Journal of Molecular Biology

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  • Bacterial voltage-gated sodium channels (BacNaVs) from the soil, sea, and salt lakes enlighten molecular mechanisms of electrical signaling and pharmacology in the brain and heart
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Jian Payandeh , Daniel L. Minor Jr.

    Voltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of NaVs have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts.
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    Categories: Journal Articles
  • A Structural Portrait of the PDZ Domain Family
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Andreas Ernst , Brent A. Appleton , Ylva Ivarsson , Yingnan Zhang , David Gfeller , Christian Wiesmann , Sachdev S. Sidhu

    PDZ (PSD-95/Discs-large/ZO-1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signalling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side chain and main chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide to understanding the structural basis for the diverse specificities across the family.
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  • The PHD finger of p300 influences its ability to acetylate histone and non-histone targets
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): Johannes G.M. Rack , Timo Lutter , Gro E.K. Bjerga , Corina Guder , Christine Ehrhardt , Signe Värv , Mathias Ziegler , Rein Aasland

    In enzymes that regulate chromatin structure, the combinatorial occurrence of modules that alter and recognise histone modifications is a recurrent feature. In this study, we explored the functional relationship between the acetyltransferase domain and the adjacent bromodomain/PHD finger region of the transcriptional coactivator p300. We found that the bromo/PHD region of p300 can bind to the acetylated catalytic domain in vitro and augment the catalytic activity of the enzyme. Deletion of the PHD finger, but not the bromodomain, impaired the ability of the enzyme to acetylate histones in vivo, while it enhanced p300 self-acetylation. A Rubinstein-Taybi syndrom-related point mutation in the p300 PHD finger resulted in increased self-acetylation, but retained the ability to acetylate histones. Hence, the PHD finger appears to negatively regulate self-acetylation. Furthermore, our data suggest that the PHD finger has a role in the recruitment of p300 to chromatin.
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  • The Enigmatic Cytoplasmic Regions of KCNH Channels
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Molecular Biology

    Author(s): João H. Morais-Cabral , Gail A. Robertson

    KCNH channels are expressed across a vast phylogenetic and evolutionary spectrum. In humans they function in a wide range of tissues and serve as biomarkers and targets for diseases such as cancer and cardiac arrhythmias. These channels share a general architecture with other voltage-gated ion channels but are distinguished by the presence of an N-terminal Per-Arnt-Sim (PAS) domain and a C-terminal domain with homology to cyclic nucleotide binding domains (referred to as the CNBh domain). Cytosolic regions outside these domains show little conservation between KCNH families but within a family are strongly conserved across species, likely reflecting variability that confers specificity to individual channel types. PAS and CNBh domains participate in channel gating, but at least twice in evolutionary history the PAS domain has been lost, and in one family it is omitted by alternate transcription to create a distinct channel subunit. In this focused review we present current knowledge of the structure and function of these cytosolic regions, discuss their evolution as modular domains, and provide our perspective on the important questions moving forward.
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  • Molecular architecture of photoreceptor phosphodiesterase elucidated by chemical cross-linking and integrative modeling
    [Aug 2014]

    Publication date: Available online 19 August 2014
    Source:Journal of Molecular Biology

    Author(s): Xiaohui Zeng-Elmore , Xiong-Zhuo Gao , Riccardo Pellarin , Dina Schneidman-Duhovny , Xiu-Jun Zhang , Katie A. Kozacka , Yang Tang , Andrej Sali , Robert J. Chalkley , Rick H. Cote , Feixia Chu

    Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from the high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for a differential binding of the γ-subunit to the GAFa domains of the α- and β-subunit are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.
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    Categories: Journal Articles
  • Coevolution of specificity determinants in eukaryotic glutamyl- and glutaminyl-tRNA synthetases
    [Aug 2014]

    Publication date: Available online 19 August 2014
    Source:Journal of Molecular Biology

    Author(s): Andrew Hadd , John J. Perona

    The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNAGln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNAGln and tRNAGlu with glutamate. This ancient GluRS also separately differentiated to exclude tRNAGln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNAGln and tRNAGlu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model, and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p-GluRS complex upon the diauxic shift to respiratory conditions.
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  • Functional Suppression of HAMP Domain Signaling Defects in the E. coli Serine Chemoreceptor
    [Aug 2014]

    Publication date: Available online 15 August 2014
    Source:Journal of Molecular Biology

    Author(s): Run-Zhi Lai , John S. Parkinson

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric E. coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA, bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: Unmodified E residues promote kinase-off output; methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer three of the HAMP bundle and followed in vivo CheA activity with a FRET-based assay. We found that an alanine or serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells, but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E, S255A) shifted Tsr output toward the kinase-off state, but two others (S255G, L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.
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    Categories: Journal Articles
  • Ryanodine Receptors: Allosteric Ion Channel Giants
    [Aug 2014]

