DOCSLIB.ORG

Regulation of Antibiotic-Resistance by Non-Coding Rnas in Bacteria Dar and Sorek 113

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Antibiotic-Resistance by Non-Coding Rnas in Bacteria Dar and Sorek 113 Available online at www.sciencedirect.com ScienceDirect Regulation of antibiotic-resistance by non-coding RNAs in bacteria Daniel Dar and Rotem Sorek Antibiotic resistance genes are commonly regulated by overcome antibiotics through various mechanisms, such sophisticated mechanisms that activate gene expression in as the ejection of antibiotics from the cell via efflux response to antibiotic exposure. Growing evidence suggest pumps, enzymatic deactivation of the antibiotic mole- that cis-acting non-coding RNAs play a major role in regulating cules, and protection of antibiotic cellular targets via the expression of many resistance genes, specifically those chemical modifications (e.g. modifying the ribosome or which counteract the effects of translation-inhibiting the cell wall) [4]. Together, such resistance mechanisms 0 antibiotics. These ncRNAs reside in the 5 UTR of the regulated threaten the continued efficacy of antibiotics in medicine gene, and sense the presence of the antibiotics by recruiting [3]. translating ribosomes onto short upstream open reading frames (uORFs) embedded in the ncRNA. In the presence of translation-inhibiting antibiotics ribosomes arrest over the Although resistance genes provide bacteria with a distinct uORF, altering the RNA structure of the regulator and switching advantage during exposure to antibiotic, they are gener- the expression of the resistance gene to ‘ON’. The specificity of ally thought to carry a fitness burden at times when these riboregulators is tuned to sense-specific classes of antibiotics are not applied [2,5,6]. For example methyl- antibiotics based on the length and composition of the ation of specific residues in the 23S ribosomal RNA respective uORF. Here we review recent work describing new protects bacteria from macrolide antibiotics, but also types of antibiotic-sensing RNA-based regulators and causes cell-wide disruption in protein synthesis, which elucidating the molecular mechanisms by which they function lead to major fitness defects [7 ]. Therefore, to mitigate to control antibiotic resistance in bacteria. the negative effects of antibiotic resistance, bacteria employ regulatory mechanisms that can sense the antibi- otic molecule, and then selectively activate the expres- Address sion of the relevant antibiotic resistance genes only dur- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel ing exposure to the antibiotic, effectively circumventing the fitness burden and facilitating the long term mainte- Corresponding author: Sorek, Rotem ([email protected]) nance of such genes in the genome [2,5,6,7 ]. Current Opinion in Microbiology 2017, 36:111–117 This review comes from a themed issue on Cell regulation One of the common mechanisms underlying antibiotic- dependent activation of resistance genes is via specific Edited by Petra Dersch and Michael T Laub transcription factors that can sense the presence of the antibiotic [4]. In Gram-negative bacteria such regulation is manifested, for example in the context of the tetracy- http://dx.doi.org/10.1016/j.mib.2017.02.005 cline efflux pump, tetA. In the absence of tetracycline, the transcriptional repressor TetR constitutively binds the 1369-5274/ã 2017 Elsevier Ltd. All rights reserved. tetA promoter and inhibits the expression of the tetA resistance gene [8]. When tetracycline antibiotic is pres- ent in the cell, direct binding of tetracycline to the tetR repressor leads to its dissociation from the DNA and drives tetA expression, leading to antibiotic resistance Introduction [8] (Figure 1a). Transcription regulation has also been detected in the vancomycin resistance operon, vanHAX, The discovery of antibiotic compounds had made a commonly found in Enterococci [9]. In this case, however, profound impact on modern medicine, extending both the presence of vancomycin is detected by a membrane life span and life quality. Presently, antibiotics are perva- sensory kinase (VanS) which, in turn, phosphorylates and sively administered in clinical and in veterinary care, as activates VanR, a transcription regulator that drives the well as in agricultural applications, resulting in a