DOCSLIB.ORG

Guanine Riboswitch Variants from Mesoplasma Florum Selectively Recognize 2؅-Deoxyguanosine

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Guanine Riboswitch Variants from Mesoplasma Florum Selectively Recognize 2؅-Deoxyguanosine Guanine riboswitch variants from Mesoplasma florum selectively recognize 2؅-deoxyguanosine Jane N. Kim†, Adam Roth‡, and Ronald R. Breaker†‡§¶ †Department of Molecular, Cellular, and Developmental Biology, §Department of Molecular Biophysics and Biochemistry, and ‡Howard Hughes Medical Institute, Yale University, P.O. Box 208103, New Haven, CT 06520-8103 Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved August 21, 2007 (received for review June 22, 2007) Several mRNA aptamers have been identified in Mesoplasma florum witch aptamers for the modified nucleobase 7-aminomethyl-7- that have sequence and structural features resembling those of deazaguanine have been identified that require as few as 34 guanine and adenine riboswitches. Two features distinguish these nucleotides to form a selective and high-affinity binding pocket RNAs from established purine-sensing riboswitches. All possess short- (31). Moreover, there are three classes of aptamers for S- ened hairpin-loop sequences expected to alter tertiary contacts adenosylmethionine (SAM) (23, 24, 32) whose representatives are known to be critical for aptamer folding. The RNAs also carry nucle- more rare in bacteria than the SAM-I class of riboswitches com- otide changes in the core of each aptamer that otherwise is strictly monly found in Gram-positive bacteria (33–35). These findings conserved in guanine and adenine riboswitches. Some aptamers indicate that a far greater diversity of metabolite-sensing ribos- retain the ability to selectively bind guanine or adenine despite these witches exists that might be difficult for existing search strategies to mutations. However, one variant type exhibits selective and high- definitively identify and classify. affinity binding of 2؅-deoxyguanosine, which is consistent with its One possibility is that some organisms will have recently evolved occurrence in the 5؅ untranslated region of an operon containing riboswitch classes with aptamers that are unique in architecture or ribonucleotide reductase genes. The identification of riboswitch vari- ligand specificity. If such boutique riboswitches can easily emerge ants that bind nucleosides and reject nucleobases reveals that natural through evolution, there could be far more riboswitches than have metabolite-sensing RNA motifs can accrue mutations that expand the been discovered to date. In this report, we describe a series of diversity of ligand detection in bacteria. aptamers that are exceedingly rare among sequenced genomes and have been identified only in the bacterial species Mesoplasma allosteric RNA ͉ aptamer ͉ metabolite ͉ ribonucleotide reductase ͉ florum. One subclass of these RNAs is selective for 2Ј- transcription termination deoxyguanosine (2Ј-dG). Our findings highlight the capacity for metabolite-binding RNAs to evolve specificities toward structurally ene-control elements called riboswitches (1) are mRNA related derivatives and further demonstrate that exceptionally rare G motifs typically found in the 5Ј untranslated regions of riboswitch classes are likely to be present in some organisms. bacterial mRNAs. Riboswitches selectively bind small mole- Results and Discussion cules, and structural changes within the 5Ј untranslated regions Consensus Sequences and Structures of Guanine- and Adenine-Sens- are usually harnessed to control the expression of the adjoining ing Riboswitches. Guanine-sensing riboswitches usually reside ORF. The architectures of these RNAs are commonly formed upstream of genes involved in purine biosynthesis, salvage, and from a metabolite-binding aptamer domain and an expression transport (26). The guanine