    Publication date: Available online 15 August 2014
    Source:Journal of Molecular Biology

    Author(s): Filip Van Petegem

    The endoplasmic (ER) and sarcoplasmic reticulum (SR) form major intracellular Ca2+ stores. Ryanodine Receptors (RyRs) are large tetrameric ion channels in the SR and ER membranes that can release the Ca2+ upon triggering. With molecular weights exceeding 2.2 MDa, they represent the pinnacle of ion channel complexity. RyRs have adopted long-range allosteric mechanisms, with pore opening resulting in conformational changes over 200Å away. Together with the tens of protein and small molecule modulators, RyRs have adopted rich and complex regulatory mechanisms. Structurally related to inositol-1,4,5-trisphosphate receptors (IP3Rs), RyRs have been studied extensively using cryo-electron microscopy. Along with more recent X-ray crystallographic analyses of individual domains, these have resulted in pseudo-atomic models. Over 500 mutations in RyRs have been linked to severe genetic disorders, which underscore their role in the contraction of cardiac and skeletal muscle. Most of these have been linked to gain-of-function phenotypes, resulting in premature or prolonged leak of Ca2+ in the cytosol. This review outlines our current knowledge on the structure of RyRs at high and low resolution, their relationship to IP3Rs, an overview of the most commonly studied regulatory mechanisms, and models that relate disease-causing mutations to altered channel function.
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    Categories: Journal Articles
  • DNA Looping Provides for “Intersegmental Hopping” by Proteins: A Mechanism for Long-Range Site Localization
    [Aug 2014]

    Publication date: Available online 15 August 2014
    Source:Journal of Molecular Biology

    Author(s): Adam J. Pollak , Aaron T. Chin , Frank L.H. Brown , Norbert O. Reich

    Studies of how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites which controls the expression of virulence genes in several human pathogens. Provocatively, we find Dam travels between its recognition sites most efficiently when those sites are ~500 base pairs apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), invoking recent reports that demonstrate DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.
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    Categories: Journal Articles
  • Chromatin regulation of DNA damage repair and genome integrity in the central nervous system
    [Aug 2014]

    Publication date: Available online 14 August 2014
    Source:Journal of Molecular Biology

    Author(s): Ling Pan , Jay Penney , Li-Huei Tsai

    With the continued extension of lifespan, aging and age related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons’ DNA. When the accumulation of DNA damage exceeds a neuron’s capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.
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    Categories: Journal Articles
  • Editorial Board
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16









    Categories: Journal Articles
  • Contents List
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16









    Categories: Journal Articles
  • Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Cuiling Xu , Brian A. Maxwell , Zucai Suo

    Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.
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    Categories: Journal Articles
  • Undesigned Selection for Replication Termination of Bacterial Chromosomes
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Nobuaki Kono , Kazuharu Arakawa , Mitsuru Sato , Hirofumi Yoshikawa , Masaru Tomita , Mitsuhiro Itaya

    The oriC DNA replication origin in bacterial chromosomes, the location of which appears to be physically identified, is genetically regulated by relevant molecular machinery. In contrast, the location of the terminus remains obscure for many bacterial replicons, except for terC, the proposed and well-studied chromosome termination site in certain bacteria. The terC locus, which is composed of specific sequences for its binding protein, is located at a site opposite from oriC, exhibiting a symmetric structure around the oriC–terC axis. Here, we investigated Bacillus subtilis 168 strains whose axes were hindered and found that the native terC function was robust. However, eradication of terminus region specific binding protein resulted in the natural terC sites not being used for termination; instead, new termini were selected at a location exactly opposite to oriC. We concluded that replication generally terminates at the loci where the two approaching replisomes meet. This site was automatically selected, and two replisomes moving at the same rate supported symmetrical chromosome structures relative to oriC. The rule, which was even validated by artificial chromosomes irrespective of oriC, should be general for replicons administered by two replisomes.
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    Categories: Journal Articles
  • Sus1p Facilitates Pre-Initiation Complex Formation at the SAGA-Regulated Genes Independently of Histone H2B De-Ubiquitylation
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Geetha Durairaj , Rwik Sen , Bhawana Uprety , Abhijit Shukla , Sukesh R. Bhaumik

