Key Concepts and Challenges in Archaeal Transcription
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Complete Architecture of the Archaeal RNA Polymerase Open Complex from Single-Molecule FRET and NPS
ARTICLE Received 18 Aug 2014 | Accepted 21 Dec 2014 | Published 30 Jan 2015 DOI: 10.1038/ncomms7161 Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS Julia Nagy1, Dina Grohmann2, Alan C.M. Cheung3, Sarah Schulz2, Katherine Smollett3, Finn Werner3 & Jens Michaelis1 The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Fo¨rster resonance energy transfer and the Bayesian parameter estimation- based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH. 1 Biophysics Institute, Ulm University, Albert-Einstein-Allee 11, Ulm 89069, Germany. 2 Institut fu¨r Physikalische und Theoretische Chemie—NanoBioSciences, Technische Universita¨t Braunschweig, Hans-Sommer-Strae 10, 38106 Braunschweig, Germany. 3 Division of Biosciences, Institute for Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK. Correspondence and requests for materials should be addressed to J.M. -
Cyclic Di-AMP Signaling in Listeria Monocytogenes by Aaron Thomas
Cyclic di-AMP signaling in Listeria monocytogenes By Aaron Thomas Whiteley A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Infectious Diseases and Immunity in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Daniel A. Portnoy, Chair Professor Sarah A. Stanley Professor Russell E. Vance Professor Kathleen R. Ryan Summer 2016 Abstract Cyclic di-AMP signaling in Listeria monocytogenes by Aaron Thomas Whiteley Doctor of Philosophy in Infectious Diseases and Immunity University of California, Berkeley Professor Daniel A. Portnoy, Chair The Gram positive facultative intracellular pathogen Listeria monocytogenes is both ubiquitous in the environment and is a facultative intracellular pathogen. A high degree of adaptability to different growth niches is one reason for the success of this organism. In this dissertation, two facets of L. monocytogenes, growth and gene expression have been investigated. The first portion examines the function and necessity of the nucleotide second messenger c-di-AMP, and the second portion of this dissertation examines the signal transduction network required for virulence gene regulation. Through previous genetic screens and biochemical analysis it was found that the nucleotide second messenger cyclic di-adenosine monophosphate (c-di-AMP) is secreted by the bacterium during intracellular and extracellular growth. Depletion of c-di- AMP levels in L. monocytogenes and related bacteria results in sensitivity to cell wall acting antibiotics such as cefuroxime, decreased growth rate, and decreased virulence. We devised a variety of bacterial genetic screens to identify the function of this molecule in bacterial physiology. The sole di-adenylate cyclase (encoded by dacA) responsible for catalyzing synthesis of c-di-AMP in L. -
Bacterial RNA Polymerase Can Retain Σ Throughout Transcription
Bacterial RNA polymerase can retain σ70 throughout transcription Timothy T. Hardena,b, Christopher D. Wellsc, Larry J. Friedmanb, Robert Landickd,e, Ann Hochschildc, Jane Kondeva,1, and Jeff Gellesb,1 aDepartment of Physics, Brandeis University, Waltham, MA 02454; bDepartment of Biochemistry, Brandeis University, Waltham, MA 02454; cDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; dDepartment of Biochemistry, University of Wisconsin, Madison, WI 53706; and eDepartment of Bacteriology, University of Wisconsin, Madison, WI 53706 Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved December 10, 2015 (received for review July 15, 2015) Production of a messenger RNA proceeds through sequential stages early elongation pause. This early elongation pause, in turn, al- of transcription initiation and transcript elongation and termination. lows loading of an antitermination factor that enables tran- During each of these stages, RNA polymerase (RNAP) function is scription of the late gene operon (10, 13–15). Similar promoter- regulated by RNAP-associated protein factors. In bacteria, RNAP- proximal pause elements are also associated with many E. coli associated σ factors are strictly required for promoter recognition promoters (16–19), but the function of these elements is yet and have historically been regarded as dedicated initiation factors. unknown. Furthermore, σ70 interaction sites on core RNAP par- However, the primary σ factor in Escherichia coli, σ70,canremain tially overlap with those of transcription elongation factors such as associated with RNAP during the transition from initiation to elon- NusA, NusG, and RfaH (20–23). This and other evidence raises the gation, influencing events that occur after initiation. Quantitative possibility that σ70 retained in TECs sterically occludes the binding studies on the extent of σ70 retention have been limited to com- of other factors, which in turn could affect processes modulated by plexes halted during early elongation. -
High-Resolution RNA 3 -Ends Mapping of Bacterial Rho-Dependent
