomere with ~100-bp telomeric DNA in experimental strain (short) or ~300-bp telomere (wild-type length, WT) in control strain. Restriction enzyme sites: X, XhoI; S, StuI; V, EcoRV; P, PstI; H, HindIII. () We arrested experimental and control strains expressing Mre11-Myc, Rad50-Myc or Xrs2-Myc, induced FLP and removed cells from both alpha factor and galactose (0 min) to proceed through synchronous cell cycle. Squares indicate mean ± s.d. fold enrichment of tagged protein to short (open squares) or wild-type (closed squares) VII-L telomere compared with binding to non-telomeric ARO1 locus in the same sample. Untag, untagged protein. () Using the same samples as in , we determined the mean ± s.d. binding of indicated protein to wild-type length VI-R telomere in experimental (open triangl! es) and control (closed triangles) strains. The binding of each protein to VI-R telomeres in the two strains was not significantly different at any time point (P ≥ 0.12 for all). () Xrs2 binds preferentially to short telomeres for at least two cell cycles. Experimental strain expressing Xrs2-Myc was synchronized. Samples were taken over two cell cycles and processed for ChIP. Plot shows mean ± s.d. binding of Xrs2-Myc to short VII-L (open squares) and wild-type length VI-R (open triangles) telomeres. * Figure 2: Mec1-HA does not bind preferentially to short telomeres, even in tel1Δ cells. (–) The control and experimental strains expressing Mec1-HA () or Tel1-HA () were synchronized and processed as described (Fig. 1). () Mec1-HA bound to the VII-L telomeres at low levels at 60, 75 and 90 min. The timing and level of Mec1-HA binding to the short VII telomere (open squares) and the wild-type length VII-L telomere (closed squares) were indistinguishable at all time points (P = 0.19–0.96). () Binding of Mec1-HA to the wild-type length VI-R and XV-L telomeres was indistinguishable from binding to the short VII-L telomere (taken from and shown for comparison) except at 75 and 90 min (P = 0.01 and 0.03, respectively). Only data from the experimental strain are shown. () The experiment in was carried out in tel1Δ versions of the control and experimental strains expressing Mec1-HA. Binding to the short and wild-type length VII-L telomeres was indistinguishable from binding to the non-telomeric ARO1 locus in the same samples (to which the values are normalized). (! ) Experiments to determine whether Tel1-HA binding is affected by absence of Mec1 were done in an sml1Δ derivative of the wild-type and mec1Δ strains53. Binding of Tel1-HA to the short VII-L telomere or the wild-type VI-R telomeres was indistinguishable in sml1Δ (MEC1) and mec1Δ sml1Δ (mec1) cells (P = 0.08–0.84 for 30–90 min). * Figure 3: Mec1 and Rfa1 bind DSBs, even when the break is adjacent to telomeric DNA. () Schematic of chromosome VII-L end in DSB strains24. TG80-HO contains 80-bp TG1–3 on centromere side of HO site; N80-HO contains 80-bp lambda DNA. V, EcoRV sites. () Binding of Mec1-HA to DSBs. For and cells were synchronized and processed as described in Figure 1 except that an extra sample was taken from G1-arrested cells before addition of galactose. Binding of Mec1 was determined at N80-HO (closed circles) and TG80-HO (open circles) before and after HO induction. Results are mean ± s.d. fold enrichment over binding to control site ARO1. () Rfa1-Myc binding to DSBs determined at N80-HO (closed circles) and TG80-HO (open circles) before and after induction of HO. Results are mean ± s.d. percent of DNA in input. Binding of Rfa1-Myc to N80-HO was higher than at TG80-HO break (P = 0.00037–0.017) in all post-galactose samples except the 45-min time point (P = 0.093). () Samples used in were examined for binding of Rfa1-Myc to internal ARO locus in both TG80 (open trian! gles) and N80 (closed triangles) strains. The level and timing of Rfa1-Myc binding was equivalent in the two strains. () Binding of Cdc13-Myc to TG80 (open circles) and N80 (closed circles) in the first cell cycle after breakage. () Results from for the N80 strain with expanded scale. * Figure 4: Rfa1 binding and H2A phosphorylation are similar at short and wild-type length telomeres. Rfa1-Myc cells were treated as in Figure 1 (–) or immunoprecipitated with anti-γ-H2A serum (–). Data are mean ± s.d. percent immunoprecipitated (IP) DNA. () Binding of Rfa1-Myc to short and wild-type length VII-L telomeres. Mean enrichment at short VII-L was modestly higher than for wild-type length VII-L telomeres, but the difference was significant only at 37.5 min (P = 0.02; P for other time points 0.08–0.42). Rfa1-Myc bound at the time of telomere replication (60–75 min). () Binding of Rfa1-MYC to short VII-L telomere (open squares; data from ), wild-type length VI-R (open triangles) and XV-L (open circles) telomeres in experimental strain was indistinguishable except at early points (P = 0.03–0.45 for 45–90 min). Data from control strain were also indistinguishable from binding in experimental strain (P = 0.17–0.98). () Binding of Rfa1-Myc to ARO1 locus is the same in experimental (open triangles) and control (closed triangles) strains (P = 0.19–0.95).! () H2A phosphorylation was similar at short (open squares) and wild-type length (closed squares) VII-L telomeres except at 75 min (P = 0.04). () γ-H2AX levels at VI-R and XV-L telomeres were constant throughout cell cycle. Binding to telomeres is shown only for the experimental strain, but values for both telomeres were indistinguishable in the control versus experimental strains (P = 0.08–0.98). () γ-H2AX phosphorylation at RPL11A (diamonds) and ARO1 (triangles) in experimental strain. * Figure 5: Rif2 (and Rap1) but not Rif1 or Yku80 occupancy are reduced at short telomeres. () Schematic of assay. () Rif2 but not Rif1 content is lower at short than at wild-type length telomeres. Samples from three (Yku80 and Rap1) or five (Rif1 and Rif2) independent TLC1 or tlc1Δ spore clones were subjected to ChIP after ~25–30 generations of spore outgrowth. Samples expressing Myc-tagged proteins () were immunoprecipitated with an anti-Myc antibody. Samples from an untagged strain were immunoprecipitated with anti-Rap1 antibody (). Purified DNA was analyzed by quantitative PCR to determine the level of protein binding to VI-R and XV-L telomeres. Data are expressed as the mean ± s.d. fold enrichment of telomeric sequence over the non-telomeric ARO1 sequence in the same immunoprecipitate. The differences between binding of Yku80 (P = 0.354, VI-R; 0.840, XV-L) and Rif1 (P = 0.731, VI-R; 0.492, XV-L) at wild-type length and short telomeres were not significant. The binding of Rap1 (P = 0.026, VI-R; 0.009, XV-L) and Rif2 (P = 1.2 × 10−3, VI-R; 1.3 × 10−6, ! XV-L) were significantly different at wild-type length and short tlc1Δ telomeres. * Figure 6: Preferential binding of Tel1 to short telomeres is lost in rif2Δ cells. () Schematic of assay. IP, immunoprecipitation. (,) After ~30 cell generations, three to nine independent spores from tlc1Δ RIF1RIF2, tlc1Δ rif1Δ and tlc1Δ rif2Δ strains, all expressing Tel1-HA, were subjected to ChIP with anti-HA antibodies. The mean length of telomeric DNA in each sample was determined (, representative gels for telomere XV-L). Data are expressed as mean ± s.d. percent decrease in the average telomere length of the immunoprecipitate compared to length of input. Percent differences correspond to the following average absolute differences in telomere length in input versus Tel1 immunoprecipitated DNA: WT: 38 bp (VI-R) and 76 bp (XV-L); rif1Δ: 33 bp (VI-R) and 78 bp (XV-L); rif2Δ: 18 bp (VI-R) and 26 bp (XV-L). () Model for distribution of Rif1 and Rif2 along telomere. Wild-type (WT) length telomere of ~300 bp C1–3A/TG1–3 duplex DNA contains ~17 Rap1 binding sites (not all shown) and a short single-strand TG1–3 tail. Short and wild-type length t! elomeres contain equal numbers of heterodimeric Ku complexes and Cdc13–Stn1–Ten1 complexes. For simplicity, we show one Ku and one Cdc13 complex per telomere. Rif1 and Rif2 come to the telomere by interaction with Rap1. As telomeres shorten, they lose Rif2 before Rif1, suggesting that Rif1 is positioned closer to the centromere than Rif2. Thus, 300-bp wild-type length and 150-bp short telomeres would have roughly the same amount of Ku, Cdc13 and Rif1. The shorter telomere has about half as much Rap1 and Rif2 as the wild-type length telomere. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jean S McGee & * Jane A Phillips Affiliations * Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA. * Jean S McGee, * Jane A Phillips, * Angela Chan, * Michelle Sabourin, * Katrin Paeschke & * Virginia A Zakian Contributions J.S.M. did the experiments in Figures 5 and 6, J.A.P. did the experiments in Figure 3, A.C. and M.S. did experiments in Figures 1, 2 and 4, and K.P. helped with analysis of H2A phosphorylation. V.A.Z. and all other authors participated in the design and interpretation of experiments and in the preparation of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Virginia A Zakian Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–4, Supplementary Tables 1 and 2 and Supplementary Methods Additional data - Dissection of Dom34–Hbs1 reveals independent functions in two RNA quality control pathways
- Nat Struct Mol Biol 17(12):1446-1452 (2010)