riboswitch aptamer from the Bacil- platform (2–5), although more diverse assemblies of aptamers lus subtilis xpt-pbuX mRNA exhibits a K for guanine of Ϸ5 nM. and expression platforms have been found that yield more d Ligand binding to this aptamer causes transcription termination, complex gene-control characteristics (6–9). and a similar gene-control mechanism is predicted for most Each aptamer adopts a complex secondary- and tertiary- other guanine riboswitches as well. X-ray crystallography has structured fold to form a conserved receptor for the ligand (10–17). been performed on this aptamer bound to either guanine or the This demand for precise structure formation and specific molecular functional analog hypoxanthine (10, 11). In both instances, the recognition causes the aptamer domains to be highly conserved ligands are almost completely enveloped by the RNA. Similarly, even among distantly related species. In contrast, the expression a related aptamer that binds adenine by using a similar archi- platform can adopt a variety of different structures provided it tecture also engulfs the ligand (11). maintains its responsiveness to the occupation state of the aptamer The tight ligand-binding pocket of this aptamer class is formed domain. by conserved nucleotides at the junction of three stems termed P1, Computer-aided searches based on conserved RNA sequences P2, and P3 (Fig. 1A). When the ligand is bound, the aptamer adopts and structures have been used to identify representatives of nu- a conformation with the P2 and P3 stems extending parallel to one merous riboswitch classes (18–24). However, these bioinformatics another. This structure is held in place by Watson–Crick base- algorithms can fail to identify variants of known riboswitch classes pairing interactions and other hydrogen bonds formed between the that differ substantially from the established aptamer consensus. loops of these stems, called L2 and L3. Most of the highly conserved Furthermore, there could be exceedingly rare classes of riboswitch nucleotides forming the ligand-binding core of the aptamer are aptamers or exceptionally small aptamers that will be missed by existing bioinformatics algorithms because there are too few rep- resentatives for comparison or they have too few conserved fea- Author contributions: J.N.K., A.R., and R.R.B. designed research; J.N.K. and A.R. performed tures. Given these limitations, many new riboswitch classes might research; J.N.K., A.R., and R.R.B. analyzed data; and J.N.K., A.R., and R.R.B. wrote the paper. remain undiscovered, and the true number of metabolite-sensing Conflict of interest statement: R.R.B. is a cofounder of BioRelix, a biotechnology company aptamer folds could greatly exceed the many types published to that has licensed riboswitch technology from Yale University for antibiotics development. date (25). This article is a PNAS Direct Submission. Indications that there are many variant, small, or rare riboswitch Freely available online through the PNAS open access option. classes to be discovered come from several recent reports of new Abbreviations: 2Ј-dA, 2Ј-deoxyadenosine; 2Ј-dG, 2Ј-deoxyguanosine; 2Ј-d-2,6-DAP, 2Ј- riboswitch classes. For example, a single C-to-U mutation within deoxy-2,6-diathinopurine nucleoside. the core of guanine riboswitches can change the specificity of ligand ¶To whom correspondence should be addressed. E-mail: [email protected]. binding to adenine (11, 26–30). Also, two related types of ribos- © 2007 by The National Academy of Sciences of the USA 16092–16097 ͉ PNAS ͉ October 9, 2007 ͉ vol. 104 ͉ no. 41 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705884104 Downloaded by guest on September 29, 2021 Fig. 1. Sequence and structural features of guanine A J1-2L2 J2-3 L3 J3-1 riboswitch aptamers and several newly found RNAs. (A) P1 P2P2’ P3 P3’ * P1’ Sequence alignment comparing the xpt