    Sus1p is a common component of transcriptional co-activator, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and mRNA export complex, TREX-2 (Transcription-export 2), and is involved in promoting transcription and mRNA export. However, it is not clearly understood how Sus1p promotes transcription. Here, we show that Sus1p is predominantly recruited to the upstream activating sequence of a SAGA-dependent gene, GAL1, under transcriptionally active conditions as a component of SAGA to promote the formation of pre-initiation complex (PIC) at the core promoter and, consequently, transcriptional initiation. Likewise, Sus1p promotes the PIC formation at other SAGA-dependent genes and hence transcriptional initiation. Such function of Sus1p in promoting PIC formation and transcriptional initiation is not mediated via its role in regulation of SAGA's histone H2B de-ubiquitylation activity. However, Sus1p's function in regulation of histone H2B ubiquitylation is associated with transcriptional elongation, DNA repair and replication. Collectively, our results support that Sus1p promotes PIC formation (and hence transcriptional initiation) at the SAGA-regulated genes independently of histone H2B de-ubiquitylation and further controls transcriptional elongation, DNA repair and replication via orchestration of histone H2B ubiquitylation, thus providing distinct functional insights of Sus1p in regulation of DNA transacting processes.
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    Categories: Journal Articles
  • Topology, Dimerization, and Stability of the Single-Span Membrane Protein CadC
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Eric Lindner , Stephen H. White

    Under acid stress, Escherichia coli induce expression of CadA (lysine decarboxylase) and CadB (lysine/cadaverine antiporter) in a lysine-rich environment. The ToxR-like transcriptional activator CadC controls expression of the cadBA operon. Using a novel signal peptidase I (SPase I) cleavage assay, we show that CadC is a type II single-span membrane protein (S-SMP) with a cytoplasmic DNA-binding domain and a periplasmic sensor domain. We further show that, as long assumed, dimerization of the sensor domain is required for activating the cadBA operon. We prove this using a chimera in which the periplasmic domain of RodZ—a type II membrane protein involved in the maintenance of the rod shape of E. coli—replaces the CadC sensor domain. Because the RodZ periplasmic domain cannot dimerize, the chimera cannot activate the operon. However, replacement of the transmembrane (TM) domain of the chimera with the glycophorin A TM domain causes intramembrane dimerization and consequently operon activation. Using a low-expression protocol that eliminates extraneous TM helix dimerization signals arising from protein over-expression, we enhanced dramatically the dynamic range of the β-galactosidase assay for cadBA activation. Consequently, the strength of the intramembrane dimerization of the glycophorin A domain could be compared quantitatively with the strength of the much stronger periplasmic dimerization of CadC. For the signal peptidase assay, we inserted an SPase I cleavage site (AAA or AQA) at the periplasmic end of the TM helix. Cleavage occurred with high efficiency for all TM and periplasmic domains tested, thus eliminating the need for the cumbersome spheroplast-proteinase K method for topology determinations.
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    Categories: Journal Articles
  • NMR Model of PrgI–SipD Interaction and Its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Thenmalarchelvi Rathinavelan , Maria Lara-Tejero , Matthew Lefebre , Srirupa Chatterjee , Andrew C. McShan , Da-Chuan Guo , Chun Tang , Jorge E. Galan , Roberto N. De Guzman

    Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI–SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein—the N-terminal α-helical hairpin—has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs; thus, our results have wider implication in understanding other needle-tip complexes.
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    Categories: Journal Articles
  • Rtr1 Is a Dual Specificity Phosphatase That Dephosphorylates Tyr1 and Ser5 on the RNA Polymerase II CTD
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Peter L. Hsu , Fan Yang , Whitney Smith-Kinnaman , Wen Yang , Jae-Eun Song , Amber L. Mosley , Gabriele Varani

    The phosphorylation state of heptapeptide repeats within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (PolII) controls the transcription cycle and is maintained by the competing action of kinases and phosphatases. Rtr1 was recently proposed to be the enzyme responsible for the transition of PolII into the elongation and termination phases of transcription by removing the phosphate marker on serine 5, but this attribution was questioned by the apparent lack of enzymatic activity. Here we demonstrate that Rtr1 is a phosphatase of new structure that is auto-inhibited by its own C-terminus. The enzymatic activity of the protein in vitro is functionally important in vivo as well: a single amino acid mutation that reduces activity leads to the same phenotype in vivo as deletion of the protein-coding gene from yeast. Surprisingly, Rtr1 dephosphorylates not only serine 5 on the CTD but also the newly described anti-termination tyrosine 1 marker, supporting the hypothesis that Rtr1 and its homologs promote the transition from transcription to termination.
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    Categories: Journal Articles
  • The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix
    [Aug 2014]

    Publication date: 12 August 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 16

    Author(s): Minttu T. Virkki , Christoph Peters , Daniel Nilsson , Therese Sörensen , Susana Cristobal , Björn Wallner , Arne Elofsson

    The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.
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    Categories: Journal Articles
  • Hydrophobic Gating in Ion Channels
    [Aug 2014]

    Publication date: Available online 12 August 2014
    Source:Journal of Molecular Biology

    Author(s): Prafulla Aryal , Mark S.P. Sansom , Stephen J. Tucker

    Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid–vapor transitions. These transient vapor states are “dewetted”, i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed “hydrophobic gating”, was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.
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    Categories: Journal Articles