Hot off the presses! Feb 04 mol cell

The Feb 04 issue of the mol cell is now up on Pubget (About mol cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Ultrasensitivity and Positive Feedback to Promote Sharp Mitotic Entry
  - mol cell 41(3):243-244 (2011)
  In this issue, Trunnell et al. (2011) show that in mitotic entry the positive feedback that drives the activation of cyclin-dependent kinase (Cdk) involves a very ultrasensitive step of phosphorylation of Cdc25C by Cdk, thus strongly contributing to the switch-like behavior of this essential cell-cycle transition.
• Sweet Business: Spot42 RNA Networks with CRP to Modulate Catabolite Repression
  - mol cell 41(3):245-246 (2011)
  Spot42 is a paradigm for small RNAs that fine-tune carbon metabolism. In this issue of Molecular Cell, Beisel and Storz (2011) reveal that this conserved RNA acts through a multioutput feedforward loop to modulate the global dynamics of sugar consumption.
• Peptides in the Ribosomal Tunnel Talk Back
  - mol cell 41(3):247-248 (2011)
  In this issue of Molecular Cell, Ramu et al. demonstrate that nascent peptides located within the ribosomal tunnel can talk back to the peptidyl transferase center to induce translational stalling by restricting the species of aminoacyl-tRNAs that can bind there.
• Single-Molecule Studies of RNA Polymerase: One Singular Sensation, Every Little Step It Takes
  - mol cell 41(3):249-262 (2011)
  Transcription is the first of many biochemical steps that turn the genetic information found in DNA into the proteins responsible for driving cellular processes. In this review, we highlight certain advantages of single-molecule techniques in the study of prokaryotic and eukaryotic transcription, and the specific ways in which these techniques complement conventional, ensemble-based biochemistry. We focus on recent literature, highlighting examples where single-molecule methods have provided fresh insights into mechanism. We also present recent technological advances and outline future directions in the field.
• Ultrasensitivity in the Regulation of Cdc25C by Cdk1
  - mol cell 41(3):263-274 (2011)
  Cdc25C is a critical component of the interlinked positive and double-negative feedback loops that constitute the bistable mitotic trigger. Computational studies have indicated that the trigger's bistability should be more robust if the individual legs of the loops exhibit ultrasensitive responses. Here, we show that in Xenopus extracts two measures of Cdc25C activation (hyperphosphorylation and Ser 287 dephosphorylation) are highly ultrasensitive functions of the Cdk1 activity; estimated Hill coefficients were 11 to 32. Some of Cdc25C's ultrasensitivity can be reconstituted in vitro with purified components, and the reconstituted ultrasensitivity depends upon multisite phosphorylation. The response functions determined here for Cdc25C and previously for Wee1A allow us to formulate a simple mathematical model of the transition between interphase and mitosis. The model shows how the continuously variable regulators of mitosis work collectively to generate a switch-like,! hysteretic response.
• Viral-Mediated Noisy Gene Expression Reveals Biphasic E2f1 Response to MYC
  - mol cell 41(3):275-285 (2011)
  Gene expression mediated by viral vectors is subject to cell-to-cell variability, which limits the accuracy of gene delivery. When coupled with single-cell measurements, however, such variability provides an efficient means to quantify signaling dynamics in mammalian cells. Here, we illustrate the utility of this approach by mapping the E2f1 response to MYC, serum stimulation, or both. Our results revealed an underappreciated mode of gene regulation: E2f1 expression first increased, then decreased as MYC input increased. This biphasic pattern was also reflected in other nodes of the network, including the miR-17-92 microRNA cluster and p19Arf. A mathematical model of the network successfully predicted modulation of the biphasic E2F response by serum and a CDK inhibitor. In addition to demonstrating how noise can be exploited to probe signaling dynamics, our results reveal how coordination of the MYC/RB/E2F pathway enables dynamic discrimination of aberrant and normal l! evels of growth stimulation.