Nucleic Acids Research, 2018 1 doi: 10.1093/nar/gky274 High-resolution RNA 3-ends mapping of bacterial Rho-dependent transcripts Daniel Dar and Rotem Sorek* Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel Received February 21, 2018; Revised March 29, 2018; Editorial Decision March 31, 2018; Accepted April 04, 2018 ABSTRACT defined according to their dependence on the proteinaceous termination factor, Rho (4). Transcription termination in bacteria can occur ei- Rho-independent terminators, also known as intrinsic ther via Rho-dependent or independent (intrinsic) terminators, efficiently destabilize the elongating RNA- mechanisms. Intrinsic terminators are composed of polymerase (RNAP) complex in the absence of Rho and a stem-loop RNA structure followed by a uridine are encoded by a short ∼30nt DNA sequence downstream stretch and are known to terminate in a precise man- of the protein-coding region of the gene, as well as in some ner. In contrast, Rho-dependent terminators have regulatory 5 mRNA leaders (5,6). These terminators are more loosely defined characteristics and are thought composed of two main modules: a GC-rich sequence that to terminate in a diffuse manner. While transcripts folds into an energetically stable stem-loop RNA structure ending in an intrinsic terminator are protected from and a 7–8 nt uridine rich sequence (7–10). Termination ini- 3-5 exonuclease digestion due to the stem-loop tiates when the U-rich tract is transcribed and occupies the transcription bubble, causing the RNAP to pause and al- structure of the terminator, it remains unclear what lowing the upstream stem-loop forming sequence to nucle- protects Rho-dependent transcripts from being de- ate, which disrupts the transcription complex (8,9,11). -
Regulation of Stringent Factor by Branched-Chain Amino Acids
Regulation of stringent factor by branched-chain amino acids Mingxu Fanga and Carl E. Bauera,1 aMolecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405 Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved May 9, 2018 (received for review February 21, 2018) When faced with amino acid starvation, prokaryotic cells induce a Under normal growth conditions, the synthetase activity of Rel is stringent response that modulates their physiology. The stringent thought to be self-inhibited; however, during times of amino acid response is manifested by production of signaling molecules starvation, Rel interacts with stalled ribosomes, which activates guanosine 5′-diphosphate,3′-diphosphate (ppGpp) and guanosine synthetase activity to produce (p)ppGpp. The regulation of hy- 5′-triphosphate,3′-diphosphate (pppGpp) that are also called drolase activity is less understood but may involve one or more alarmones. In many species, alarmone levels are regulated by a downstream domains called the TGS and ACT domains. The TGS multidomain bifunctional alarmone synthetase/hydrolase called domain of SpoT has been shown to interact with an acyl carrier Rel. In this enzyme, there is an ACT domain at the carboxyl region protein, so it is presumed to sense the status of fatty acid metab- that has an unknown function; however, similar ACT domains are olism in E. coli (4). The function of the ACT domain is not as clear; present in other enzymes that have roles in controlling amino acid however, recent cryo-EM structures of E. coli RelA show that this metabolism. In many cases, these other ACT domains have been domain is involved in binding deacyl-tRNA as well as the ribosome shown to allosterically regulate enzyme activity through the bind- (5–7). -
The Alarmone (P)Ppgpp Is Part of the Heat Shock Response of Bacillus
bioRxiv preprint doi: https://doi.org/10.1101/688689; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 The alarmone (p)ppGpp is part of the heat shock response of Bacillus 2 subtilis 3 4 Heinrich Schäfer1,2, Bertrand Beckert3, Wieland Steinchen4, Aaron Nuss5, Michael 5 Beckstette5, Ingo Hantke1, Petra Sudzinová6, Libor Krásný6, Volkhard Kaever7, Petra 6 Dersch5,8, Gert Bange4*, Daniel Wilson3* & Kürşad Turgay1,2* 7 8 Author affiliations: 9 1 Institute of Microbiology, Leibniz Universität Hannover, Herrenhäuser Str. 2, D-30419, 10 Hannover, Germany. 11 2 Max Planck Unit for the Science of Pathogens, Charitéplatz 1, 10117 Berlin, Germany 12 3 Institute for Biochemistry and Molecular Biology, Martin-Luther-King Platz 6, University of 13 Hamburg, 20146 Hamburg, Germany. 14 4 Philipps-University Marburg, Center for Synthetic Microbiology (SYNMIKRO) and 15 Department of Chemistry, Hans-Meerwein-Strasse, 35043 Marburg, Germany. 16 5 Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, 17 Braunschweig, Germany 18 6 Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20, Prague, 19 Czech Republic. 20 7 Hannover Medical School, Research Core Unit Metabolomics, Carl-Neuberg-Strasse 1, 21 30625, Hannover, Germany. 22 8 Institute of Infectiology, Von-Esmarch-Straße 56, University of Münster, Münster, Germany 23 24 Short title: The interplay of heat shock and stringent response 25 1 bioRxiv preprint doi: https://doi.org/10.1101/688689; this version posted July 1, 2019. -
(P)Ppgpp Synthesis by the Ca2+-Dependent Rela-Spot Homolog