guanine ribos- xpt (phosphoribosyltransferase) GAACACUCAUAUAAUCGCGUGGAUA-UGGCACGCAAGUUUCUACCGGGUA-CCGUAAAUGUCCGACUAUGGGUGAGCA witch aptamer sequence from the xpt-pbuX mRNA from I-A (ribonucleotide reductase) AGAAACUUAUACAG-GGUAGC-AUAAUGGGCUACU-GACCCCGCCUUCAAACCUA--UUUGGAGACUAUAAGUGAAAA I-B (unknown lipoprotein) AGAAACUUAUACAG-GGUAGC-AUAAUGGGCUACU-GACCCCGCCAUGAAACCUA--UUUCAUGACUAUAGGUCUUUA B. subtilis with related sequences from Mesoplasma flo- II-A (phosphate transporter) AAAAACUUAUACAG-GGUAGC-AUAAUGGGCUACU-GAA-CCGCCCCGGGACCAA--UCUCGGGACUAUAAGUGUGUA rum (types I, II, III, and IV) and from Oenococcus oeni, II-B (unknown lipoprotein) AAAAACUUAUACAG-GGUAGC-AUAUUGGGCUACU-GUU-CCGCCUCAAGACCAA--UCUUGAGACUAUAAGUGUAAA Vibrio sp., Vibrio splendidus, and Leuconostoc mesen- III-A (glycerol-3-P transport) AUUAACUUAUACAU-GAUAAC-AUAUCGGGUUGUC-GAC-CUGCCUUAAGACCGA--UCUUAAGACUAUAAGAAAAUA teroides (types V-A, V-B1, V-B2, and V-C, respectively). III-B (GMP synthase) AAAAACUUAUACAU-GACAAC-AUAUUGGGUUGUC-GAC-CUGCCUCUGGACCUA--UCCUUAGACUAUAAGCGU--- The known or putative functions of the genes immedi- III-C (xylulose transporter) AAAAACUUAUACAU-GACAAC-AUAACGGGUUGUC-GAC-CUGCCUUAGGACCCA--UCCAAAGACUAUAAGCGCAGA ately downstream of each sequence are noted as pre- IV-A (xanthine/uracil permease) GAAAACUUGUAUAA-UCCUUC-AUAUCGGGAAGGA-GUCUCUACCUAACA- CCAA--UGUUAG-AUUAUGAGUUUUAU dicted elsewhere (54). Nucleotides corresponding to V-A (IMP dehydrogenase) CCAAGCAGGUAUAUC-GUCGG-AUAAUGGCUGACA-GUUUCUACCCAACA- CCAA--UGUUGG-ACUAUCUGUGGAUG pairing regions P1, P2, and P3 are shaded blue, green, V-B1 (xanthine/uracil permease) UAAAUGCUGUAUAUAUCUAGUGAUA-UGGACUGGAUGUUUCUACUACCGAGCCUAC-CUUGGUGACUACAGUUUUUAG V-B2 (xanthine/uracil permease) UAAAUGCUGUAUAUAUCUAGUGGUA-UGGACUAGAUGUUUCUACUACCGAGCCUAC-CUUGGUGACUACAGUUUUUAG and orange, respectively. Nucleotides corresponding to V-C (xanthine/uracil permease) UUAGC-UGAUAUAGU-AUCGA-AUAAUGGUCGAUU-GUUUCUAGCCAGCA- CCCA--UGCUGGAACUAUCAUAAACA- loop regions (L) and joining regions (J) also are identi- fied. The asterisk
Load more

Recommended publications
  • Modeling and Searching for Non-Coding RNA
    Modeling and Searching for Non-Coding RNA W.L. Ruzzo http://www.cs.washington.edu/homes/ruzzo Outline • Why RNA? • Examples of RNA biology • Computational Challenges – Modeling – Search – Inference The “Central Dogma” DNA RNA Protein gene Protein DNA (chromosome) RNA (messenger) cell “Classical” RNAs • mRNA • tRNA • rRNA • snRNA (small nuclear - spli cing) • snoRNA (small nucleolar - guides for t/rRNA modifications) • RNAseP (tRNA maturation; ribozyme in bacteria) • SRP (signal recognition particle; co-translational targeting of proteins to membranes) • telomerases Non-coding RNA • Messenger RNA - codes for proteins • Non-coding RNA - all the rest – Before, say, mid 1990’s, 1-2 dozen known (critically important, but narrow roles: e.g. tRNA) • Since mid 90’s dramatic discoveries – Regulation, transport, stability/degradation – E.g. “microRNA”: ≈ 100’s in humans • By some estimates, ncRNA >> mRNA RNA Secondary Structure: RNA makes helices too U C A A Base pairs G C C G A A U G C U A C G C G A U G C A A CA AU RNA on the Rise • In humans – more RNA- than DNA-binding proteins? – much more conserved DNA than coding – MUCH more transcribed DNA than coding • In bacteria – regulation of MANY genes involves RNA – dozens of classes & thousands of new examples in just last 5 years Human Predictions • Evofold – S Pedersen, G Bejerano, A Siepel, K Rosenbloom, K Lindblad-Toh, ES Lander, J Kent, W Miller, D Haussler, "Identification and classification of conserved RNA secondary structures in the human genome." PLoS Comput. Biol., 2, #4 (2006) e33. – 48,479 candidates (~70% FDR?) • RNAz – S Washietl, IL Hofacker, M Lukasser, A H殳 tenhofer, PF Stadler, "Mapping of conserved RNA secondary structures predicts thousands of functional noncoding RNAs in the human genome." Nat.