Nature Structural & Molecular Biology | Article Dissection of Dom34–Hbs1 reveals independent functions in two RNA quality control pathways * Antonia M G van den Elzen1, 2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Julien Henri3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Noureddine Lazar3 Search for this author in: * NPG journals * PubMed * Google Scholar * María Eugenia Gas1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dominique Durand3 Search for this author in: * NPG journals * PubMed * Google Scholar * François Lacroute2 Search for this author in: * NPG journals * PubMed * Google Scholar * Magali Nicaise3 Search for this author in: * NPG journals * PubMed * Google Scholar * Herman van Tilbeurgh3 Search for this author in: * NPG journals * PubMed * Google Scholar * Bertrand Séraphin1, 2 Contact Bertrand Séraphin Search for this author in: * NPG journals * PubMed * Google Scholar * Marc Graille3 Contact Marc Graille Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1446–1452Year published:(2010)DOI:doi:10.1038/nsmb.1963Received23 April 2010Accepted29 October 2010Published online21 November 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Eukaryotic cells have several quality control pathways that rely on translation to detect and degrade defective RNAs. Dom34 and Hbs1 are two proteins that are related to translation termination factors and are involved in no-go decay (NGD) and nonfunctional 18S ribosomal RNA (rRNA) decay (18S NRD) pathways that eliminate RNAs that cause strong ribosomal stalls. Here we present the structure of Hbs1 with and without GDP and a low-resolution model of the Dom34–Hbs1 complex. This complex mimics complexes of the elongation factor and transfer RNA or of the translation termination factors eRF1 and eRF3, supporting the idea that it binds to the ribosomal A-site. We show that nucleotide binding by Hbs1 is essential for NGD and 18S NRD. Mutations in Hbs1 that disrupted the interaction between Dom34 and Hbs1 strongly impaired NGD but had almost no effect on 18S NRD. Hence, NGD and 18S NRD could be genetically uncoupled, suggesting that mRNA and rRNA in a stalled translation complex! may not always be degraded simultaneously. View full text Figures at a glance * Figure 1: Hbs1 structure representation. () Ribbon representation of the Hbs1dN134 structure. For the GTPase, II and III domains of Hbs1, the r.m.s. deviation values with the corresponding domains from EF-Tu and eRF3 are ~2.8–3 Å over 175–190 Cα atoms (25–38% sequence identity), ~1–1.3 Å over 80–90 Cα atoms (20–30% sequence identity) and ~1.4–1.7 Å over 100–110 Cα atoms (18–27% sequence identity), respectively. The P-loop and switches I and II are colored yellow, red and green, respectively. () Conformational changes of switch regions upon GDP binding. For both apo and GDP-bound forms of Hbs1dN134, the crystal asymmetric unit contains two molecules organized as a non-crystallographic dimer, yielding four independent sets of coordinates. These four copies are virtually identical (r.m.s. deviation ~0.4–0.5 Å) except for the switch regions, which adopt different conformations. For clarity, only the secondary structure elements for Hbs1dN134 bound to GDP are shown (beige). The P-loop and swit! ches I and II in the Hbs1–GDP complex are colored yellow, red and green, respectively. The conformation of these regions in the Hbs1dN134 apo forms I and II are blue and pink, respectively. GDP is shown as balls and sticks. () Comparison of the GDP-binding mode as observed in Hbs1dN134 (colors as in ) and EF-Tu (gray). The GDP bound to EF-Tu is shown as black sticks. () Superimposition of Dom34 C-terminal domain (violet) and Hbs1 domain III (yellow) from yeast proteins onto the corresponding domains from human eRF1 and eRF3 as observed in the eRF1–eRF3 complex (gray). Mutated residues are shown as sticks. * Figure 2: SAXS analysis of the Dom34–Hbs1dN134 complex. () Comparison of the scattering curves calculated from the coordinates of the yeast-like (blue), archaea-like (green) and SASREF Dom34–Hbs1dN134 models (red) with the experimental SAXS curve (black). () Ribbon representation of the optimized Dom34–Hbs1dN134 model. Dom34 N-terminal, central and C-terminal domains are colored light green, green and dark green, respectively. Hbs1 GTPase, II and III domains are colored light blue, cyan and dark blue, respectively. Loops A, B and C from Dom34, which are functionally important in NGD18, are colored yellow, orange and red, respectively. () Ribbon representation of the bacterial EF-Tu–tRNA complex26. For clarity, EF-Tu and Hbs1 are shown with the same orientation. EF-Tu GTPase, II and III domains are colored pink, magenta and purple, respectively. * Figure 3: Effects of Hbs1 and Dom34 mutations on NGD. () Northern blot analysis of the steady-state levels of degradation intermediates of the NGD substrate PGK1-SL in a ski7Δhbs1Δ S. cerevisiae strain transformed with Hbs1 mutants. The PGK1-SL reporter was expressed from plasmid pRP1251 and detected with oligonucleotide oRP132 (ref. 6). () Northern blot analysis of the steady state levels of PGK1-SL degradation intermediates in a ski7Δdom34Δ S. cerevisiae strain transformed with Dom34 mutants. (,) Quantification of and , respectively. For each mutant the ratio of 5′ intermediate over full length PGK1-SL reporter was calculated and then standardized for the ratios calculated for Hbs1-proteinA or Dom34-3HA. Mean ± s.d. of three biological replicates is shown. * Figure 4: Effects of Hbs1 and Dom34 mutants on 18S NRD and growth in ribosomal protein deletion context. () Northern blot analysis of the steady-state levels of 18S A1492C NRD substrate in a hbs1Δ strain transformed with Hbs1 mutants. ScR1 RNAs were used as loading controls. The 18S A1492C reporter was expressed from plasmid pWL160-A1492C and detected by oligonucleotide FL1258. () Analysis of the steady-state levels of 18S A1492C NRD substrate in a dom34Δ strain transformed with Dom34 mutants. ScR1 RNAs are presented as loading controls. (,) Quantification of and , respectively. For each mutant, the ratio of 18S A1492C over scR1 signal was calculated and then standardized for the ratios calculated for Hbs1-proteinA or Dom34-3HA. Mean ± s.d. of three biological replicas is shown. () Strains deleted for both small ribosomal subunit protein Rps28A and Hbs1 (rps28AΔhbs1Δ, left) or Dom34 (rps28AΔdom34Δ, right) transformed with Hbs1 mutants or Dom34 mutants were spotted in six ten-fold dilutions on YPDA plates and grown at 16 °C (8 days). Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3P26 * 3P27 * 3P26 * 3P27 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Antonia M G van den Elzen & * Julien Henri Affiliations * Equipe Labellisée La Ligue, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGMBC), Illkirch, France; Centre National de la Recherche Scientifique (CNRS) UMR7104, Illkirch, France; Inserm, U964, Illkirch, France; Université de Strasbourg, Strasbourg, France. * Antonia M G van den Elzen, * María Eugenia Gas & * Bertrand Séraphin * Centre de Génétique Moléculaire (CGM), CNRS FRE3144, Gif-sur-Yvette, France. * Antonia M G van den Elzen, * María Eugenia Gas, * François Lacroute & * Bertrand Séraphin * Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC), CNRS UMR8619 Bat 430 Université Paris-Sud, Orsay, France. * Julien Henri, * Noureddine Lazar, * Dominique Durand, * Magali Nicaise, * Herman van Tilbeurgh & * Marc Graille Contributions J.H., A.M.G.v.d.E., M.G. and B.S. designed experiments. J.H., A.M.G.v.d.E., N.L., D.D., F.L., M.N. and M.E.G. performed experiments. J.H., A.M.G.v.d.E., D.D., H.v.T., M.G. and B.S. analyzed data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Bertrand Séraphin or * Marc Graille Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (9M) Supplementary Methods, Supplementary Data, Supplementary Table 1 and Supplementary Figures 1–5 Additional data - Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription
- Nat Struct Mol Biol 17(12):1453-1460 (2010)
Nature Structural & Molecular Biology | Article Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription * Shixin Liu1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Bryan T Harada2, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer T Miller3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stuart F J Le Grice3 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaowei Zhuang1, 4, 5 Contact Xiaowei Zhuang Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1453–1460Year published:(2010)DOI:doi:10.1038/nsmb.1937Received15 June 2010Accepted23 September 2010Published online21 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Human immunodeficiency virus (HIV) initiates reverse transcription of its viral RNA (vRNA) genome from a cellular tRNA3Lys primer. This process is characterized by a slow initiation phase with specific pauses, followed by a fast elongation phase. We report a single-molecule study that monitors the dynamics of individual initiation complexes, comprised of vRNA, tRNA and HIV reverse transcriptase (RT). RT transitions between two opposite binding orientations on tRNA–vRNA complexes, and the prominent pausing events are related to RT binding in a flipped orientation opposite to the polymerization-competent configuration. A stem-loop structure within the vRNA is responsible for maintaining the enzyme predominantly in this flipped orientation. Disruption of the stem-loop structure triggers the initiation-to-elongation transition. These results highlight the important role of the structural dynamics of the initiation complex in directing transitions between early reverse transcri! ption phases. View full text Figures at a glance * Figure 1: Single-molecule FRET assay for probing the structural dynamics of the initiation complex. () The vRNA template (orange) is labeled with a FRET acceptor (Cy5, red star) near the PBS (blue), annealed to a tRNA primer (gray) and immobilized to the PEG-coated surface via a streptavidin-biotin linkage. The surface-anchored tRNA–vRNA substrates are immersed in a solution containing RT (yellow) labeled with the FRET donor (Cy3, green star). The fingers and RNase H domains of RT are indicated by F and H, respectively. Fluorescence signal from single tRNA–vRNA–RT complexes are detected using a TIRF microscope. () FRET analysis of RT binding events. Top, the fluorescence signals from Cy3 (green) and Cy5 (red) under 532-nm illumination and the signal from Cy5 directly excited by 635-nm illumination (purple). Binding of RT to the substrate (highlighted by yellow) results in an increase in the total fluorescence signals from Cy3 and Cy5 under 532-nm illumination due to excitation of the FRET donor, but does not affect the Cy5 signal from direct excitation by 635-nm ligh! t. Middle, FRET values during the binding event. Bottom, FRET histogram of the binding event. () Sequences of the vRNA and simple RNA PBS templates studied in this work. () Sequences of the natural tRNA3Lys, synthetic tRNA (syn-tRNA), and oligoribonucleotide (ORN) and oligodeoxyribonucleotide (ODN) primers. * Figure 2: RNA-dependent DNA polymerase activity of RT correlates with its binding