tremen- expression of the vanHAX resistance operon [9] dous flow of antibiotics into the environment [1]. The (Figure 1b). Analogous forms of transcription regulation immense selective pressure caused by exposure to anti- occur in many bacteria, where they play important roles in biotic has driven the rapid spread and evolution of anti- controlling the expression of antibiotic resistance genes biotic-resistance genes among pathogenic and commen- [2,5]. sal bacteria [2,3]. These resistance genes allow bacteria to www.sciencedirect.com Current Opinion in Microbiology 2017, 36:111–117 112 Cell regulation Figure 1 (a) Regulation by the TetR repressor (b) Regulation by the VanS/VanR two-component system TetR No transcription Vancomycin tetA DNA Cell wall tetA promoter VanS Plasma Tetracycline binding releases the TetR repressor membrane TetR VanR P tetracycline P Activated VanR RNA-polymerase RNA-polymerase Active transcription Active transcription P tetA DNA vanJKHAX DNA tetA promoter (c ) Regulation by translational attenuation “OFF”Inhibition of translation initiation “ON” Antibiotics RBS is sequestered RBS is free uORF by RNA structure RBS Translation can inititate ermC mRNA ermC mRNA Riboregulator RBS Ribosome arrests over uORF (d) Regulation by transcriptional attenuation “OFF” Premature transcription termination “ON” Antibiotics uORF Terminator stem-loop formation Terminator is bypassed leading leads to premature termination to transcription readthrough UUUUU UUUUU resistance gene mRNA Riboregulator Ribosome arrests over uORF Current Opinion in Microbiology Control of antibiotic resistance by protein or RNA-based regulators. (a) The TetR DNA-binding repressor targets the promoter of tetA and blocks transcription. Upon binding to tetracycline, the repressor dissociates, promoting tetA transcription initiation. (b) The membrane protein VanS senses vancomycin and then phosphorylates the response regulator VanR, which activates the transcription of the vancomycin resistance gene operon. (c) Schematic representation of translational attenuation. In the absence of antibiotics the riboregulator folds into an RBS sequestering structure, such that translation of the mRNA of the resistance gene cannot be initiated. Subsequent exposure to translation inhibiting antibiotics causes the ribosome to stall over a specific position in the uORF, resulting in structural reshaping of the riboregulator such that the RBS is released, thus enabling translation to initiate. (d) Transcriptional attenuation regulates expression by controlling the formation of a premature transcription terminator—a stem-loop structure immediately followed by a poly uridine tract. In the absence of antibiotics, transcription begins, yet terminates prematurely. Ribosome stalling over the uORF inhibits terminator stem-loop formation and promotes transcription of the resistance gene. RNA-mediated regulation of antibiotic ncRNAs, also known as riboregulators, are structured 0 resistance RNA elements that reside within 5 untranslated regions 0 Growing evidence show that, in addition to classic tran- (5 UTRs) of antibiotic resistance genes, in particular scription-factor mediated gene regulation, bacteria fre- those that provide resistance to ribosome-inhibiting anti- quently employ cis-regulatory non-coding RNAs biotics. In the absence of antibiotics these RNA regula- (ncRNAs) that sense the presence of antibiotics and tors inhibit the expression of the resistance gene in cis by regulate resistance genes accordingly [10–12,13 ]. These either masking the ribosome binding site (RBS) or Current Opinion in Microbiology 2017, 36:111–117 www.sciencedirect.com Regulation of antibiotic-resistance by non-coding RNAs in bacteria Dar and Sorek 113 generating a premature transcriptional terminator within dependent ribosome stalling over the short uORF dis- 0 the 5 UTR [14,15] (Figure 1c,d). Conversely, when the rupts the terminator stem-loop base-pairing, enabling antibiotic is present in the cell, the RNA structure of transcriptional readthrough and synthesis of the bmrCD these regulators is altered, resulting in alternative base- mRNA [28] (Figure 1d). Transcriptional attenuation has pairing patterns that do not attenuate transcription or been implicated in the regulation of several antibiotic translation, and so activate the expression of the regulated resistance genes including