    [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]
  • An Internal Residue Mimicking the Purine Ligand Vanessa Delfosse1, Patricia Bouchard1, Eric Bonneau1, Pierre Dagenais1, Jean-Franc¸ Ois Lemay2, Daniel A
    Published online 18 December 2009 Nucleic Acids Research, 2010, Vol. 38, No. 6 2057–2068 doi:10.1093/nar/gkp1080 Riboswitch structure: an internal residue mimicking the purine ligand Vanessa Delfosse1, Patricia Bouchard1, Eric Bonneau1, Pierre Dagenais1, Jean-Franc¸ ois Lemay2, Daniel A. Lafontaine2,* and Pascale Legault1,* 1De´ partement de Biochimie, Universite´ de Montre´ al, C.P. 6128, Succursale Centre-Ville, Montre´ al, Que´ bec, H3C 3J7 and 2Groupe ARN/RNA Group, De´ partement de Biologie, Faculte´ des Sciences, Universite´ de Sherbrooke, Sherbrooke, Que´ bec, J1K 2R1, Canada Received September 16, 2009; Revised November 3, 2009; Accepted November 4, 2009 ABSTRACT modulate gene expression in response to the concentration level of their cognate ligand (1–3). They are positioned The adenine and guanine riboswitches regulate in the 50-UTR of bacterial mRNA and are composed of gene expression in response to their purine ligand. two overlapping domains: a purine-binding aptamer X-ray structures of the aptamer moiety of these domain and a downstream expression platform. Binding riboswitches are characterized by a compact fold of the ligand stabilizes the formation of a compact in which the ligand forms a Watson–Crick base aptamer structure, which prevents the formation of pair with residue 65. Phylogenetic analyses an alternate structure and thereby modulates gene revealed a strict restriction at position 39 of the expression. aptamer that prevents the G39–C65 and A39–U65 Depending on the expression platform composition, combinations, and mutational studies indicate that gene expression control takes place at the levels of tran- aptamers with these sequence combinations are scription or translation, and ligand binding, either posi- tively or negatively, regulates gene expression (1–3).
    [Show full text]
  • Bioinformatic Analysis of Riboswitch Structures Uncovers Variant Classes
    Bioinformatic analysis of riboswitch structures PNAS PLUS uncovers variant classes with altered ligand specificity Zasha Weinberga,1,2, James W. Nelsonb,1,3, Christina E. Lünseb,4, Madeline E. Sherlockc, and Ronald R. Breakera,b,c,5 aHoward Hughes Medical Institute, Yale University, New Haven, CT 06520; bDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; and cDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520 Contributed by Ronald R. Breaker, February 2, 2017 (sent for review December 8, 2016; reviewed by Robert T. Batey and Elena Rivas) Riboswitches are RNAs that form complex, folded structures that Several variant riboswitches share a number of characteristics selectively bind small molecules or ions. As with certain groups of that could have been exploited in a bioinformatic search for such protein enzymes and receptors, some riboswitch classes have RNAs. We chose to apply three important properties common to evolved to change their ligand specificity. We developed a pro- the guanine/adenine (15) and c-di-GMP-I/c-AMP-GMP (13, 14) cedure to systematically analyze known riboswitch classes to find riboswitch sets, among other variants. The first of these proper- additional variants that have altered their ligand specificity. This ties is that some variant riboswitches with altered ligand speci- approach uses multiple-sequence alignments, atomic-resolution ficity will remain somewhat close in both sequence and structure structural information, and riboswitch gene associations. Among to the predominant or “parent” class. For example, the initial the discoveries are unique variants of the guanine riboswitch class collections of representatives for guanine (23) and c-di-GMP-I ′ that most tightly bind the nucleoside 2 -deoxyguanosine.
    [Show full text]
  • Synthetic Ligands for Preq1 Riboswitches Provide Structural and Mechanistic Insights Into Targeting RNA Tertiary Structure
    ARTICLE https://doi.org/10.1038/s41467-019-09493-3 OPEN Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure Colleen M. Connelly1, Tomoyuki Numata2,3, Robert E. Boer1, Michelle H. Moon1, Ranu S. Sinniah1, Joseph J. Barchi1, Adrian R. Ferré-D’Amaré 2 & John S. Schneekloth Jr. 1 1234567890():,; Riboswitches are naturally occurring RNA aptamers that regulate gene expression by binding to specific small molecules. Riboswitches control the expression of essential bacterial genes and are important models for RNA-small molecule recognition. Here, we report the discovery of a class of synthetic small molecules that bind to PreQ1 riboswitch aptamers. These molecules bind specifically and reversibly to the aptamers with high affinity and induce a conformational change. Furthermore, the ligands modulate riboswitch activity through tran- scriptional termination despite no obvious chemical similarity to the cognate ligand. X-ray crystallographic studies reveal that the ligands share a binding site with the cognate ligand but make different contacts. Finally, alteration of the chemical structure of the ligand causes changes in the mode of RNA binding and affects regulatory function. Thus, target- and structure-based approaches can be used to identify and understand the mechanism of synthetic ligands that bind to and regulate complex, folded RNAs. 1 Chemical Biology Laboratory, National Cancer Institute, Frederick, MD 21701, USA. 2 Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA. 3 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan. These authors contributed equally: Colleen M.