• The Base-Pairing RNA Spot 42 Participates in a Multioutput Feedforward Loop to Help Enact Catabolite Repression in Escherichia coli
  - mol cell 41(3):286-297 (2011)
  Bacteria selectively consume some carbon sources over others through a regulatory mechanism termed catabolite repression. Here, we show that the base-pairing RNA Spot 42 plays a broad role in catabolite repression in Escherichia coli by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse nonpreferred carbon sources. Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multioutput feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. Our results suggest that base-pairing RNAs in feedforward loops can help shape the steady-state levels and dynamics of gene expression.
• p38 MAPK Controls Prothrombin Expression by Regulated RNA 3′ End Processing
  - mol cell 41(3):298-310 (2011)
  Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 3′ end processing components and phosphorylates the RNA-binding proteins FBP2 and FBP3, which inhibit 3′ end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 3′UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 3′ end processing. We provide in vivo evidence suggesting the importance of this mechanism in inflammatory hype! rcoagulation and tumor invasion. Regulated 3′ end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications.
• Structural Basis for Dimerization and Activity of Human PAPD1, a Noncanonical Poly(A) Polymerase
  - mol cell 41(3):311-320 (2011)
  Poly(A) polymerases (PAPs) are found in most living organisms and have important roles in RNA function and metabolism. Here, we report the crystal structure of human PAPD1, a noncanonical PAP that can polyadenylate RNAs in the mitochondria (also known as mtPAP) and oligouridylate histone mRNAs (TUTase1). The overall structure of the palm and fingers domains is similar to that in the canonical PAPs. The active site is located at the interface between the two domains, with a large pocket that can accommodate the substrates. The structure reveals the presence of a previously unrecognized domain in the N-terminal region of PAPD1, with a backbone fold that is similar to that of RNP-type RNA binding domains. This domain (named the RL domain), together with a β-arm insertion in the palm domain, contributes to dimerization of PAPD1. Surprisingly, our mutagenesis and biochemical studies show that dimerization is required for the catalytic activity of PAPD1.
• Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-Site of the Peptidyl Transferase Center
  - mol cell 41(3):321-330 (2011)
  The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a cri! tical role.
• The Structural Basis for Tight Control of PP2A Methylation and Function by LCMT-1
  - mol cell 41(3):331-342 (2011)
  Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crystal structures of human LCMT-1 in isolation and in complex with PP2A stabilized by a cofactor mimic. The structures show that the LCMT-1 active-site pocket recognizes the carboxyl terminus of PP2A, and, interestingly, the PP2A active site makes extensive contacts to LCMT-1. We demonstrated that activation of the PP2A active site stimulates methylation, suggesting a mechanism for efficient conversion of activated PP2A into substrate-specific holoenzymes, thus minimizing unregulated phosphatase activity or formation of inactive holoenzymes. A dominant-negative LCMT-1 mutant attenuates the cell cycle without causing cell death, li! kely by inhibiting uncontrolled phosphatase activity. Our studies suggested mechanisms of LCMT-1 in tight control of PP2A function, important for the cell cycle and cell survival.
• SecA Interacts with Ribosomes in Order to Facilitate Posttranslational Translocation in Bacteria
  - mol cell 41(3):343-353 (2011)
  In Escherichia coli, translocation of exported proteins across the cytoplasmic membrane is dependent on the motor protein SecA and typically begins only after synthesis of the substrate has already been completed (i.e., posttranslationally). Thus, it has generally been assumed that the translocation machinery also recognizes its protein substrates posttranslationally. Here we report a specific interaction between SecA and the ribosome at a site near the polypeptide exit channel. This interaction is mediated by conserved motifs in SecA and ribosomal protein L23, and partial disruption of this interaction in vivo by introducing mutations into the genes encoding SecA or L23 affects the efficiency of translocation by the posttranslational pathway. Based on these findings, we propose that SecA could interact with its nascent substrates during translation in order to efficiently channel them into the "posttranslational" translocation pathway.
• Linear Ubiquitin Assembly Complex Negatively Regulates RIG-I- and TRIM25-Mediated Type I Interferon Induction
  - mol cell 41(3):354-365 (2011)
  Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25! degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway.
• Common Design Principles in the Spliceosomal RNA Helicase Brr2 and in the Hel308 DNA Helicase
  - mol cell 41(3):366 (2011)