ribosome methylating gene (Figure 1c,d). enzymes [15], antibiotic efflux pumps [28,30], and ribo- some rescue factors [31,32]. The majority of antibiotic responsive riboregulators have been found to sense the presence of translation-inhibiting Until recently, most antibiotic-responsive riboregulation antibiotics by directly measuring the activity the ribo- was thought to function via RBS-mediated translational some, that is the target of the antibiotic [11]. This attenuation, with transcriptional attenuation (regulation regulatory mechanism, also known as attenuation, is via the generation of a premature transcriptional termi- 0 enabled by the utilization of short upstream open reading nator in the 5 UTR) considered relatively rare [11,15]. A frames (uORF) that recruit translating ribosomes to the recent study, which employed term-seq, a high-through- 0 regulatory 5 UTR element. In the presence of certain put RNA-sequencing method for detecting premature antibiotics, translating ribosomes
Load more

Recommended publications
  • Engineering a Circular Riboregulator in Escherichia Coli
    bioRxiv preprint doi: https://doi.org/10.1101/008987; this version posted September 25, 2014. 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. Engineering a circular riboregulator in Escherichia coli William Rostain1,2, Shensi Shen2, Teresa Cordero1, Guillermo Rodrigo2,3 and Alfonso Jaramillo1,2,* 1 School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom. 2 Institute of Systems and Synthetic Biology, CNRS - Université d’Evry val d’Essonne, 91000 Évry, France. 3 Instituto de Biologia Molecular y Celular de Plantas, CSIC – Universidad Politécnica de Valencia, 46022 Valencia, Spain. * Corresponding author. School of Life Sciences, University of Warwick. Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom. Tel: +44 (0)24 765 73432, E-mail: [email protected] Type: Letter Running title: Circular Riboregulator. Keywords: Biotechnology, Riboregulators, Splicing, Synthetic Biology. 1 bioRxiv preprint doi: https://doi.org/10.1101/008987; this version posted September 25, 2014. 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 Circular RNAs have recently been shown to be important gene expression regulators in mammalian cells. However, their role in prokaryotes remains elusive. Here, we engineered a synthetic riboregulator that self-splice to produce a circular molecule, exploiting group I permuted intron-exon (PIE) sequences. We demonstrated that the resulting circular riboregulator can activate gene expression, showing increased dynamic range compared to the linear form. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker.
    [Show full text]
  • Exploring the Structure of Long Non-Coding Rnas, J
    IMF YJMBI-63988; No. of pages: 15; 4C: 3, 4, 7, 8, 10 1 2 Rise of the RNA Machines: Exploring the Structure of 3 Long Non-Coding RNAs 4 Irina V. Novikova, Scott P. Hennelly, Chang-Shung Tung and Karissa Y. Sanbonmatsu Q15 6 Los Alamos National Laboratory, Los Alamos, NM 87545, USA 7 Correspondence to Karissa Y. Sanbonmatsu: [email protected] 8 http://dx.doi.org/10.1016/j.jmb.2013.02.030 9 Edited by A. Pyle 1011 12 Abstract 13 Novel, profound and unexpected roles of long non-coding RNAs (lncRNAs) are emerging in critical aspects of 14 gene regulation. Thousands of lncRNAs have been recently discovered in a wide range of mammalian 15 systems, related to development, epigenetics, cancer, brain function and hereditary disease. The structural 16 biology of these lncRNAs presents a brave new RNA world, which may contain a diverse zoo of new 17 architectures and mechanisms. While structural studies of lncRNAs are in their infancy, we describe existing 18 structural data for lncRNAs, as well as crystallographic studies of other RNA machines and their implications 19 for lncRNAs. We also discuss the importance of dynamics in RNA machine mechanism. Determining 20 commonalities between lncRNA systems will help elucidate the evolution and mechanistic role of lncRNAs in 21 disease, creating a structural framework necessary to pursue lncRNA-based therapeutics. 22 © 2013 Published by Elsevier Ltd. 24 23 25 Introduction rather than the exception in the case of eukaryotic 50 organisms. 51 26 RNA is primarily known as an intermediary in gene LncRNAs are defined by the following: (i) lack of 52 11 27 expression between DNA and proteins.