    [Show full text]
  • Loss of Conserved Noncoding Rnas in Genomes of Bacterial Endosymbionts
    GBE Loss of Conserved Noncoding RNAs in Genomes of Bacterial Endosymbionts Dorota Matelska1, Malgorzata Kurkowska1, Elzbieta Purta1, Janusz M. Bujnicki1,2,*and Stanislaw Dunin-Horkawicz1,* 1Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland 2Laboratory of Structural Bioinformatics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland *Corresponding author: E-mail: [email protected]; [email protected]. Accepted: January 11, 2016 Abstract The genomes of intracellular symbiotic or pathogenic bacteria, such as of Buchnera, Mycoplasma,andRickettsia, are typically smaller compared with their free-living counterparts. Here we showed that noncoding RNA (ncRNA) families, which are conserved in free- living bacteria, frequently could not be detected by computational methods in the small genomes. Statistical tests demonstrated that their absence is not an artifact of low GC content or small deletions in these small genomes, and thus it was indicative of an independent loss of ncRNAs in different endosymbiotic lineages. By analyzing the synteny (conservation of gene order) between the reduced and nonreduced genomes, we revealed instances of protein-coding genes that were preserved in the reduced genomes but lost cis-regulatory elements. We found that the loss of cis-regulatory ncRNA sequences, which regulate the expression of cognate protein-coding genes, is characterized by the reduction of secondary structure formation propensity, GC content, and length of the corresponding genomic regions. Key words: endosymbionts, noncoding RNA loss, covariance models, Rfam. Introduction The length of fully sequenced bacterial genomes ranges from 2001), and chromosomes undergo rearrangements (Belda 110 kb (Bennett and Moran 2013)to15Mb(Han et al.
    [Show full text]
  • Core Requirements of the Adenine Riboswitch Aptamer for Ligand Binding
    JOBNAME: RNA 13#3 2007 PAGE: 1 OUTPUT: Wednesday February 7 07:09:20 2007 csh/RNA/131630/rna1420 Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Core requirements of the adenine riboswitch aptamer for ligand binding JEAN-FRANCxOIS LEMAY and DANIEL A. LAFONTAINE De´partement de biologie, Faculte´ des sciences, Universite´ de Sherbrooke, Que´bec, J1K 2R1, Canada ABSTRACT The adenine riboswitch aptamer, the A box, positively regulates gene expression upon adenine binding. To provide insight into structure–function relationships, important for the adenine riboswitch aptamer, we have created alignments for six aptamer sequences that reveal the core requirements. In addition, 2-aminopurine (2AP) binding studies have been used to test the consensus sequence derived from the alignment. Overall, the consensus secondary structure is consistent with 2AP binding studies. However, a position in the core, previously identified as variable, shows restriction in nucleotide sequence. Further- more, this restriction is found to be related with the ligand specificity of the riboswitch. The implications of this relationship for the riboswitch gene regulation mechanism are discussed. Keywords: riboswitch; RNA structure; RNA folding; aptamer; molecular recognition; fluorescence spectroscopy INTRODUCTION 2003), glucosamine-6-phosphate (Winkler et al. 2004), glycine (Mandal et al. 2004), lysine (Grundy et al. 2003; Riboswitches are regulatory elements found predominantly Sudarsan et al. 2003b), intracellular magnesium (Cromie in 59-untranslated regions of messenger RNAs (mRNAs). et al. 2006), S-adenosylmethionine (Epshtein et al. 2003; These genetic switches are highly structured domains that McDaniel et al. 2003; Winkler et al. 2003), and thiamine can bind cellular metabolites and regulate the expression of pyrophosphate (Mironov et al.