orientation in the initiation complex. () Cartoon illustrating RT bound to the tRNA–vRNA complex in the polymerase-competent (left) and flipped (right) orientations. () FRET distribution obtained when Cy3-labeled RT binds to Cy5-labeled tRNA+n–vRNA complexes in the presence of 200 μM cognate dNTP. In the case of tRNA (n = 0), the FRET distribution in the absence of dNTP (red) is also shown. The FRET distributions for other n values in the absence of dNTP are shown in Supplementary Figure 5. () Representative time trace of an RT binding event (highlighted in yellow) shows spontaneous transitions between the high- and low-FRET states. () The equilibrium constants K between the polymerase-competent and the flipped binding orientations of RT in the presence of 200 μM cognate dNTP. Error bars are s.e.m. from at least three independent experiments. () Primer extension rates on the vRNA template. The primer extension rate is highly correlated with the binding orientation equilibrium K, showing a correlation coeffi! cient of 0.94. Error bars are s.d. from at least three independent experiments. * Figure 3: The stem-loop structure upstream of the PBS causes the major pauses during initiation and governs the initiation-to-elongation transition. () Left, cartoon of RT bound to tRNA+3–simple PBS substrate. Middle, FRET histograms for RT bound to tRNA+3 primers annealed to the vRNA template (blue) and simple PBS template (red). Right, rates of single-nucleotide addition to various tRNA primers on the simple PBS template. () Left, cartoon of RT bound to tRNA+3–vRNAh– substrate. Middle, FRET histograms for RT bound to tRNA+3 primers annealed to wild-type (WT) vRNA (blue) and vRNAh– (red) templates. Right, rates of single-nucleotide addition to tRNA+3 primers annealed to WT vRNA (blue) and vRNAh– (red) templates. () Left, cartoon of RT bound to tRNA+6–vRNAh+ substrate. Middle, FRET histograms for RT bound to tRNA+6 primers annealed to WT vRNA (blue) and vRNAh+ (red) templates. Right, rates of single-nucleotide addition to tRNA+6 primers annealed to WT vRNA (blue) and vRNAh+ (red) templates. The nucleotides mutated to create the vRNAh– and vRNAh+ constructs are shown in green. Error bars are s.d. from at lea! st three independent experiments. * Figure 4: Disruption of the stem-loop structure upstream of the PBS occurs upon addition of the sixth nucleotide to the tRNA primer. () Diagram of the doubly labeled construct used to monitor the folding of the stem-loop structure. Cy3 (green star) and Cy5 (red star) are attached to vRNA positions U132 and U177. (–) The FRET distributions obtained when 100 nM RT was added to the doubly labeled template annealed to the tRNA (), tRNA+3 (), tRNA+5 () and tRNA+6 () primers. * Figure 5: HIV-1 NC destabilizes the stem-loop structure. () Left, cartoon of the doubly labeled tRNA–vRNA construct complexed with NC (green). Upper right, FRET distributions obtained from the doubly labeled tRNA–vRNA substrate in the absence (red) or presence (blue) of 1 μM NC. Lower right, representative FRET time trace in the presence of 1 μM NC. () Left, cartoon of RT bound to tRNA+3–vRNA in the presence of NC. Right, FRET distribution obtained for Cy3-labeled RT bound to Cy5-labeled tRNA+3–vRNA complexes in the presence of 10 nM NC. () Single-nucleotide extension kinetics of tRNA+3–vRNA complex in the presence of 20 nM RT and various concentrations of NC. Left, the fraction of tRNA primers extended by a single nucleotide. Right, primer extension rate constants. * Figure 6: Structural dynamics of the HIV-1 initiation complex regulate the early phases of reverse transcription. RT binds to the initiation complex in two orientations—a polymerase-competent orientation and a flipped, polymerase-inactive orientation. RT spends a large portion of the time bound to the tRNA–vRNA substrate in the flipped orientation. Addition of the first couple of deoxyribonucleotides to the tRNA primer shifts the RT binding equilibrium toward the polymerase-competent orientation and increases the DNA synthesis rate. The synthesis rate drops markedly at position +3, where RT encounters a stem-loop structure in the vRNA template that forces the enzyme to bind predominantly in the flipped orientation, thereby increasing the probability of pausing. Strand-displacement synthesis until the +6 position eventually leads to unfolding of the stem-loop, which allows RT to reorient into the polymerase-competent binding mode and enter the fast, processive elongation phase of DNA synthesis. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Shixin Liu & * Bryan T Harada Affiliations * Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. * Shixin Liu & * Xiaowei Zhuang * Graduate Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA. * Bryan T Harada * HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland, USA. * Jennifer T Miller & * Stuart F J Le Grice * Department of Physics, Harvard University, Cambridge, Massachusetts, USA. * Xiaowei Zhuang * Howard Hughes Medical Institute, Cambridge, Massachusetts, USA. * Xiaowei Zhuang Contributions S.L., B.T.H. and X.Z. designed the experiments; S.L. and B.T.H. performed the experiments and analyzed the data; S.L., B.T.H. and X.Z. interpret the data and wrote the paper; J.T.M. made the enzyme and some of the tRNA constructs; S.F.J.L.G. contributed to discussion, data interpretation and manuscript preparation. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Xiaowei Zhuang Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 Additional data - BRCA2 acts as a RAD51 loader to facilitate telomere replication and capping
- Nat Struct Mol Biol 17(12):1461-1469 (2010)
Nature Structural & Molecular Biology | Article BRCA2 acts as a RAD51 loader to facilitate telomere replication and capping * Sophie Badie1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Jose M Escandell1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Bouwman2, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Ana Rita Carlos1 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Thanasoula1 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria M Gallardo3 Search for this author in: * NPG journals * PubMed * Google Scholar * Anitha Suram4 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabel Jaco3 Search for this author in: * NPG journals * PubMed * Google Scholar * Javier Benitez5 Search for this author in: * NPG journals * PubMed * Google Scholar * Utz Herbig4 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria A Blasco3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jos Jonkers2 Search for this author in: * NPG journals * PubMed * Google Scholar * Madalena Tarsounas1 Contact Madalena Tarsounas Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1461–1469Year published:(2010)DOI:doi:10.1038/nsmb.1943Received21 April 2010Accepted01 October 2010Published online14 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The tumor suppressor protein BRCA2 is a key component of the homologous recombination pathway of DNA repair, acting as the loader of RAD51 recombinase at sites of double-strand breaks. Here we show that BRCA2 associates with telomeres during the S and G2 phases of the cell cycle and facilitates the loading of RAD51 onto telomeres. Conditional deletion of Brca2 and inhibition of Rad51 in mouse embryonic fibroblasts (MEFs), but not inactivation of Brca1, led to shortening of telomeres and accumulation of fragmented telomeric signals—a hallmark of telomere fragility that is associated with replication defects. These findings suggest that BRCA2-mediated homologous recombination reactions contribute to the maintenance of telomere length by facilitating telomere replication and imply that BRCA2 has an essential role in maintaining telomere integrity during unchallenged cell proliferation. Mouse mammary tumors that lacked Brca2 accumulated telomere dysfunction–induced foci. Hum! an breast tumors in which BRCA2 was mutated had shorter telomeres than those in which BRCA1 was mutated, suggesting that the genomic instability in BRCA2-deficient tumors was due in part to telomere dysfunction. View full text Figures at a glance * Figure 1: BRCA2 is required to recruit RAD51 to the telomeres during the S and G2 phases. () ChIP analysis of HeLa 1.2.11 extracts prepared 0, 2, 4, 6 and 8 h after release from double-thymidine block or untreated cells (ASY). Error bars represent s.d. of three independent experiments. P.I., preimmune serum. () ChIP analysis of extracts from HeLa 1.2.11 cells collected 6 d after the first transfection with control GFP or BRCA2 siRNAs. * Figure 2: Conditional deletion of Brca2 causes telomere shortening. () Representation of the Brca2+ and Brca2sko alleles. Wild-type and sko alleles of Brca2 were visualized on a Southern blot of mouse genomic DNA. Cleavage of the Brca2sko allele after Cre treatment was detected by genomic PCR with the pair of primers indicated. () Cell extracts were prepared from TBX2-immortalized Brca2sko/− MEFs at the indicated times after selection and analyzed by western blotting as indicated. SMC1 was used as a loading control. (–) Q-FISH analysis of telomere length distribution in Brca2sko/− MEFs treated with Cre (+ Cre) and control (− Cre) retroviruses and analyzed 2, 6 and 10 d after the start of selection. Mean telomere lengths given as mean ± s.e.m. n, number of telomeres analyzed for each sample. Red bars mark the 20–60-kb interval. * Figure 3: BRCA2 and RAD51 are required for maintenance of telomere length in MEFs. () Trp53−/− MEFs or Terc−/− G4 MEFs immortalized by p53 knockdown were infected with retroviruses that expressed control GFP or RAD51 shRNAs, then selected with puromycin. Cell extracts prepared 6 d after the start of selection were analyzed by western blotting as indicated. Recombinant human RAD51-His6 was used as control. () Q-FISH analysis of telomere length distribution in cells treated as in . Mean telomere length values (white bar) are shown with s.e.m. Statistical analyses were performed using the Wilcoxon signed-rank test. ***P < 0.0001. * Figure 4: Increased telomere fragility in BRCA2- and RAD51-deficient MEFs. () Representative images of fragile telomeres identified by the presence of multiple telomeric signals (MTSs, yellow arrowheads) in Brca2-deleted and RAD51 shRNA-depleted MEFs. Scale bar, 10 μm. (–) MEFs of the indicated genotypes were treated with retroviruses that encoded Cre or shRNAs in the presence (APH) or absence (DMSO) of aphidicolin. The frequency of MTSs was quantified in metaphase spreads after colcemid arrest. Error bars represent s.d. of three independent experiments. n, number of chromosomes scored for each sample. * Figure 5: Homologous recombination activities and the telomeric factor TRF1 act independently in facilitating telomere replication. () Western blot detection of mouse TRF1 and RAD51 in LT-immortalized TRF1F/F MEFs treated with Cre (+ Cre) and control (− Cre) retroviruses 3 d after selection. α-Tubulin was used as loading control. () MTS quantification in metaphase spreads after colcemid arrest of cells treated as in . Error bars represent s.d. of three independent experiments. n, number of chromosomes scored for each sample. Statistical analyses were performed on total MTSs using an unpaired two-tailed t-test; P < 0.0001. () Cell proliferation assays for cells treated as in . Error bars represent s.d. of three independent experiments. * Figure 6: Telomere dysfunction–induced foci (TIFs) accumulate in BRCA2- and RAD51-deficient MEFs and in Brca2-deficient mammary tumors. () Immunofluorescence detection of γH2AX (green) combined with FISH staining of the telomeres (red) in Brca2sko/− MEFs immortalized by TBX2 overexpression and treated with either empty vector (− Cre) or vector encoding Cre recombinase (+ Cre). Enlarged image (bottom right) depicts the area marked with yellow rectangle. Yellow arrowheads indicate TIFs (sites of γH2AX colocalization with telomeres). Scale bar, 10 μm. () The percentage of metaphase nuclei with <3 or ≥3 TIFs was determined for at least 50 metaphases prepared as in and collected 6 d after selection. Error bars represent s.d. of two independent experiments. Statistical analyses were performed using an unpaired two-tailed t-test. **P < 0.001. () As , but TIFs were analyzed in Trp53−/− MEFs treated with control GFP shRNA and an shRNA against mouse RAD51. Error bars represent s.d. of two independent experiments. Statistical analyses were performed using an unpaired two-tailed t-test. *P < 0.05. () Immuno! fluorescence detection of 53BP1 (green) combined with telomeric FISH staining (red) in sections from a K14Cre-Trp53F/FBrca2F/F mammary tumor. Scale bar, 10 μm. () Enlarged images of TIFs, marking sites of colocalization of 53BP1 with telomeres, or non-TIFs from mouse mammary tumors. Scale bar, 3 μm. () The percentage of cells containing one or more 53BP1 foci that colocalized with telomeres into TIFs was determined relative to the total number of cells analyzed (% of TIF positive cells) using paraffin-embedded sections from mouse K14Cre-Trp53F/F, K14Cre-Trp53F/FBrca1F/F or K14Cre-Trp53F/FBrca2F/F tumors. At least 100 cells were scored for each tumor. Error bars represent s.d. P values were obtained using an unpaired two-tailed t-test. ***P < 0.0001. * Figure 7: The function of BRCA2, but not that of BRCA1, is essential for telomere maintenance in human breast tumors. () Conditional deletion of Brca2 but not Brca1 triggers telomere shortening in MEFs. Q-FISH analysis of telomere length distribution in Brca1SCo/− and Brca2sko/− MEFs treated with Cre (+ Cre) and control (− Cre) retroviruses and analyzed 6 d after start of selection. Mean telomere length values, indicated by white bar, are shown with s.e.m. Statistical analyses were performed using the Wilcoxon signed-rank test. NS, P > 0.05; ***P < 0.0001. () Quantification of MTS frequency in metaphase spreads from cells treated as in . Error bars represent s.d. from two independent experiments. n, number of chromosomes scored for each sample. () Q-FISH analysis of a breast tumor microarray collection consisting of 12 BRCA1-null and 10 BRCA2-null human breast tumors. The intensity of FISH telomeric signal was quantified for at least 6,000 cells for each type of tumor. a.u., arbitrary fluorescence units. Mean telomere intensity values, indicated by white bar, are shown with s.e.m. Sta! tistical analyses were performed using the Wilcoxon signed-rank test. ***P < 0.0001. () Quantification of the relative changes in the frequency of cells with short telomeres (<20 a.u.) in human breast tumors with mutations in BRCA1 or BRCA2. Error bars represent s.d. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sophie Badie, * Jose M Escandell & * Peter Bouwman Affiliations * Telomere and Genome Stability Group, The Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK. * Sophie Badie, * Jose M Escandell, * Ana Rita Carlos, * Maria Thanasoula & * Madalena Tarsounas * Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. * Peter Bouwman & * Jos Jonkers * Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Center (CNIO), Madrid, Spain. * Maria M Gallardo, * Isabel Jaco & * Maria A Blasco * Department of Microbiology and Molecular Genetics and New Jersey Medical School–University Hospital Cancer Center, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA. * Anitha Suram & * Utz Herbig * Human Cancer Genetics Program, CNIO, Madrid, Spain. * Javier Benitez Contributions M. Tarsounas, S.B. and J.M.E. designed and planned the experiments. S.B. and J.M.E. performed most of the experiments. P.B. and J.J. generated the Brca2sko conditional mouse model, established immortalized MEFs and contributed to the results in Figure 2a. A.R.C. contributed the results in Figure 1a,b and Supplementary Figure 6a. M. Thanasoula performed the IF-FISH experiments in Figure 6a–c, Supplementary Figure 1d and Supplementary Figure 6b,c. M.M.G., J.B. and M.A.B. performed the experiments in Figure 7b,c. A.S. and U.H. contributed to the results in Figure 6d–f. I.J. designed and validated the shRNA against mouse RAD51. M. Tarsounas made the figures and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Madalena Tarsounas Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (776K) Supplementary Figures 1–6 and Supplementary Methods Additional data - Correlated conformational events in EF-G and the ribosome regulate translocation
- Nat Struct Mol Biol 17(12):1470-1477 (2010)
Nature Structural & Molecular Biology | Article Correlated conformational events in EF-G and the ribosome regulate translocation * James B Munro1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Wasserman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Roger B Altman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Leyi Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott C Blanchard1 Contact Scott C Blanchard Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1470–1477Year published:(2010)DOI:doi:10.1038/nsmb.1925Received10 February 2010Accepted09 September 2010Published online07 November 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In bacteria, the translocation of tRNA and mRNA with respect to the ribosome is catalyzed by the conserved GTPase elongation factor-G (EF-G). To probe the rate-determining features in this process, we imaged EF-G–catalyzed translocation from two unique structural perspectives using single-molecule fluorescence resonance energy transfer. The data reveal that the rate at which the ribosome spontaneously achieves a transient, 'unlocked' state is closely correlated with the rate at which the tRNA-like domain IV-V element of EF-G engages the A site. After these structural transitions, translocation occurs comparatively fast, suggesting that conformational processes intrinsic to the ribosome determine the rate of translocation. Experiments conducted in the presence of non-hydrolyzable GTP analogs and specific antibiotics further reveal that allosterically linked conformational events in EF-G and the ribosome mediate rapid, directional substrate movement and EF-G release. View full text Figures at a glance * Figure 1: Structural models of the ribosome and EF-G. Left, the pre-translocation ribosome complex, showing the large (50S) and small (30S) subunits and the A, P and E sites. The rRNA is shown in gray, the 50S proteins in blue and the 30S proteins in tan. The A- and P-site tRNAs are in red. The GTPase activating center (GAC) and L1 stalk are indicated. Center, EF-G with structural domains and GTP labeled. EF-G binds at the GAC of the pre-translocation complex, hydrolyzes GTP and promotes formation of the post-translocation complex shown at right, in which the tRNAs have moved to the P and E sites, and EF-G domains IV-V protrude into the A site41. Structural models of the ribosome and EF-G were constructed from PDB accession codes 2WRI and 2WRJ. The A-site tRNA is from PDB 1GIX. * Figure 2: Observation of the translocation reaction from two unique structural perspectives. () The dynamics of the ribosome complex with P-site tRNAfMet(Cy3-s4U8), A-site fMet-Phe-tRNAPhe and L1(Cy5-S55C) following the addition of 10 μM EF-G and 1 mM GTP. () The apparent formation of an EF-G–ribosome interaction obtained by delivering 0.2 μM C-terminally labeled EF-G and 1 mM GTP to pre-translocation complexes with P-site tRNAfMet and A-site fMet-Phe-tRNAPhe(Cy3-acp3U47). The intervals Δt, Δtarr and ΔtFRET were used to estimate the apparent rate of translocation, the apparent on-rate and the apparent off-rate , respectively. Left, cartoon diagrams indicating the sites of labeling (Cy3, green star; Cy5, red star) and the putative dynamic elements. Right, single-molecule fluorescence (Cy3, green; Cy5, red) and FRET (blue) trajectories. Overlaid on the FRET traces are the idealizations (red) generated during kinetic analysis (in only). * Figure 3: The kinetics of unlocked-state formation and decay and EF-G–ribosome interactions are correlated. (,) Shown are the distributions of dwell times leading to the formation of the unlocked state (black) and of those leading to the formation of the EF-G–ribosome interaction determined from delivery of labeled EF-G to complexes with labeled A-site tRNA (blue). Also shown are the distribution of the lifetime of the stable unlocked state (red) and that of the EF-G–ribosome interaction (cyan). Overlaid on the distributions are exponential functions with the rate constants shown in Supplementary