bioRxiv preprint doi: https://doi.org/10.1101/767004; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Plastidial (p)ppGpp synthesis by the Ca2+-dependent RelA-SpoT homolog regulates the adaptation of chloroplast gene expression to darkness in Arabidopsis Sumire Ono1,4, Sae Suzuki1,4, Doshun Ito1 Shota Tagawa2, Takashi Shiina2, and Shinji Masuda3,5 1School of Life Science & Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan 2Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan 3Center for Biological Resources & Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan 4These authors contributed equally to the study 5Corresponding author: E-mail, [email protected] Abbreviations: CRSH, Ca2+-activated RSH; flg22, flagellin 22; (p)ppGpp, 5'-di(tri)phosphate 3'-diphosphate; PAMPs; pathogen-associated molecular patterns; RSH, RelA-SpoT homolog; qRT-PCR, quantitative real-time PCR 1 bioRxiv preprint doi: https://doi.org/10.1101/767004; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract In bacteria, the hyper-phosphorylated nucleotides, guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp), function as secondary messengers in the regulation of various metabolic processes of the cell, including transcription, translation, and enzymatic activities, especially under nutrient deficiency. The activity carried out by these nucleotide messengers is known as the stringent response. -
Tetracycline Modulates the Valine Induced Stringent Response and Decreases Expressed Rpos in Escherichia Coli
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 15: 117 – 124 Copyright © April 2011, M&I UBC Tetracycline Modulates the Valine Induced Stringent Response and Decreases Expressed RpoS in Escherichia coli Natasha Castillon, Krysta Coyle, June Lai, and Cindy Yang Department of Microbiology & Immunology, University of British Columbia The stringent response in Escherichia coli is a physiological response to stress characterized by accumulation of the alarmone (p)ppGpp and the down-regulation of various processes involved in cellular growth and protein synthesis. It has been previously shown that activation of the stringent response by amino acid starvation is able to confer protection against various classes of antibiotics, including tetracycline and ampicillin. However, no observations have been made on partial activation of stringency. This investigation attempted to repress the level of bacterial stringent response in E. coli CP78 through the use of tetracycline. Tetracycline is an antibiotic which has been shown to restrict the cellular accumulation of (p)ppGpp in CP78 Escherichia coli. The effectiveness of the starvation by valine was monitored by measuring turbidity. The modulation of stringent response was monitored by the level of RpoS, since RpoS production is known to be enhanced by activated stringent response. As measured by turbidity, we successfully established an amino acid starvation condition with valine treatment as seen in the inhibited growth of CP78 cells as compared to untreated cells. The valine inhibition of the growth of the bacteria appeared to be reduced by the addition of tetracycline. However, cultures treated with high concentrations of tetracycline seemed to become more turbid than the untreated control even though they had similar levels of protein. -
RNA Polymerase Clamp Movement Aids Dissociation from DNA but Is Not Required for RNA Release at Intrinsic Terminators
bioRxiv preprint doi: https://doi.org/10.1101/453969; this version posted October 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. RNA polymerase clamp movement aids dissociation from DNA but is not required for RNA release at intrinsic terminators Michael J Bellecourta, Ananya Ray-Sonia,1, Alex Harwiga, Rachel Anne Mooneya, Robert Landicka,b* Departments of aBiochemistry and bBacteriology, University of Wisconsin–Madison, Madison, WI 53706, USA 1Present address: Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA *To whom correspondence should be addressed; Tel: +1 (608) 265-8475; Fax: +1 (608) 890- 2415; E-mail: [email protected] Highlights • The contributions of RNAP conformation changes during termination are ill-defined • Restricting RNAP clamp movement affects elongation rate, but not termination rate • RNA but not DNA can release at terminators when clamp movement is restricted • Inhibiting TL conformational flexibility impairs both RNA and DNA release Keywords: Escherichia coli, Termination commitment, Transcription elongation, Transcription pausing, Trigger loop Abbreviations: EC, elongating transcription complex; fxlink, crosslinking efficiency; Cys-pair, cysteine-pair; MccJ25, microcin J25; NA, nucleic acid; RNAP, RNA polymerase; TE, termination (commitment) efficiency; Thp, terminator hairpin; TL, trigger loop Declarations of interest: none Bellecourt et al. Page 1 10/25/2018 bioRxiv preprint doi: https://doi.org/10.1101/453969; this version posted October 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. -
Functional Analysis of Archaeal MBF1 by Complementation Studies in Yeast Jeannette Marrero Coto1,3, Ann E Ehrenhofer-Murray2, Tirso Pons3, Bettina Siebers1*