    [Show full text]
  • Gene Expression Control by Bacillus Anthracis Purine Riboswitches
    Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Kirchner and Schneider 1 Gene expression control by Bacillus anthracis purine riboswitches Marion Kirchner* and Sabine Schneider* Center for Integrated Protein Science at the Department of Chemistry Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany * To whom correspondence should be addressed. Tel: +49 89 289 13336; Fax: +49 89 289 13363; Email: [email protected]; [email protected] Running Title: Purine riboswitches of Bacillus anthracis Keywords (max 6): purine riboswitches, Bacillus anthracis, purine biosynthesis, nucleobase salvage, nucleotide metabolism ABSTRACT In all kingdoms of life, cellular replication relies on the presence of nucleosides and nucleotides, the building blocks of nucleic acids and the main source of energy. In bacteria, the availability of metabolites sometimes directly regulates the expression of enzymes and proteins involved in purine salvage, biosynthesis and uptake through riboswitches. Riboswitches are located in bacterial mRNAs and can control gene expression by conformational changes in response to ligand binding. We have established an inverse reporter gene system in Bacillus subtilis that allows us to monitor riboswitch-controlled gene expression. We used it to investigate the activity of five potential purine riboswitches from B. anthracis in response to different purines and pyrimidines. Furthermore, in vitro studies on the aptamer domains of the riboswitches reveal their variation in guanine binding affinity ranging from nM to µM. These data do not only provide insight into metabolite sensing but can also aid to engineer artificial cell regulatory systems. Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Kirchner and Schneider 2 INTRODUCTION Riboswitches are structural elements in the 5’ untranslated region of mRNAs, which consist of an aptamer domain and an expression platform (Fig.
    [Show full text]
  • BMC Genomics Biomed Central
    BMC Genomics BioMed Central Research article Open Access Abundance and functional diversity of riboswitches in microbial communities Marat D Kazanov*1, Alexey G Vitreschak1 and Mikhail S Gelfand1,2 Address: 1Institute for Information Transmission Problems (the Kharkevich Institute) RAS, Bolshoi Karetnyi per. 19, Moscow, 127994, Russia and 2Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia Email: Marat D Kazanov* - [email protected]; Alexey G Vitreschak - [email protected]; Mikhail S Gelfand - [email protected] * Corresponding author Published: 1 October 2007 Received: 13 July 2007 Accepted: 1 October 2007 BMC Genomics 2007, 8:347 doi:10.1186/1471-2164-8-347 This article is available from: http://www.biomedcentral.com/1471-2164/8/347 © 2007 Kazanov et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Several recently completed large-scale enviromental sequencing projects produced a large amount of genetic information about microbial communities ('metagenomes') which is not biased towards cultured organisms. It is a good source for estimation of the abundance of genes and regulatory structures in both known and unknown members of microbial communities. In this study we consider the distribution of RNA regulatory structures, riboswitches, in the Sargasso Sea, Minnesota Soil and Whale Falls metagenomes. Results: Over three hundred riboswitches were found in about 2 Gbp metagenome DNA sequences. The abundabce of riboswitches in metagenomes was highest for the TPP, B12 and GCVT riboswitches; the S-box, RFN, YKKC/YXKD, YYBP/YKOY regulatory elements showed lower but significant abundance, while the LYS, G-box, GLMS and YKOK riboswitches were rare.
    [Show full text]
  • A Dissertation Submitted for Ph.D. Degree in Pharmaceutical Biotechnology
    Faculty of Pharmacy Tabriz University of Medical Sciences A Dissertation submitted for Ph.D. degree in Pharmaceutical Biotechnology Entitled: Cloning and sequencing of riboswitches of Alishewanella tabrizica strain RCRI4 and comparative studies on similar riboswitches in other bacteria By: Elnaz Mehdizadeh Aghdam Supervisors: Dr Mohammad Saeid Hejazi Dr Abolfazl Barzegar Advisor: Dr Jörg S. Hartig September 1026 Thesis No: 00 Clarification and Copyright Declaration I hereby declare that I am the sole author of this thesis. I take full responsibility of this work and declare that the results of this study are original and based on my personal work. All scientific materials used in this thesis are fully referenced. All rights are transferred to Faculty of pharmacy, Tabriz University of Medical sciences. The Faculty of pharmacy is authorized to lend or reproduce this thesis, in total or in part. Professors’ names: Dr. Mohammad Saeid Hejazi; Dr Abolfazl Barzegar Signature: Name: Elnaz Mehdizadeh Aghdam Student No: 0020210102 Date: Signature: I would like to dedicate this thesis to my dear husband Dr Hamid Reza Heidari For his endless patience, kindness and love Acknowledgments I would like to express my deepest acknowledgments to my supervisor, Prof. Mohammad Saeid Hejazi, for the patient guidance, encouragement and advice he has provided throughout my time as his student. I have been extremely lucky to have a supervisor who cared so much about my work, and who responded to my questions and queries so promptly. I believe he has been more than a supervisor to me, as he has guided me patiently and constantly all through my academic life and I am and will be grateful to him for the rest of my life.