Table 2. Data are presented for complexes with P-site tRNAfMet and A-site fMet-Phe-tRNAPhe (), and P-site tRNAPhe and A-site NAcPhe-Lys-tRNALys (). * Figure 4: A step-like increase in Cy3 fluorescence accompanies peptidyl-tRNA movement to the P site. () Single-molecule fluorescence trajectories (Cy3, green; Cy5, red) obtained during delivery of 10 μM unlabeled EF-G and 1 mM GTP to complexes with P-site tRNAfMet and A-site fMet-Phe-tRNAPhe(Cy3-acp3U47). () Rate at which the increase in Cy3 fluorescence is observed in complexes with either (blue) P-site tRNAfMet and A-site fMet-Phe-tRNAPhe(Cy3-acp3U47) or (red) P-site tRNAPhe and A-site NAcPhe-Lys-tRNALys(Cy3-acp3U47), across a range of EF-G concentrations (0.05–25 μM). The data were fit to the hyperbolic function rate = ratemax [EF – G]/(K1/2 + [EF – G] with ratemax = 1.0 ± 0.1 s−1 and K1/2 = 0.9 ± 0.1 μM for the case of P-site tRNAfMet, and ratemax = 1.7 ± 0.1 s−1 and K1/2 = 0.6 ± 0.1 μM for P-site tRNAPhe. () Distribution of time over which the increase in Cy3 fluorescence occurs for complexes containing P-site tRNAfMet. In agreement with a previous report36, the distribution is well fit (R2 ≈0.95) by the distribution predicted for a model with three! successive steps with equal rate constants (f(t) = (k3t2/2) exp(−kt), k = 37 ± 3 s−1). () The distribution obtained from complexes containing P-site tRNAPhe was fit to the same function with k = 35 ± 3 s−1 (R2 ≈0.94). Error bars represent the standard error. * Figure 5: Translocation in the presence of inhibitors. (–) Single-molecule fluorescence (Cy3, green; Cy5, red) and FRET (blue) trajectories acquired during delivery of labeled EF-G to complexes with labeled A-site tRNAPhe in the presence of 200 μM viomycin (), GDPNP (), 50 μM fusidic acid () or 5 mM spectinomycin (). * Figure 6: Schematic of the translocation mechanism highlighting points of antibiotic inhibition. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 2WRI * 2WRJ * 1GIX * 2WRI * 2WRJ * 1GIX Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, New York, USA. * James B Munro, * Michael R Wasserman, * Roger B Altman, * Leyi Wang & * Scott C Blanchard Contributions J.B.M. and S.C.B. designed the experiments. J.B.M. and M.R.W. conducted the experiments and analyzed the data. R.B.A. and L.W. prepared reagents. J.B.M., M.R.W., R.B.A. and S.C.B. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Scott C Blanchard Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–12 and Supplementary Tables 1–3 Additional data - Mre11–Rad50–Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks
- Nat Struct Mol Biol 17(12):1478-1485 (2010)
Nature Structural & Molecular Biology | Article Mre11–Rad50–Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks * Matthew L Nicolette1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kihoon Lee2 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhi Guo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mridula Rani3 Search for this author in: * NPG journals * PubMed * Google Scholar * Julia M Chow1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sang Eun Lee2 Search for this author in: * NPG journals * PubMed * Google Scholar * Tanya T Paull1 Contact Tanya T Paull Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1478–1485Year published:(2010)DOI:doi:10.1038/nsmb.1957Received22 December 2009Accepted14 October 2010Published online21 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The repair of DNA double-strand breaks (DSBs) by homologous recombination is essential for genomic stability. The first step in this process is resection of 5′ strands to generate 3′ single-stranded DNA intermediates. Efficient resection in budding yeast requires the Mre11–Rad50–Xrs2 (MRX) complex and the Sae2 protein, although the role of MRX has been unclear because Mre11 paradoxically has 3′→5′ exonuclease activity in vitro. Here we reconstitute resection with purified MRX, Sae2 and Exo1 proteins and show that degradation of the 5′ strand is catalyzed by Exo1 yet completely dependent on MRX and Sae2 when Exo1 levels are limiting. This stimulation is mainly caused by cooperative binding of DNA substrates by Exo1, MRX and Sae2. This work establishes the direct role of MRX and Sae2 in promoting the resection of 5′ strands in DNA DSB repair. View full text Figures at a glance * Figure 1: MRX and Sae2 promote 5′ strand degradation at a DNA break. () dsDNA (5.6 kb, pTP407) linearized with BseRI (0.3 nM) was incubated with Exo1 (0.5 nM), MRX (5 nM) and Sae2 (5 nM) for 60 min at 30 °C. DNA was stained with SYBR green. M, molecular weight markers. () Resection assays were carried out with a 4.4-kb plasmid DNA substrate (pNO1) linearized with SphI and analyzed as in with SYBR green staining (top), and with nondenaturing Southern hybridization with a strand-specific RNA probe for the 3′ strand at one end (see diagram). Reactions contained 14 nM MRX, 3.5 nM Sae2, and 0.4, 0.8, 1.6, 3.2, 6.4 and 32 nM Exo1 and were incubated for 60 min at 30 °C. () Resection assays were done as in with 4 nM Exo1. The reactions were split and separated in parallel in a nondenaturing gel, followed by nondenaturing Southern hybridization and probed separately for single-stranded 3′ strand (left) or 5′ strand (right) DNA adjacent to the break site. Denatured plasmid DNA was used as a marker for the ssDNA (ss); ds, position of the unresec! ted plasmid. () Quantification of the reactions in , in addition to reactions from four other independent experiments, by phosphorimager analysis of total counts in each lane. Within each experiment, the signal from the reactions containing wild-type MRX, Exo1 and Sae2 was set to 100%, with the other values shown relative to this (mean ± s.d.). * Figure 2: Characterization of products of cooperative DNA resection by Exo1, MRX and Sae2. () Diagram of end of pNO1 DNA substrate cut with SphI, with locations of NciI sites, PCR primers and qPCR 6-FAM-TAMRA probes (asterisks). NciI digests dsDNA (top) but leaves ssDNA intact (bottom). () Standard curves using undigested DNA with the primer sets for the 29-bp and 1025-bp NciI sites. () qPCR analysis of resection assays (done as in Fig. 1b) to assess level of DNA resection at each site: 29 nt from the end (top) and 1025 nt from the end (bottom). From each reaction, undigested aliquots were analyzed and compared with digested aliquots to obtain a ΔCT value, which was used to calculate the percentage of ssDNA in the reaction as described in Methods. Each resection reaction was done in triplicate and a qPCR analysis done for each (values are mean ± s.e.m.). * Figure 3: Digestion of linear DNA by Exo1 produces both single-nucleotide and oligonucleotide products. () Resection assays were done as in Figure 1b except with 700-bp DNA substrate internally labeled with 32P, with Exo1 (0.2, 0.4 nM), MRX (1.6 nM) and Sae2 (0.6 nM) or T7 exonuclease (1 unit per reaction). Reactions were stopped with SDS and EDTA and separated by thin-layer chromatography; migration of the labeled dAMP product is indicated. T7 exonuclease was used as a positive control to generate single-nucleotide products. () Resection assays were done as in except that the 700-bp DNA substrate was labeled on one 5′ strand with Cy5. Reactions contained 2.5 nM (lanes 2 and 3), 5 nM (lanes 4 and 5) and 10 nM (lanes 6, 7 and 10) Exo1, 7.5 nM MRX and 1.5 nM Sae2, with 25 nM MRX and 3 nM Sae2 in the reaction in lane 12. Arrows at right, positions of cleavage products (solid line, Exo1; dashed line, MRX and Sae2). * Figure 4: Mutations in Exo1 and Rad50 prevent DNA end processing. () Resection assays were carried out and analyzed (as in Fig. 1b) using nondenaturing Southern hybridization with a 3′ strand probe and with D173A Exo1. Concentrations of wild-type (WT) and mutant Exo1 were 4 nM and 8 nM. () Resection assays were done (as in Fig. 1b) with the probe specific for the 3′ strand but with MRX complexes containing Mre11 H125N. Total counts in each lane from three experiments were quantified using phosphorimager analysis (values are mean ± s.d.). In each experiment, the signal from the reactions containing wild-type MRX, Exo1 and Sae2 was the highest and set to 100%, with the signals in other lanes shown relative to this value. () Resection assays were carried out, analyzed and presented as in but with the MR(K81I)X mutant complex. Reactions from three independent experiments were quantified. * Figure 5: Mutations in Sae2 reduce the efficiency of Exo1-mediated DSB resection in vitro and in vivo. () Resection assays were carried out, analyzed and presented as in Figure 4b but with the Sae2 mutants ΔC (deletion of residues 251–345) and ΔN (deletion of residues 21–172). WT, wild type. Reactions from two independent experiments were quantified (values are mean). () Schematic of the MAT locus containing an HO endonuclease cut site, and locations of PCR primers used to assess the levels of RPA in sae2Δ strains carrying plasmids expressing a vector control, wild type, ΔN or ΔC truncations of Sae2. mre11Δ and exo1Δ strains are also shown for comparison. Chromatin immunoprecipitation (ChIP) assays were carried out using an antibody to RPA as described47. () Quantification of PCR signals from a primer set that anneals 0.2 kb (P1-P2) from an HO break at different durations of HO expression (values are mean ± s.d.). * Figure 6: MRX and Sae2 promote Exo1 DNA binding. () Gel mobility shift assays were done with wild-type MRX (2.5 nM), Sae2 (2.5 nM) and Exo1 D173A (4 nM) proteins and a 32P-labeled, double-stranded oligonucleotide substrate containing 4-nt 3′ overhangs on both ends. Reactions were incubated for 15 min on ice before separation on a native acrylamide gel. () MRX and Exo1 D173A proteins were incubated with biotinylated, blunt 100-bp duplex DNA, cross-linked with formaldehyde, and proteins bound to the DNA were isolated using streptavidin-coated magnetic beads.Bound protein was visualized by SDS-PAGE and western blotting with antibody to Flag for Exo1 and Rad50. () Protein-DNA binding assays were done with a 90-bp blunt DNA substrate, containing five azide groups (N3) on the 5′ ends of the 5′ strands or the 3′ ends of the 3′ strands. Both DNA substrates were labeled with 32P (asterisk). Proteins were