DOCSLIB.ORG

Life Without Trna[Superscript Ile]-Lysidine Synthetase: Translation of the Isoleucine Codon AUA in Bacillus Subtilis Lacking the Canonical Trna[Ile Over 2]

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Life Without Trna[Superscript Ile]-Lysidine Synthetase: Translation of the Isoleucine Codon AUA in Bacillus Subtilis Lacking the Canonical Trna[Ile Over 2] Life without tRNA[superscript Ile]-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA[Ile over 2] The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Kohrer, C., D. Mandal, K. W. Gaston, H. Grosjean, P. A. Limbach, and U. L. RajBhandary. “Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile.” Nucleic Acids Research (November 4, 2013). As Published http://dx.doi.org/10.1093/nar/gkt1009 Publisher Oxford University Press Version Final published version Citable link http://hdl.handle.net/1721.1/83245 Detailed Terms http://creativecommons.org/licenses/by-nc/3.0/ Nucleic Acids Research Advance Access published November 14, 2013 Nucleic Acids Research, 2013, 1–12 doi:10.1093/nar/gkt1009 Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis Ile lacking the canonical tRNA2 Caroline Ko¨ hrer1, Debabrata Mandal1, Kirk W. Gaston2, Henri Grosjean3, Patrick A. Limbach2 and Uttam L. RajBhandary1,* 1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, 2Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, OH 45221, USA and 3Centre de Ge´ ne´ tique Mole´ culaire, CNRS, Gif-sur-Yvette, F-91198, France Received August 29, 2013; Revised October 3, 2013; Accepted October 5, 2013 Downloaded from ABSTRACT Crick proposes how a single tRNA with G in the first position of the anticodon (also called the wobble base) Translation of the isoleucine codon AUA in most can read codons ending in U or C and how a tRNA prokaryotes requires a modified C (lysidine or with U (or a modified U) can read codons ending in A Ile http://nar.oxfordjournals.org/ agmatidine) at the wobble position of tRNA2 to or G (3–5). The AUN codon box specifying isoleucine and base pair specifically with the A of the AUA codon methionine is unique in that three of the four codons, but not with the G of AUG. Recently, a Bacillus AUU, AUC and AUA, specify isoleucine, whereas the subtilis strain was isolated in which the essential fourth codon, AUG, specifies methionine. This organiza- gene encoding tRNAIle-lysidine synthetase was tion raises the question of how the AUA codon is read by deleted for the first time. In such a strain, C34 at an isoleucine tRNA without also reading the AUG codon Ile the wobble position of tRNA2 is expected to for methionine. remain unmodified and cells depend on a mutant The strategy used by various organisms to read isoleu- Ile cine codons is kingdom-specific. Most eukaryotic cells at MIT Libraries on December 9, 2013 suppressor tRNA derived from tRNA1 , in which G34 has been changed to U34. An important contain two isoleucine tRNAs, the one with the anticodon IAU (tRNAIle I = inosine) reads all three isoleucine question, therefore, is how U34 base pairs with A IAU; codons following the Wobble hypothesis (3), whereas the without also base pairing with G. Here, we show other with the anticodon cAc (tRNAIle ; c = pseudo- (i) that unlike U34 at the wobble position of all ÉAÉ uridine) is thought to read only AUA (6). A possible ex- B. subtilis tRNAs of known sequence, U34 in the planation for the presence of two tRNAs which can read mutant tRNA is not modified, and (ii) that the AUA in eukaryotes is inefficient decoding of AUA by mutant tRNA binds strongly to the AUA codon Ile tRNAIAU (7,8). Prokaryotes, which also contain two iso- on B. subtilis ribosomes but only weakly to AUG. leucine tRNAs, have, however, evolved a different These in vitro data explain why the suppressor strategy for reading the three isoleucine codons. In most strain displays only a low level of misreading AUG bacteria and archaea, a tRNA with the anticodon GAU Ile codons in vivo and, as shown here, grows at a rate (tRNA1GAU), reads two of the isoleucine codons (AUU comparable to that of the wild-type strain. and AUC) following the Wobble hypothesis, whereas another tRNA with the anticodon C*AU reads the third isoleucine codon AUA. C* is derived from C and has been INTRODUCTION identified as lysidine in bacterial isoleucine tRNA Ile The genetic code consists of 16 four-codon boxes in which (tRNA2LAU; L = lysidine) (9,10) and agmatidine in 0 Ile + the four codons in a box differ from one another in the 3 archaeal isoleucine tRNA (tRNA2C+AU;C = agmatidine) terminal nucleotide. In 14 of the 16 boxes, all four codons (11–13). In both cases, an amino acid, lysine (in bacteria) either specify the same amino acid or are split into two and a decarboxylated arginine (in archaea), replaces the sets of two codons; those ending in pyrimidines specifying C2-oxo group of C34, the wobble base. The modification Ile one amino acid and those ending in purines specifying of C34 to lysidine or agmatidine in tRNA2 results in a different amino acid (1,2). The Wobble hypothesis of a dual specificity switch of the tRNA in aminoacylation *To whom correspondence should be addressed. Tel: +1 617 253 4702; Fax: +1 617 252 1556; Email: [email protected] ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Nucleic Acids Research, 2013 and in codon binding: while the unmodified tRNA with modification in the anticodon and has the expected C34 is aminoacylated in vitro with methionine by aminoacylation and codon binding properties. methionyl-tRNA synthetase (MetRS) and reads the AUG codon, the modified tRNA is aminoacylated with isoleucine by isoleucyl-tRNA synthetase (IleRS) and reads MATERIALS AND METHODS the AUA codon (14–16). Isolation of total tRNA from B. subtilis Why have bacteria and archaea evolved a mechanism to Bacillus subtilis strains were grown in LB medium at 37C use an isoleucine tRNA with a modified C34 in the anti- with aeration; cells were harvested by centrifugation at an codon to exclusively base pair with A instead of using an OD600 of 1.5–1.8 and immediately used for RNA isola- isoleucine tRNA with U34? Is it because a tRNA which tion. All steps were carried out at 4C unless otherwise contains U or a modified U in the wobble position cannot noted. Bacillus subtilis cells from a 3 L-culture were read the AUA codon without also misreading the AUG pelleted and resuspended in 90 ml of extraction buffer codon (4)? A possible answer to these questions could [1 mM Tris–HCl or 4-(2-hydroxyethyl)piperazine-1- come from the analysis of codon recognition properties ethanesulfonic acid (HEPES) pH 7.5, 10 mM of isoleucine tRNAs from the very few bacterial and Mg(OAc)2]. A total of 100 ml of acid phenol (Ambion) archaeal species, whose genomes encode an isoleucine was added and the suspension was placed on a nutator Ile Downloaded from tRNA with the anticodon UAU (tRNAUAU) but not for 30 min. The mixture was centrifuged for 30 min at C*AU, such as Nanoarchaeum equitans, Korarchaeum 10 000g, the aqueous layer was adjusted to 1 M lithium sp., Mycoplasma mobile, Bifidobacterium adolescentis, chloride and left on ice for 2 h. After centrifugation Neorickettsia sennetsu and others [summarized in (17)]. (30 min, 10 000g), tRNAs were recovered from the super- These selected organisms are also distinguished by the natant by precipitation with ethanol. The tRNA was absence of genes encoding tRNAIle-lysidine synthetase washed extensively with 70% ethanol and resuspended http://nar.oxfordjournals.org/ (TilS) in bacteria or tRNAIle-agmatidine synthetase in 10 mM HEPES, pH 7.5. Typically 250 to 350 A260 (TiaS) in archaea, responsible for the biosynthesis of units of total tRNA, mostly free of rRNA, were lysidine or agmatidine, respectively. obtained from a 3 L culture. The quality of the total Ile The recent isolation of a mutant tRNA1 gene in tRNA preparation was confirmed by agarose gel electro- Bacillus subtilis in which the anticodon sequence GAT phoresis and in vitro aminoacylation with isoleucine or has been mutated to TAT (17) has provided us with the methionine. For isolation of total tRNA under acidic con- opportunity to study the properties, including the codon ditions, cells were resuspended in ice-cold 0.1 M sodium binding properties, of an isoleucine tRNA carrying U in acetate, pH 5.0. After extraction of total RNA using the wobble position. This mutant tRNA, henceforth called Trizol (Invitrogen) following the manufacturer’s instruc- at MIT Libraries on December 9, 2013 Ile mutant tRNA1UAU, was isolated as a suppressor in a tions, large rRNAs were removed by precipitation with B. subtilis strain in which the gene encoding TilS had lithium chloride as described above. After several washes been deleted. In the absence of TilS, the wobble base of with ethanol, the tRNA was resuspended in 10 mM the isoleucine tRNA containing the CAU anticodon is sodium acetate, pH 5.0, and stored at À80C. A portion expected to remain unmodified and cells depend on the of the material was subjected to deacylation by the Ile mutant tRNA1UAU for translation of the AUA codon. addition of Tris–HCl, pH 9.5, to a final concentration of The availability of B. subtilis strains carrying the suppres- 0.1 M. The deacylation reaction was performed at 37C sor mutation in the isoleucine tRNA gene has allowed us for 90–120 min; deacylated tRNAs were re-precipitated to investigate (i) whether U34 in the mutant tRNA is with ethanol and stored in 10 mM HEPES, pH 7.5.
Load more