    [Show full text]
  • Dynamic Signal Processing by Ribozyme-Mediated RNA Circuits to Control Gene Expression
    bioRxiv preprint doi: https://doi.org/10.1101/016915; this version posted March 23, 2015. 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. Dynamic signal processing by ribozyme- mediated RNA circuits to control gene expression Shensi Shen1,†, Guillermo Rodrigo1,†, Satya Prakash3, Eszter Majer2, Thomas E. Landrain1, Boris Kirov1, José-Antonio Daròs2, and Alfonso 1,3,* Jaramillo 1 Institute of Systems and Synthetic Biology, Université d’Évry Val d’Essonne, CNRS, F-91000 Évry, France 2 Instituto de Biología Molecular y Celular de Plantas, CSIC - Universidad Politécnica de Valencia, 46022 Valencia, Spain 3 School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom † Equal contribution to the work. * Correspondence: School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. E-mail: Alfonso.Jaramillo at warwick.ac.uk Manuscript information Type: Article. Subject category: RNA -- Synthetic Biology & Chemistry. Manuscript size: about 8,100 words (total). Abstract size: 144 words. Number of Figures: 6 (color). With Supplementary Information. 1" bioRxiv preprint doi: https://doi.org/10.1101/016915; this version posted March 23, 2015. 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. Abstract Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression.
    [Show full text]
  • RNA Regulatory Elements Play a Significant Role in Gene
    Ab initio IDENTIFICATION OF REGULATORY RNAS USING INFORMATION-THEORETIC UNCERTAINTY by AMIRHOSSEIN MANZOUROLAJDAD (Under the Direction of Dr. Jonathan Arnold) ABSTRACT RNA regulatory elements play a significant role in gene regulation. Riboswitches are regulatory elements which function by forming a ligand-induced alternative fold that controls access to ribosome binding sites or other regulatory sites in RNA. Traditionally, riboswitches have been identified based on sequence and structural homology. In this work, in an attempt to devise an ab initio method for identification of regulatory elements, mainly riboswitches, we derive and implement Shannon’s entropy of the SCFG ensemble on an RNA sequence in polynomial time for both structurally ambiguous and unambiguous grammars. We then evaluate the significance of this new measure of structural entropy in identifying riboswitches. Finally, sim- ple lightweight stochastic context-free grammar folding models assign significant values to long extensive secondary structures in Bacillus subtilis. INDEX WORDS: Riboswitch, RNA Secondary Structure, Stochastic Context-free Grammars, Information Theory Ab initio IDENTIFICATION OF REGULATORY RNAS USING INFORMATION-THEORETIC UNCERTAINTY by AMIRHOSSEIN MANZOUROLAJDAD BS, The University of Guilan, Rasht, Iran, 2004 MS, Isfahan University of Technology, Isfahan, Iran, 2007 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS,GEORGIA 2014 ⃝c 2014 Amirhossein Manzourolajdad All Rights Reserved Ab initio IDENTIFICATION OF REGULATORY RNAS USING INFORMATION-THEORETIC UNCERTAINTY by AMIRHOSSEIN MANZOUROLAJDAD Approved: Major Professor: Jonathan Arnold Committee: Sidney Kushner Russell L. Malmberg Jan Mrazek Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2014 I dedicate this work to my mother, Roya iv ACKNOWLEGEMENTS I will be eternally indebted to my major advisor Dr.