incubated with the DNA substrates on ice, UV irradiated, separated by SDS-PAGE and transferred to a PVDF membrane to re! move all uncross-linked DNA before phosphorimager analysis. M, molecular weight markers. * Figure 7: MRX and Sae2 facilitate Exo1 activity through distinct 'processing' and 'recruitment' pathways. () Resection reaction was carried out (as in Fig. 1a) using pTP407 plasmid DNA linearized with BseRI except that the reaction was done in two stages. The first incubation (Inc. 1), included MRX (50 nM) and Sae2 (26 nM) but the DNA was deproteinized with SDS and proteinase K, followed by ethanol precipitation. The DNA from each reaction was incubated in a second reaction (Inc. 2) that included Exo1 (1 nM). Reactions were separated by native agarose gel electrophoresis and DNA stained with SYBR green. M, molecular weight markers. () Two-stage reactions were carried out as in except that T7 exonuclease (1 unit) was used in the first incubation and MRX (5 nM) and Sae2 (5 nM) were also added in the second incubation. () Two-stage reactions were carried out as in except with pNO1 plasmid DNA linearized with Sph1 and analyzed by nondenaturing Southern hybridization using a probe specific for the 3′ strand (as in Fig. 1b). Reactions included MRX (14 nM), Sae2 (3.5 nM) and Exo1 (4 ! nM). M, molecular weight marker. () Working model for association and cleavage of DNA ends by MRX, Sae2 and Exo1. We hypothesize a DNA-unwinding step followed by inefficient cleavage by MRX and Sae2, Exo1 recruitment and further excision catalyzed by Exo1 (see text for details). Author information * Abstract * Author information * Supplementary information Affiliations * The Howard Hughes Medical Institute and the Department of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas, USA. * Matthew L Nicolette, * Zhi Guo, * Julia M Chow & * Tanya T Paull * The Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA. * Kihoon Lee & * Sang Eun Lee * Department of Chemical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA. * Mridula Rani Contributions M.L.N. expressed and purified recombinant proteins, performed resection experiments in vitro, analyzed the data and helped to edit the manuscript. K.L. performed resection experiments in vivo in S. cerevisiae. Z.G. carried out quantitative PCR analysis of resection products in vitro and contributed to the editing of the manuscript. M.R. performed the SPR experiment and analyzed the SPR data. J.M.C. expressed and purified Sae2 proteins used in this study. S.E.L. helped to design the in vivo experiments with sae2 mutants and helped in the analysis of the data and editing of the manuscript. T.T.P. performed the DNA binding and some of the resection experiments in vitro, analyzed the data, and wrote and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tanya T Paull Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–8 and Supplementary Methods Additional data - Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-neutralizing antibodies
- Nat Struct Mol Biol 17(12):1486-1491 (2010)
Nature Structural & Molecular Biology | Article Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-neutralizing antibodies * Gary Frey1, 2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jia Chen1, 2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Sophia Rits-Volloch1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael M Freeman1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Zolla-Pazner3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Bing Chen1, 2 Contact Bing Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1486–1491Year published:(2010)DOI:doi:10.1038/nsmb.1950Received10 August 2010Accepted06 October 2010Published online14 November 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg HIV-1 envelope glycoprotein gp41 undergoes large conformational changes to drive fusion of viral and target cell membranes, adopting at least three distinct conformations during the viral entry process. Neutralizing antibodies against gp41 block HIV-1 infection by targeting gp41′s membrane-proximal external region in a fusion-intermediate state. Here we report biochemical and structural evidence that non-neutralizing antibodies, capable of binding with high affinity to an immunodominant segment adjacent to the neutralizing epitopes in the membrane-proximal region, recognize a gp41 conformation that exists only when membrane fusion is complete. We propose that these non-neutralizing antibodies are induced in HIV-1–infected individuals by gp41 in a triggered, postfusion form and contribute to production of ineffective humoral responses. These results have important implications for gp41-based vaccine design. View full text Figures at a glance * Figure 1: HIV-1 envelope constructs and GCN4-gp41-inter. Top, schematic representation of HIV-1 envelope glycoprotein gp160, the full-length precursor. Segments of gp120 and gp41 are designated as follows: C1–C5, conserved regions 1–5; V1–V5, variable regions 1–5; F, fusion peptide; HR1, heptad repeat 1; C-C loop, the immunodominant loop with a conserved disulfide bond; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail. Glycans are represented by tree-like symbols. HIV-1 envelope constructs used in this study include gp140, the uncleaved ectodomain of gp160 with a trimerization foldon (Fd) tag and a histidine tag at its C terminus; gp41-post, gp41 in the six-helix conformation with partial MPER; gp41-inter, HR2 peptide– and foldon tag–trapped gp41 in the prehairpin-intermediate conformation; GCN4-gp41-inter, gp41-inter with the six-helix-bundle portion replaced with a trimeric GCN4 coiled coil36 (in light blue). Bottom, diagrams representing three-dimensional organization of gp41-inter and GCN4-gp41-! inter. The trimeric GCN4 with its heptad repeat in the same register as HR1 replaces the HR2-linker-HR1 of gp41-inter. The coordinates of HR1 (ref. 11) and GCN4 (ref. 36) coiled coils are shown in yellow and light blue, respectively. * Figure 2: HIV-1 gp41 cluster II antibodies preferentially bind gp41 in its postfusion conformation. Human mAbs 1281, 98-6D, 126-7D, 167D and 1379 were analyzed by SPR for binding to the HIV-1 gp41 constructs gp140 (sensorgrams in black), GCN4-gp41-inter (blue) and gp41-post (red). RU, response units. GCN4-gp41-inter or gp41-post was immobilized on CM5 chips; gp140 was captured on a Ni-NTA chip. Each IgG at 50 nM was passed over each surface individually. Data with the antibodies immobilized on a Protein A chip are shown in Supplementary Figure 3. * Figure 3: Analysis of interactions of 1281 Fab with various gp41 constructs. Fab fragment derived from mAb 1281 was tested by SPR for binding to gp41 constructs. RU, response units. () The recorded sensorgram for gp41-post is in red, gp140 in black and GCN4-gp41-inter in blue. () To confirm no detectable binding of 1281 Fab to GCN4-gp41-inter, solutions of 1281 Fab at various concentrations were flowed over the GCN4-gp41-inter surface. The sensorgrams are shown in various colors. (,) 1281 Fab at various concentrations was passed over the surfaces immobilized with gp41-post or gp41-inter containing the six-helix bundle. Binding kinetics were evaluated using a 1:1 Langmuir binding model; binding constants are summarized in Supplementary Table 1. The sensorgrams are shown in black and the fits in green. All injections were carried out in duplicate, and duplicates gave essentially identical results. Only one of the duplicates is shown. * Figure 4: Crystal structure of the complex of gp41-post and the Fab fragment of cluster II antibody 1281. Side () and top () views of the overall structure of the postfusion conformation of HIV-1 gp41 in complex with the Fab derived from an anti-gp41 cluster II mAb 1281 are shown in ribbon representation. Dark green, heavy chain of the antibody; light green, light chain; yellow, HR1 of gp41; blue, HR2; red, part of MPER. The Fab mainly grips HR2 but also makes direct contacts with HR1 via CDR loops from both the heavy and light chains, suggesting the six-helix-bundle conformation of gp41 is crucial for 1281 binding. The MPER part, in red, contains the 2F5 epitope (residues 663–669), which is α-helical in the postfusion conformation. * Figure 5: Close-up of major contacts between gp41 and 1281 Fab. () gp41 and1281 Fab shown as ribbon diagrams. () gp41 shown in surface representation and the Fab as a ribbon diagram. Coloring is as in Figure 4; surface-exposed residues in HR2 are labeled in white. The CDR H1 and L2 loops of the antibody contact the HR2 helix in gp41-post; the CDR H3 reaches out and interacts with both the HR1 and HR2 helices. The footprint of the antibody covers residues 643–661, consistent with the previous epitope-mapping data27, 33, 34. The 2F5 in red is spatially close to the cluster II epitope. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3P30 * 3P30 Referenced accessions Protein Data Bank * 1AIK * 1AIK Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Gary Frey & * Jia Chen Affiliations * Division of Molecular Medicine, Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Gary Frey, * Jia Chen, * Sophia Rits-Volloch, * Michael M Freeman & * Bing Chen * Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA. * Gary Frey, * Jia Chen, * Sophia Rits-Volloch, * Michael M Freeman & * Bing Chen * Department of Pathology, New York University School of Medicine, New York, New York, USA. * Susan Zolla-Pazner * New York Veterans Affairs Medical Center, New York, New York, USA. * Susan Zolla-Pazner Contributions G.F., J.C. and B.C. designed research; G.F., J.C., S.R.-V. and M.M.F. performed the experiments; S.Z.-P. provided antibodies; G.F., J.C., S.R.-V., M.M.F. and B.C. analyzed data; and G.F., J.C., S.Z.-P. and B.C. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bing Chen Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Methods Additional data - Crystal structure of a non-neutralizing antibody to the HIV-1 gp41 membrane-proximal external region
- Nat Struct Mol Biol 17(12):1492-1494 (2010)