    [Show full text]
  • Riboswitches and the RNA World
    Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Riboswitches and the RNA World Ronald R. Breaker Department of Molecular, Cellular and Developmental Biology; Department of Molecular Biophysics and Biochemistry; Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8103 Correspondence: [email protected] SUMMARY Riboswitches are structured noncoding RNA domains that selectively bind metabolites and control gene expression (Mandal and Breaker 2004a; Coppins et al. 2007; Roth and Breaker 2009). Nearly all examples of the known riboswitches reside in noncoding regions of messen- ger RNAs where they control transcription or translation. Newfound classes of riboswitches are being reported at a rate of about three per year (Ames and Breaker 2009), and these have been shown to selectively respond to fundamental metabolites including coenzymes, nucleobases or their derivatives, amino acids, and other small molecule ligands. The characteristics of some riboswitches suggest they could be modern descendents of an ancient sensory and regulatory system that likely functioned before the emergence of enzymes and genetic factors made of protein (Nahvi et al. 2002; Vitreschak et al. 2004; Breaker 2006). If true, then some of the riboswitch structures and functions that serve modern cells so well may accurately reflect the capabilities of RNA sensors and switches that existed in the RNAWorld. This article will address some of the characteristics of modern riboswitches that may be rele- vant to ancient versions of these metabolite-sensing RNAs. Outline 1 Riboswitches and Their Moving Parts 6 Something Special About SAM? 2 Modern Mechanisms for Riboswitch- 7 Increasing Capabilities by Stacking Riboswitch Mediated Gene Control Components 3 Possible Mechanisms for Riboswitch 8 The Origin of Riboswitches Control of Ribozymes 9 Conclusions 4 Ligand Binding Affinity and Kinetics of Riboswitch Function References 5 How Many Riboswitch Classes Currently Exist? Editors: John F.
    [Show full text]
  • Folding of a Transcriptionally Acting Preq Riboswitch
    Folding of a transcriptionally acting PreQ1 riboswitch Ulrike Riedera,b, Christoph Kreutza,b, and Ronald Micuraa,b,1 aInstitute of Organic Chemistry and bCenter for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 52a, 6020 Innsbruck, Austria Edited* by Dinshaw J. Patel, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved May 11, 2010 (received for review December 30, 2009) 7-Aminomethyl-7-deazaguanine (preQ1) sensitive mRNA domains complex were reported by three other groups and disclosed the belong to the smallest riboswitches known to date. Although molecular details of its three-dimensional architecture (13–15). recent efforts have revealed the three-dimensional architecture The question remains how nature uses the same aptamer of the ligand–aptamer complex less is known about the molecular domain to modulate the expression platform in different ways details of the ligand-induced response mechanism that modulates in various organisms. The key for understanding lies in the apta- gene expression. We present an in vitro investigation on the mer’s downstream sequence and the sequence overlap between ligand-induced folding process of the preQ1 responsive RNA ele- aptamer and expression platform. Sequence conservation of ment from Fusobacterium nucleatum using biophysical methods, the expression platform is significantly less compared to sequence including fluorescence and NMR spectroscopy of site-specifically la- conservation of the aptamer. Therefore, it becomes a complex beled riboswitch variants. We provide evidence that the full-length task to reveal general rules for the molecular response mechan- riboswitch domain adopts two different coexisting stem-loop ism of riboswitches. Our understanding will significantly grow if the ligand-induced folding process is dissected at the base-pair structures in the expression platform.
    [Show full text]