    [Show full text]
  • Characterization of the Smk Box: a Riboswitch
    CHARACTERIZATION OF THE SMK BOX: A RIBOSWITCH THAT BINDS S-ADENOSYLMETHIONINE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ryan T. Fuchs, B.S. Graduate Program in Microbiology The Ohio State University 2009 Dissertation Committee: Professor Tina M. Henkin, Adviser Professor Juan D. Alfonzo Professor Charles J. Daniels Professor Kurt Fredrick ABSTRACT Riboswitches are conserved RNA sequences that regulate downstream gene expression via changes in the RNA structure. Riboswitches usually respond to an environmental stimulus, typically binding of an effector molecule, that causes a structural rearrangement without additional trans-acting factors. The structural rearrangement can cause or prevent the formation of an intrinsic transcriptional terminator to regulate the gene at the level of premature transcription termination, or it can modulate the accessibility of the ribosome binding site to regulate at the level of translation initiation. Previous work has shown that there are multiple classes of riboswitches that bind S- adenosylmethionine (SAM) and regulate genes involved in biosynthesis of methionine, cysteine and SAM. The S box (or SAM-I) riboswitches are found in a wide range of bacteria while the SAM-II riboswitch is found primarily in Proteobacteria. Both riboswitches recognize SAM in a similar manner, but they are very different in terms of sequence, secondary and tertiary structure. This demonstrates that it is possible for bacteria to evolve very different regulatory RNAs to respond to the same metabolite. We discovered a third SAM binding riboswitch motif (called the SMK box or SAM-III) upstream of the metK gene (encoding SAM synthetase) in many Lactobacillales species.
    [Show full text]
  • Signal Integration: Applications of RNA Riboregulator Capabilities Kyliah Clarkson, Natasha Tuskovich, Derek Jacoby, Chris Tuttle, Layne Woodfin
    Signal Integration: Applications of RNA Riboregulator Capabilities Kyliah Clarkson, Natasha Tuskovich, Derek Jacoby, Chris Tuttle, Layne Woodfin Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC Introduction Background – Biothermometer Background - Ribolock and Ribokey Self-complementary messenger RNA has a high potential for tunable repression An RNA hairpin will also unfold when exposed to a sequence of higher of translation. A self-complementary hairpin which includes the Shine-Dalgarno site An RNA hairpin will unfold when exposed to temperatures past the melting point for the sequence. This permits the temperature-sensitive expression of the specificity. When the expression of such a complementary sequence is controlled by in the stem will greatly reduce protein expression until the necessary conditions are a separate promoter, this permits a condition-sensitive expression of the downstream met for the hairpin to completely unfold. downstream gene. The TUDelft 2008 iGEM team retrieved natural RNA thermometers from three species, then sequenced and redesigned them to test for a gene. The condition may be the presence or absence of a metabolite, of an antibiotic modified temperature range. We worked with their 32°C thermometer as it was or toxin, or of various wavelengths of light. The Berkeley 2006 iGEM team began found to be their most effective. with the ribosome binding site hairpin (the "ribolock") and the highly specific complementary sequence (the "ribokey") produced by Collins et al., then redesigned the lock and key sequences to reduce background transcription and increase activated transcription. Figure 1. Temperature sensitive hairpin loop Figure 2. The 32°C temperature sensitive hairpin part of TUDelft Figure 3.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2007/0136827 A1 Collins Et Al
    US 2007013 6827A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0136827 A1 Collins et al. (43) Pub. Date: Jun. 14, 2007 (54) CSFTRANS RIBOREGULATORS Publication Classification (51) Int. Cl. (75) Inventors: James J. Collins, Newton, MA (US); AOIK 67/027 (2006.01) Farren J. Isaacs, Brookline, MA (US); C7H 2L/04 (2006.01) Charles R. Cantor, Del Mar, CA (US); CI2N 15/09 (2006.01) Daniel J. Dwyer, Brookline, MA (US) CI2N 5/06 (2006.01) (52) U.S. Cl. ............................ 800/14; 435/325; 435/455; 536/23.2 Correspondence Address: CHOATE, HALL & STEWART LLP (57) ABSTRACT TWO INTERNATIONAL PLACE BOSTON, MA 02110 (US) The present invention provides nucleic acid molecules, DNA constructs, plasmids, and methods for post-transcrip tional regulation of gene expression using RNA molecules to (73) Assignee: TRUSTEES OF BOSTON UNIVER both repress and activate translation of an open reading SITY, Boston, MA (US) frame. Repression of gene expression is achieved through the presence of a regulatory nucleic acid element (the cis-repressive RNA or crRNA) within the 5' untranslated (21) Appl. No.: 10/535,128 region (5' UTR) of an mRNA molecule. The nucleic acid element forms a hairpin (stem/loop) structure through complementary base pairing. The hairpin blocks access to (22) PCT Fed: Nov. 14, 2003 the mRNA transcript by the ribosome, thereby preventing translation. In particular, in embodiments of the invention PCT No.: PCT/USO3A36506 designed to operate in prokaryotic cells, the stem of the (86) hairpin secondary structure sequesters the ribosome binding S 371(c)(1), site (RBS).