Marrero Coto et al. Biology Direct 2011, 6:18 http://www.biology-direct.com/content/6/1/18 RESEARCH Open Access Functional analysis of archaeal MBF1 by complementation studies in yeast Jeannette Marrero Coto1,3, Ann E Ehrenhofer-Murray2, Tirso Pons3, Bettina Siebers1* Abstract Background: Multiprotein-bridging factor 1 (MBF1) is a transcriptional co-activator that bridges a sequence-specific activator (basic-leucine zipper (bZIP) like proteins (e.g. Gcn4 in yeast) or steroid/nuclear-hormone receptor family (e. g. FTZ-F1 in insect)) and the TATA-box binding protein (TBP) in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH) domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. The main structural difference between archaeal MBF1 (aMBF1) and eukaryotic MBF1 is the presence of a Zn ribbon motif in aMBF1. In addition MBF1 interacting activators are absent in the archaeal domain. To study the function and therefore the evolutionary conservation of MBF1 and its single domains complementation studies in yeast (mbf1Δ) as well as domain swap experiments between aMBF1 and yMbf1 were performed. Results: In contrast to previous reports for eukaryotic MBF1 (i.e. Arabidopsis thaliana, insect and human) the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile Thermoproteus tenax and MMBF1 from the mesophile Methanosarcina mazei were not functional for complementation of an Saccharomyces cerevisiae mutant lacking Mbf1 (mbf1Δ). Of twelve chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1. -
The RNA Hairpin (Escherichia Coli/RNA Polymerase/Rho-Independent Termination) KEVIN S
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8793-8797, September 1995 Biochemistry Transcription termination at intrinsic terminators: The role of the RNA hairpin (Escherichia coli/RNA polymerase/rho-independent termination) KEVIN S. WILSONt AND PETER H. VON HIPPEL Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403 Contributed by Peter H. von Hippel, April 28, 1995 ABSTRACT Intrinsic termination of transcription in protein-dependent antitermination (Rees et al., unpublished Escherichia coli involves the formation of an RNA hairpin in data) and is likely to be quite general. In this view transcript the nascent RNA. This hairpin plays a central role in the termination is considered to be possible, in principle, at every release of the transcript and polymerase at intrinsic termi- template position. In practice, however, termination does not nation sites on the DNA template. We have created variants of occur at most template positions because the stability of the the AtR2 terminator hairpin and examined the relationship elongation complex results in characteristic half-times for between the structure and stability of this hairpin and the complex dissociation and RNA release of hours or days, while template positions and efficiencies of termination. The results the average dwell-time for elongation at a given template were used to test the simple nucleic acid destabilization model position at saturating NTP concentrations is 10-50 msec (3). of Yager and von Hippel and showed that this model must be It has been shown for the AtR2 terminator (2) and confirmed modified to provide a distinct role for the rU-rich sequence in for the AtR' terminator (Rees et al., unpublished data) that the the nascent RNA, since a perfect palindromic sequence that is termination pathway becomes accessible at intrinsic termina- sufficiently long to form an RNA hairpin that could destabilize tors because the transcription complex is massively destabi- the entire putative 12-bp RNADNA hybrid does not trigger lized in the vicinity of these sites. -
1 Evolutionary Origins of Two-Barrel RNA Polymerases and Site-Specific
Evolutionary origins of two-barrel RNA polymerases and site-specific transcription initiation Thomas Fouqueau*, Fabian Blombach* & Finn Werner Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, UK. email: [email protected]; [email protected] * These authors contributed equally to this work Abstract Evolutionary related multi-subunit RNA polymerases (RNAPs) carry out RNA synthesis in all domains life. While their catalytic cores and fundamental mechanisms of transcription elongation are conserved, the initiation stage of the transcription cycle differs substantially between bacteria and archaea/eukaryotes in terms of the requirements for accessory factors and details of the molecular mechanisms. This review focuses on recent insights into the evolution of the transcription apparatus with regard to (i) the surprisingly pervasive double-Ψ β-barrel active site configuration among different nucleic acid polymerase families, (ii) the origin and phylogenetic distribution of TBP, TFB and TFE transcription factors, and (iii) the functional relation between transcription- and translation initiation mechanisms in terms of TSS selection and RNA structure. Keywords Multisubunit RNA polymerases, Evolution, LUCA, Translation initiation 1 Contents Introduction 3 PolD and Qde1 contain double-Ψ β-barrels 4 Evolutionary insights from viral two-barrel RNAPs 6 The origins of the barrels in the RNA world? 7 The search for the evolutionary origins of the general transcription factors 9 Are bacterial sigma and archaeo-eukaryotic TFB/TFIIB factors evolutionary related? 11 Analogous initiation mechanisms in the three domains of life 11 Clues from unorthodox RNAPs from bacteriophages and eukaryotic viruses 14 The connection between transcription initiation and translation initiation 16 Conclusion 19 2 Introduction Nucleic acid polymerases carry out key functions in DNA replication, -repair and – recombination, as well as RNA transcription.