Latest Articles Include:
- Related Mechanisms for End Processing at Telomeres and DNA Double-Strand Breaks
- Mol Cell 35(2):137-138 (2009)
In a recent issue of Molecular Cell, Bonetti et al. (2009) identify in the yeast Saccharomyces cerevisiae that the molecular activities that generate 3′ overhangs at telomeric DNA ends are the same as those that resect DNA at double-strand breaks.
- MicroRNAs Cross the Line: The Battle for mRNA Stability Enters the Coding Sequence
- Mol Cell 35(2):139-140 (2009)
A study in this issue of Molecular Cell (Elcheva et al., 2009) shows the inherent instability of the βTrCP1 mRNA to be caused by microRNA-183 targeting the coding sequence; interestingly, this action is directly opposed by the RNA-binding protein CRD-BP.
- Ras and the FAK Paradox
- Mol Cell 35(2):141-142 (2009)
In a recent issue of Molecular Cell, Zheng et al. (2009) describe a surprising set of findings that highlight an unexpected negative regulation of FAK by oncogenic Ras and its consequences for cancer cell migration and invasion.
- A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors and Controls Bacterial Pathogenicity
- Mol Cell 35(2):143-153 (2009)
Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene expression changes. Here, we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protect! s Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development.
- Rejuvenation of CcdB-Poisoned Gyrase by an Intrinsically Disordered Protein Domain
- Mol Cell 35(2):154-163 (2009)
Toxin-antitoxin modules are small regulatory circuits that ensure survival of bacterial populations under challenging environmental conditions. The ccd toxin-antitoxin module on the F plasmid codes for the toxin CcdB and its antitoxin CcdA. CcdB poisons gyrase while CcdA actively dissociates CcdB:gyrase complexes in a process called rejuvenation. The CcdA:CcdB ratio modulates autorepression of the ccd operon. The mechanisms behind both rejuvenation and regulation of expression are poorly understood. We show that CcdA binds consecutively to two partially overlapping sites on CcdB, which differ in affinity by six orders of magnitude. The first, picomolar affinity interaction triggers a conformational change in CcdB that initiates the dissociation of CcdB:gyrase complexes by an allosteric segmental binding mechanism. The second, micromolar affinity binding event regulates expression of the ccd operon. Both functions of CcdA, rejuvenation and autoregulation, are mechanisti! cally intertwined and depend crucially on the intrinsically disordered nature of the CcdA C-terminal domain.
- Two-Site Phosphorylation of EPRS Coordinates Multimodal Regulation of Noncanonical Translational Control Activity
- Mol Cell 35(2):164-180 (2009)
Glutamyl-prolyl tRNA synthetase (EPRS) is a component of the heterotetrameric γ-interferon-activated inhibitor of translation (GAIT) complex that binds 3′UTR GAIT elements in multiple interferon-gamma (IFN-γ)-inducible mRNAs and suppresses their translation. Here, we elucidate the specific EPRS phosphorylation events that regulate GAIT-mediated gene silencing. IFN-γ induces sequential phosphorylation of Ser886 and Ser999 in the noncatalytic linker connecting the synthetase cores. Phosphorylation of both sites is essential for EPRS release from the parent tRNA multisynthetase complex. Ser886 phosphorylation is required for the interaction of NSAP1, which blocks EPRS binding to target mRNAs. The same phosphorylation event induces subsequent binding of ribosomal protein L13a and GAPDH and restores mRNA binding. Finally, Ser999 phosphorylation directs the formation of a functional GAIT complex that binds initiation factor eIF4G and represses translation. Thus, two-sit! e phosphorylation provides structural and functional pliability to EPRS and choreographs the repertoire of activities that regulates inflammatory gene expression.
- Translation Initiation from the Ribosomal A Site or the P Site, Dependent on the Conformation of RNA Pseudoknot I in Dicistrovirus RNAs
- Mol Cell 35(2):181-190 (2009)
Translation initiation of the second ORF of insect dicistrovirus RNA depends on an internal ribosomal entry site (IRES) in its intergenic region (IGR) and is exceptional in using a codon other than AUG and in not using the canonical initiator methionine tRNA. Studies in vitro suggest that pseudoknot I (PKI) immediately preceding the initiation codon occupies the ribosomal P site and that an elongator tRNA initiates translation from the ribosomal A site. Using dicistronic reporters carrying mutations in the initiation codon of the second ORF and mutant elongator or initiator tRNAs capable of reading these codons, we provide direct evidence for initiation from the A site in mammalian cells and, under certain conditions, also from the P site. Initiation from the A but not the P site requires PKI. Thus, PKI structure may be dynamic, and optimal IGR IRES-mediated translation of dicistroviral RNAs may require trans-acting factors to stabilize PKI.
- Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes
- Mol Cell 35(2):191-205 (2009)
Multiple RNA polymerase II (RNAPII) molecules can transcribe a gene simultaneously, but what happens when such polymerases collide—for example due to polymerase pausing or DNA damage? Here, RNAPII collision was characterized using a reconstituted system for simultaneous transcription by two polymerases. When progression of leading polymerase is obstructed, rear-end collision entails a transient state in which the elongation complexes interact, followed by substantial backtracking of trailing polymerase. Elongation complexes remain stable on DNA, with their activity and the integrity of transcription bubbles remaining intact. Subsequent TFIIS-stimulated transcript cleavage allows resumed forward translocation, resulting in trailing polymerase oscillating at the obstruction. Conversely, if leading polymerase is merely stalled at a pause site, collision and TFIIS cooperate to drive it through. We propose that dynamic interactions between RNAPII elongation complexes help! regulate polymerase traffic and that their conformational flexibility buffers the effect of collisions with objects on DNA, thereby maintaining stability in the face of obstacles to transcription.
- Phosphorylation of Mcm2 by Cdc7 Promotes Pre-replication Complex Assembly during Cell-Cycle Re-entry
- Mol Cell 35(2):206-216 (2009)
Cyclin E has been shown to have a role in pre-replication complex (Pre-RC) assembly in cells re-entering the cell cycle from quiescence. The assembly of the pre-RC, which involves the loading of six MCM subunits (Mcm2–7), is a prerequisite for DNA replication. We found that cyclin E, through activation of Cdk2, promotes Mcm2 loading onto chromatin. This function is mediated in part by promoting the accumulation of Cdc7 messenger RNA and protein, which then phosphorylates Mcm2. Consistent with this, a phosphomimetic mutant of Mcm2 can bypass the requirement for Cdc7 in terms of Mcm2 loading. Furthermore, ectopic expression of both Cdc6 and Cdc7 can rescue the MCM loading defect associated with expression of dominant-negative Cdk2. These results are consistent with a role for cyclin E-Cdk2 in promoting the accumulation of Cdc6 and Cdc7, which is required for Mcm2 loading when cells re-enter the cell cycle from quiescence.
- Initial Stages of V(D)J Recombination: The Organization of RAG1/2 and RSS DNA in the Postcleavage Complex
- Mol Cell 35(2):217-227 (2009)
To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.
- A Genome-wide siRNA Screen Reveals Diverse Cellular Processes and Pathways that Mediate Genome Stability
- Mol Cell 35(2):228-239 (2009)
Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (γH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased γH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological proces! ses than previously appreciated.
- CRD-BP Protects the Coding Region of βTrCP1 mRNA from miR-183-Mediated Degradation
- Mol Cell 35(2):240-246 (2009)
miRNAs are largely known to base pair with the 3′UTR of target mRNAs, downregulating their stability and translation. mRNA of βTrCP1 ubiquitin ligase is very unstable, but unlike the majority of mRNAs where 3′UTR determines the rate of mRNA turnover, βTrCP1 mRNA contains cis-acting destabilizing elements within its coding region. Here we show that degradation of mRNA of βTrCP1 is miRNA dependent and identify miR-183 as a microRNA that interacts with the coding region of βTrCP1 mRNA. Argonaute2 interacts with the same region of βTrCP1 mRNA in an miR-183-dependent manner. Inhibition of miR-183 function or disruption of the miR-183-binding site stabilizes βTrCP1 mRNA and elevates βTrCP1 levels, resulting in activation of the SCFβTrCP E3 ubiquitin ligase. We previously showed that the RNA-binding protein CRD-BP binds to the coding region of βTrCP1 mRNA and stabilizes it. Here we demonstrate that CRD-BP prevents degradation of βTrCP1 mRNA by attenuating its mi! R-183-dependent interaction with Ago2.
- Protein Occupancy Landscape of a Bacterial Genome
- Mol Cell 35(2):247-253 (2009)
Protein-DNA interactions are fundamental to core biological processes, including transcription, DNA replication, and chromosomal organization. We have developed in vivo protein occupancy display (IPOD), a technology that reveals protein occupancy across an entire bacterial chromosome at the resolution of individual binding sites. Application to Escherichia coli reveals thousands of protein occupancy peaks, highly enriched within and in close proximity to noncoding regulatory regions. In addition, we discovered extensive (>1 kilobase) protein occupancy domains (EPODs), some of which are localized to highly expressed genes, enriched in RNA-polymerase occupancy. However, the majority are localized to transcriptionally silent loci dominated by conserved hypothetical ORFs. These regions are highly enriched in both predicted and experimentally determined binding sites of nucleoid proteins and exhibit extreme biophysical characteristics such as high intrinsic curvature. Our o! bservations implicate these transcriptionally silent EPODs as the elusive organizing centers, long proposed to topologically isolate chromosomal domains.