    [Show full text]
  • Secondary Structural Entropy in RNA Switch (Riboswitch) Identification Amirhossein Manzourolajdad1,2* and Jonathan Arnold1,3
    Manzourolajdad and Arnold BMC Bioinformatics (2015) 16:133 DOI 10.1186/s12859-015-0523-2 METHODOLOGY ARTICLE Open Access Secondary structural entropy in RNA switch (Riboswitch) identification Amirhossein Manzourolajdad1,2* and Jonathan Arnold1,3 *Correspondence: [email protected] Abstract 1 Institute of Bioinformatics, Background: RNA regulatory elements play a significant role in gene regulation. University of Georgia, Davison Life Sciences Bldg, Room B118B, 120 Riboswitches, a widespread group of regulatory RNAs, are vital components of many Green St, 30602 Athens, USA bacterial genomes. These regulatory elements generally function by forming a 2 National Center for Biotechnology ligand-induced alternative fold that controls access to ribosome binding sites or other Information (NCBI), NIH, Building 38A, RM 6S614K, 8600 Rockville regulatory sites in RNA. Riboswitch-mediated mechanisms are ubiquitous across Pike, 20894 Bethesda, USA bacterial genomes. A typical class of riboswitch has its own unique structural and Full list of author information is available at the end of the article biological complexity, making de novo riboswitch identification a formidable task. Traditionally, riboswitches have been identified through comparative genomics based on sequence and structural homology. The limitations of structural-homology-based approaches, coupled with the assumption that there is a great diversity of undiscovered riboswitches, suggests the need for alternative methods for riboswitch identification, possibly based on features intrinsic to their structure. As of yet, no such reliable method has been proposed. Results: We used structural entropy of riboswitch sequences as a measure of their secondary structural dynamics. Entropy values of a diverse set of riboswitches were compared to that of their mutants, their dinucleotide shuffles, and their reverse complement sequences under different stochastic context-free grammar folding models.
    [Show full text]
  • Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus Methanolicus
    International Journal of Molecular Sciences Article Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus methanolicus Marta Irla , Sigrid Hakvåg and Trygve Brautaset * Department of Biotechnology and Food Sciences, Norwegian University of Science and Technology, 7034 Trondheim, Norway; [email protected] (M.I.); [email protected] (S.H.) * Correspondence: [email protected]; Tel.: +47-73593315 Abstract: Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a re- liable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 50 untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data. Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based biopro- cesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a Citation: Irla, M.; Hakvåg, S.; novel tool for inducible gene expression in this bacterium.