Nature Structural & Molecular Biology | Brief Communication Crystal structure of a non-neutralizing antibody to the HIV-1 gp41 membrane-proximal external region * Nathan I Nicely1 Contact Nathan I Nicely Search for this author in: * NPG journals * PubMed * Google Scholar * S Moses Dennison1 Search for this author in: * NPG journals * PubMed * Google Scholar * Leonard Spicer2 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard M Scearce1 Search for this author in: * NPG journals * PubMed * Google Scholar * Garnett Kelsoe3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshihiro Ueda3 Search for this author in: * NPG journals * PubMed * Google Scholar * Haiyan Chen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hua-Xin Liao1 Search for this author in: * NPG journals * PubMed * Google Scholar * S Munir Alam1 Search for this author in: * NPG journals * PubMed * Google Scholar * Barton F Haynes1 Contact Barton F Haynes Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1492–1494Year published:(2010)DOI:doi:10.1038/nsmb.1944Received18 June 2010Accepted08 September 2010Published online14 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The monoclonal antibody 13H11 shares part of its epitope in the HIV-1 gp41 membrane-proximal external region (MPER) with the rare, broadly neutralizing human antibody 2F5. Although 13H11 partially cross-blocked 2F5 binding, 13H11 is non-neutralizing and does not block 2F5 neutralization. We show that unlike 2F5, 13H11 binds to a well-defined helical MPER structure that is consistent with the structure of gp41 in a post-fusion six-helix bundle conformation. View full text Figures at a glance * Figure 1: Structure of 13H11 Fab. () 13H11 Fab structure (white) rendered to show surface features contributed to its idiotope by its CDRs. In this view, the presence of a large groove on the idiotope is apparent. () In a view head-on with respect to the idiotope (rotated 90° from ), it is seen that the groove is engendered by the short CDR-H3 and the long CDR-L1. * Figure 2: Structure of 13H11 bound to MPER. () 13H11 (white surface) binds the gp41652–671 peptide (gray ribbon) in an α-helical conformation. 13H11 epitope residues are highlighted (yellow). Residues in parentheses are behind the helix in this view. Sequences of the peptides gp41652–671 in the 13H11 complex structure and gp41654–670 crystallized with 2F5 (ref. 2) are indicated with respective epitope residues boxed. () To show shape and charge complementarity between Fab and peptide, the same view is shown as in but with the Fab and peptide exploded, the peptide rotated by 180° in the plane and surfaces colored by electrostatic potential. * Figure 3: Relevance to the post-fusion six-helix bundle. () Pocket binding residues (yellow highlights, labeled) on the gp41652–671 peptide (gray ribbon) orient away from 13H11 (white surface) in the complex structure. () The orientation of the pocket binding residue side chains on the face of the helix directed away from the Fab is more evident in this view looking down the helical axis of the peptide. () A recent six-helix bundle structure (orange, light orange) superimposes the helical gp41652–671 peptide with an r.m.s. deviation of 0.44 Å (ref. 21). Equivalent pocket binding residues from on the bundle structure are shown in green. Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3MNW * 3MNW Author information * Accession codes * Author information * Supplementary information Affiliations * Duke Human Vaccine Institute, Duke University School of Medicine, Durham, North Carolina, USA. * Nathan I Nicely, * S Moses Dennison, * Richard M Scearce, * Haiyan Chen, * Hua-Xin Liao, * S Munir Alam & * Barton F Haynes * Departments of Biochemistry and Radiology, Duke University, Durham, North Carolina, USA. * Leonard Spicer * Department of Immunology, Duke University and Duke Human Vaccine Institute, Duke University School of Medicine, Durham, North Carolina, USA. * Garnett Kelsoe & * Yoshihiro Ueda Contributions N.I.N. purified proteins and carried out all crystallographic processes and analyses. H.C. and H.-X.L. designed the expression system and produced proteins. S.M.D. and S.M.A. did the binding kinetics measurements and analyses. L.S. assisted in review of the data. B.F.H. and R.M.S. made the mAb 13H11. Y.U. and G.K. sequenced mAb 13H11 VH and VL genes. B.F.H. was responsible for project design and leadership. N.I.N., S.M.A. and B.F.H. wrote the manuscript. N.I.N., S.M.D. and S.M.A. generated the figures. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Nathan I Nicely or * Barton F Haynes Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (828K) Supplementary Figures 1–4, Supplementary Tables 1–3, Supplementary Results and Supplementary Methods Additional data - Reciprocal intronic and exonic histone modification regions in humans
- Nat Struct Mol Biol 17(12):1495-1499 (2010)
Nature Structural & Molecular Biology | Analysis Reciprocal intronic and exonic histone modification regions in humans * Jason T Huff1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Alex M Plocik2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christine Guthrie2 Contact Christine Guthrie Search for this author in: * NPG journals * PubMed * Google Scholar * Keith R Yamamoto1 Contact Keith R Yamamoto Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1495–1499Year published:(2010)DOI:doi:10.1038/nsmb.1924Received30 March 2010Accepted08 September 2010Published online07 November 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg While much attention has been focused on chromatin at promoters and exons, human genes are mostly composed of intronic sequences. Analyzing published surveys of nucleosomes and 41 chromatin marks in humans, we identified histone modifications specifically associated with 5′ intronic sequences, distinguishable from promoter marks and bulk nucleosomes. These intronic marks were spatially reciprocal to trimethylated histone H3 Lys36 (H3K36me3), typically transitioning near internal exons. Several marks transitioned near bona fide exons, but not near nucleosomes at exon-like sequences. Therefore, we examined whether splicing affects histone marking. Even with considerable changes in regulated alternative splicing, histone marks were stable. Notably, these findings are consistent with exon definition influencing histone marks. In summary, we show that the location of many intragenic marks in humans can be distilled into a simple organizing principle: association with 5′ intro! nic or 3′ exonic regions. View full text Figures at a glance * Figure 1: Groups of histone marks revealed by PCA. PCA of all ChIP-seq and MNase-seq data 10 kb up- and downstream of RefSeq TSSs are presented in a three-dimensional stereoscopic image. Gray points represent the PCA loadings for each of the 41 histone marks and nucleosome occupancy (Nucs). Principal components (Comp.) 1, 2 and 3 are projected with the orientation of the axes shown as gray bars radiating from the origin. Individual points representative of each component are colored: H3K4me3 (orange) for Comp. 1, Nucs (purple) for Comp. 2, and H3K79me2 (green) for Comp. 3. For reference, marks enriched at promoters are colored light orange and H3K36me3 is colored blue. Supplementary Figure 1 shows the fully labeled and separated PCA loadings and the transformed PCA scores plotted on the genome with respect to transcription start sites. * Figure 2: Intragenic histone modification regions reflect gene architecture. () H3K79me2 and H3K36me3 ChIP-seq reads plotted on two genes (UGGT1 and SMURF1) of similar length but different gene architectures. A black line in each density plot shows where a uniform distribution of sequence reads would be. The proposed 5′ intronic and 3′ exonic histone modification zones are diagrammed below each gene. () Heatmaps show the densities of H3K4me3, nucleosomes (Nucs), H3K79me2 and H3K36me3 from ChIP-seq and MNase-seq data, plotted with respect to transcription start sites (bent arrows), with genes sorted by distance to the beginning of the first internal exon (that is, the first 3′ splice site). For clarity, only the 4,286 annotated RefSeq genes that are in the top 50% of expression and with the 50% longest distances to the first internal exon are shown. Full plots are shown in Supplementary Figure 2. Each gene is displayed as a row, and columns are 1-kb bins from 10 kb upstream to 50 kb downstream of transcription start sites (as indicated at bottom! ). The value of each bin is shaded by the number of ChIP-seq or MNase-seq reads per kb, scaled to cover data between 0 reads (white) and the ninety-ninth percentile of each sequencing experiment (black), as indicated at the top right of each heatmap. At far right, the aligned transcription start sites (broken line) and the positions of first internal exons (gray line) are shown as a visual reference. * Figure 3: Histone modification profiles at the alternatively spliced exon of YPEL5 are similar between caffeine-treated SW620 cells expressing different YPEL5 mRNA isoforms. () Increasing caffeine concentrations result in greater exon inclusion of YPEL5 exon 2. Shown are products of RT-PCR of SW620 cells treated for 7 h with 0, 6 or 18 mM caffeine. Alternatively (red) and constitutively (black) spliced exons are numbered and diagrammed to the right of the corresponding RT-PCR product. () Normalized H3K79me2 and H3K36me3 profiles in SW620 cells treated with 0, 6 or 18 mM caffeine for 7 h. Points indicate log2(ratios) of H3K79me2/H3 (top) and H3K36me3/H3 (bottom), based on ChIP-qPCR measurements, relative to the lowest value, for each of two SW620 biological replicates. Vertical gray lines mark the 3′ splice site of each internal exon. Arrowheads indicate the positions of qPCR amplicons along the gene (bottom). The alternatively spliced cassette exon 2 is indicated in red. The gene schematic and markers are drawn to scale according to their genomic coordinates except for the second (green) and third (blue) data points at each amplicon, which are! offset to the right to facilitate comparison between conditions. * Figure 4: Histone modification profiles at the alternatively spliced exons of CD45 are similar between B-cell lines stably expressing different CD45 mRNA isoforms. In all panels, colored points and lines refer to matched BJAB B-cell lines expressing a short hairpin targeting knockdown of either red fluorescent protein (RFP) as a control (blue) or hnRNPLL (green), or to matched BL41 B-cell lines overexpressing hnRNPLL (orange) or mock-treated as a control (red). () BJAB and BL41 B-cell lines differ in the relative abundance of CD45 and hnRNPLL mRNAs. Points indicate mean log2(ratios) ± s.d. of CD45/RPLP0 and hnRNPLL/RPLP0 qRT-PCR measurements from n = 4 biological replicates (not detectable, ND). () Confirmation that passaged BJAB and BL41 B-cell lines express different CD45 isoforms. Alternatively (red) and constitutively (black) spliced exons are diagrammed to the right of the corresponding RT-PCR product. (,) Normalized H3K79me2 and H3K36me3 profiles in matched B-cell lines. Points indicate the mean log2(ratios) ± s.d. of H3K79me2/H3 (top) and H3K36me3/H3 (bottom) ChIP-qPCR measurements, relative to the lowest value, from BJAB (; n! = 5) and BL41 (; n = 6) biological replicates. Positions of the 3′ splice sites and qPCR amplicons along the gene are indicated with vertical gray lines and arrowheads, respectively. All positions are drawn to scale, except for a gap in the second intron and the second point at each amplicon, which is offset to the right to facilitate comparison. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jason T Huff & * Alex M Plocik Affiliations * Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA. * Jason T Huff & * Keith R Yamamoto * Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA. * Alex M Plocik & * Christine Guthrie Contributions J.T.H. and A.M.P. designed and performed the analyses and experiments. J.T.H., A.M.P., C.G. and K.R.Y. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Christine Guthrie or * Keith R Yamamoto Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figs. 1-7 and Supplementary Table 1 Additional data - H2A.Z nucleosomes enriched over active genes are homotypic
- Nat Struct Mol Biol 17(12):1500-1507 (2010)
Nature Structural & Molecular Biology | Resource H2A.Z nucleosomes enriched over active genes are homotypic * Christopher M Weber1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jorja G Henikoff1 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven Henikoff1, 3 Contact Steven Henikoff Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 17,Pages:1500–1507Year published:(2010)DOI:doi:10.1038/nsmb.1926Received30 April 2010Accepted09 September 2010Published online07 November 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nucleosomes that contain the histone variant H2A.Z are enriched around transcriptional start sites, but the mechanistic basis for this enrichment is unknown. A single octameric nucleosome can contain two H2A.Z histones (homotypic) or one H2A.Z and one canonical H2A (heterotypic). To elucidate the function of H2A.Z, we generated high-resolution maps of homotypic and heterotypic DrosophilaH2A.Z (H2Av) nucleosomes. Although homotypic and heterotypic H2A.Z nucleosomes mapped throughout most of the genome, homotypic nucleosomes were enriched and heterotypic nucleosomes were depleted downstream of active promoters and intron-exon junctions. The distribution of homotypic H2A.Z nucleosomes resembled that of classical active chromatin and showed evidence of disruption during transcriptional elongation. Both homotypic H2A.Z nucleosomes and classical active chromatin were depleted downstream of paused polymerases. Our results suggest that H2A.Z enrichment patterns result from intrinsic! structural differences between heterotypic and homotypic H2A.Z nucleosomes that follow disruption during transcriptional elongation. View full text Figures at a glance * Figure 1: Broad distribution of homotypic and heterotypic H2Av nucleosomes. () Western analyses of two replicates show that sequential affinity-purified heterotypic and homotypic nucleosomes contain the expected composition of tagged histones and that endogenous epitopes are preserved. From the heterotypic purification (Het PD), antibodies (or streptavidin (Strep)) recognize endogenous epitopes and tags from Biotag-H2A (16.9 kDa) and Flag-H2Av (18 kDa). From the homotypic purification (Hom PD), antibodies (anti-H2A (Upstate 07-146), anti-H2Av49 or anti-Flag (Sigma, F3165)) or streptavidin recognize endogenous epitopes and tags from Biotag-H2Av (18.5kDa) and Flag-H2Av (H2Av). Proteins extracted from streptavidin beads with affinity-purified nucleosomes were resolved on 18% (w/v) SDS-PAGE gels and transferred to nitrocellulose. () Agarose gel showing that input DNA (1 μg) comprises mostly mononucleosomal fragments for two replicates. Asterisks mark input samples that were sequenced. () Normalized counts in 10-bp intervals from representative regions ! of chromosome 3R, showing two biological replicates for all DNA sizes for input, homotypic and heterotypic sequenced libraries. * Figure 2: Homotypic H2Av nucleosomes are enriched downstream of gene promoters. () Length distribution of mapped paired-end reads from the mean of two biological replicates for input, heterotypic and homotypic H2Av purifications. The size range of binned reads is indicated at the top of the graph by arrows and the corresponding size class is labeled. () Average profiles for input, heterotypic and homotypic H2Av purifications for the six size-class intervals: 55 bp (35–75 bp), 90 bp (76–110 bp), 130 bp (111–140 bp), 147 bp (141–160 bp), 170 bp (161–180 bp) and >180 bp (181–333 bp). Genes were divided into quintiles on the basis of expression level for all 10,997 fully annotated genes, aligned at their TSSs and averaged in 10-bp bins. Averaging was truncated at 5′ or 3′ ends of neighboring genes. The y axis refers to mapped paired-end counts. See also Supplementary Figure 4 for 3′ ends. * Figure 3: Homotypic H2Av nucleosomes are enriched downstream of intron-exon junctions and are low-salt soluble. () Average profile for homotypic H2Av nucleosomes in the 147-bp size class aligned at intron-exon and exon-intron junctions and split into quintiles of gene expression as in Figure 2. () Single Flag-H2Av pulldown (total H2Av) profile for comparison with . () Average profile for DNA from 80-mM-soluble nucleosomes. Profiles in – represent means of biological replicates over 67,630 intron-exon junctions, divided into 10-bp bins. (–) Same as – except aligned around gene 5′ and 3′ ends. The y axis refers to mapped single-end reads, where the scales were adjusted slightly to facilitate peak comparisons between the panels. * Figure 4: Depletion of homotypic H2Av nucleosomes at genes with stalled Pol II. () Average profiles for homotypic and heterotypic H2Av and input nucleosomes in the 147-bp size class for 950 genes that have promoter-proximal enrichment of polymerase (stalled, red) and 4,061 genes that have polymerase throughout the genes but are not enriched proximal to the promoter (green). () Genes with and without stalled Pol II show displacement of homotypic H2Av nucleosomes with increasing expression level. Average profiles for homotypic H2Av nucleosomes in the 147-bp size class show shifting of peaks downstream with increasing expression for the top three quintiles, and relative depletion of the top expression quintile. Similar results are seen for the 950 stalled and the 4,061 unstalled genes. Only the +1 position is shown for stalled genes because there are fewer genes and the resolution is lower than for unstalled genes. The y axis refers to paired-end counts. * Figure 5: Low-salt extracted chromatin reveals distinctive features of RNA Pol II stalling. () Single-end sequence reads from DNA extracted from low salt–soluble nucleosomes are displayed for 950 stalled and 4,061 unstalled genes. The mapped ends of plus-strand reads were offset by adding 75 bp, and minus-strand reads were offset by subtracting 75 bp (dotted blue lines), which assumes that all reads represent one end or the other of a protected nucleosome core. Read ends were mapped separately for plus strands (green lines) and minus strands (orange lines) without offset, which assumes that all reads were derived from a small protected fragment. Average profiles are shown for low salt–soluble nucleosomes mapped over genes in the stalled and unstalled classes. The y axis refers to mapped single-end reads. As a negative control, we also performed the same analysis at intron-exon junctions, but did not find evidence of enhanced protection at the corresponding position in comparing stalled and unstalled profiles (Supplementary Fig. 10). () A biological replicate sa! mple of low salt–extracted chromatin was subjected to limited paired-end sequencing. The 35–55-bp fraction is shown in the bottom panels for quintiles of expression for genes in the stalled and unstalled classes. For comparison, the 5′ plots from panel are shown in the panels above on the same x-axis scale. The y axis refers to paired-end counts. * Figure 6: Model for the generation of H2A.Z enrichment patterns. The cartoon depicts nucleosome disruptions that occur upon RNA Pol II (RNAPII) transit. Pol II disruption causes dimer loss that is smaller for homotypic H2A.Z (purple, Z) than for either heterotypic H2A.Z or canonical H2A (blue, A), with homotypic H2A.Z being the most stable nucleosome to disruption. Homotypic H2A.Z nucleosomes are formed with multiple rounds of Pol II transit and remain relatively stable to further disruption. Heterotypic H2A.Z nucleosomes form but are relatively unstable to successive rounds of Pol II transit, leading to their depletion relative to homotypic H2A.Z nucleosomes. H2A.Z recruits remodelers, which should show the highest activity on homotypic H2A.Z, in support of transcription. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE21615 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. * Christopher M Weber, * Jorja G Henikoff & * Steven Henikoff * Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, USA. * Christopher M Weber * Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. * Steven Henikoff Contributions C.M.W. performed the experiments, J.G.H. did the analysis, S.H. supervised the work and C.M.W. and S.H. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Steven Henikoff Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–11 and Supplementary Table 1 Additional data
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