    [Show full text]
  • Structural and Mechanistic Studies of the THI Box and SMK Box Riboswitches
    Structural and Mechanistic Studies of the THI Box and SMK Box Riboswitches Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Angela Mae Smith, M.S. Graduate Program in Microbiology The Ohio State University 2009 Dissertation Committee: Professor Tina Henkin, Advisor Professor Kurt Fredrick Professor Michael Ibba Professor Ross Dalbey ABSTRACT Organisms have evolved a variety of mechanisms for regulating gene expression. Expression of individual genes is carefully modulated during different stages of cell development and in response to changing environmental conditions. A number of regulatory mechanisms involve structural elements within messenger RNAs (mRNAs) that, in response to an environmental signal, undergo a conformational change that affects expression of a gene encoded on that mRNA. RNA elements of this type that operate independently of proteins or translating ribosomes are termed riboswitches. In this work, the THI box and SMK box riboswitches were investigated in order to gain insight into the structural basis for ligand recognition and the mechanism of regulation employed by each of these RNAs. Both riboswitches are predicted to regulate at the level of translation initiation using a mechanism in which the Shine-Dalgarno (SD) sequence is occluded in response to ligand binding. For the THI box riboswitch, the studies presented here demonstrated that 30S ribosomal subunit binding at the SD region decreases in the presence of thiamin pyrophosphate (TPP). Mutation of conserved residues in the ligand binding domain resulted in loss of TPP-dependent repression in vivo. Based on these experiments two classes of mutant phenotypes were identified.
    [Show full text]
  • A Mechanism for Ligand Gated Strand Displacement in ZTP Riboswitch Transcription Regulation
    bioRxiv preprint doi: https://doi.org/10.1101/521930; this version posted January 16, 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-NC-ND 4.0 International license. A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation Eric J. Strobel1*, Luyi Cheng2,4, Katherine E. Berman2,4, Paul D. Carlson3,4, Julius B. Lucks1,4* Affiliations: 1Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA 2Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL 60208, USA 3Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA 4Center for Synthetic Biology, Northwestern University, Evanston, IL, 20208 *Correspondence to: Email: [email protected], [email protected] Abstract Cotranscriptional RNA folding forms structural intermediates that can be critically important for RNA biogenesis. This is especially true for transcriptional riboswitches that must undergo ligand-dependent structural changes during transcription to regulate the synthesis of downstream genes. Here, we systematically map the folding states traversed by the Clostridium beijerinckii pfl riboswitch as it controls transcription termination in response to the purine biosynthetic intermediate ZMP. We find that after rearrangement of a non-native hairpin to form the ZTP aptamer, cotranscriptional ZMP binding stabilizes two structural elements that lead to antitermination by tuning the efficiency of terminator hairpin nucleation and strand displacement. We also uncover biases within natural ZTP riboswitch sequences that could avoid misfolded intermediates that disrupt function.
    [Show full text]
  • Structure and Ligand Binding of the Glutamine-II Riboswitch
    Nucleic Acids Research, 2019 1 doi: 10.1093/nar/gkz539 Structure and ligand binding of the glutamine-II riboswitch 1, 1, 2 2 1,* Lin Huang †,JiaWang †,AndrewM.Watkins , Rhiju Das and David M. J. Lilley 1 Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow June 2019 26 on by guest https://academic.oup.com/nar/advance-article-abstract/doi/10.1093/nar/gkz539/5520577 from Downloaded Street, Dundee DD1 5EH, UK and 2Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA Received March 25, 2019; Revised May 31, 2019; Editorial Decision June 03, 2019; Accepted June 06, 2019 ABSTRACT GS-inactivating protein factors IF7 and IF17, encoded by gifA and gifB, respectively (7,8). Ames and Breaker (9) iden- We have determined the structure of the glutamine-II tifed two 60 nt elements in the 5′-UTRs of genes involved riboswitch ligand binding domain using X-ray crys- in nitrogen∼ metabolism in cyanobacterial and metagenomic tallography. The structure was solved using a novel sequences. One is based on a three-way helical junction with combination of homology modeling and molecular a loop E motif, termed glnA, and the other with a pseu- replacement. The structure comprises three coaxial doknot termed the downstream peptide (DP) motif. Us- helical domains, the central one of which is a pseudo- ing in-line probing they demonstrated that the RNA binds knot with partial triplex character. The major groove L-glutamine with mM affnity, but discriminated against a of this helix provides the binding site for L-glutamine, series of similar compounds.
    [Show full text]