Recommended publications
  • Identification and Codon Reading Properties of 5
    Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Mandal, D., C. Kohrer, D. Su, I. R. Babu, C. T. Y. Chan, Y. Liu, D. Soll, et al. “Identification and Codon Reading Properties of 5- Cyanomethyl Uridine, a New Modified Nucleoside Found in the Anticodon Wobble Position of Mutant Haloarchaeal Isoleucine tRNAs.” RNA 20, no. 2 (December 16, 2013): 177–188. As Published http://dx.doi.org/10.1261/rna.042358.113 Publisher Cold Spring Harbor Laboratory Press/RNA Society Version Final published version Citable link http://hdl.handle.net/1721.1/91989 Terms of Use Creative Commons Attribution-Noncommerical Detailed Terms http://creativecommons.org/licenses/by-nc/3.0/ Downloaded from rnajournal.cshlp.org on April 3, 2014 - Published by Cold Spring Harbor Laboratory Press Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs Debabrata Mandal, Caroline Köhrer, Dan Su, et al. RNA 2014 20: 177-188 originally published online December 16, 2013 Access the most recent version at doi:10.1261/rna.042358.113 Supplemental http://rnajournal.cshlp.org/content/suppl/2013/12/03/rna.042358.113.DC1.html Material References This article cites 38 articles, 17 of which can be accessed free at: http://rnajournal.cshlp.org/content/20/2/177.full.html#ref-list-1 Creative This article is distributed exclusively by the RNA Society for the first 12 months after the Commons full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml).
    [Show full text]
  • Characterization of a B. Subtilis Minor Isoleucine Trna Deduced from Tdna Having a Methionine Anticodon CAT1
    J. Biochem. 119, 811-816 (1996) Characterization of a B. subtilis Minor Isoleucine tRNA Deduced from tDNA Having a Methionine Anticodon CAT1 Jitsuhiro Matsugi,2 Katsutoshi Murao, and Hisayuki Ishikura Laboratory of Chemistry, Jichi Medical School, 3311-1 Minamikawachi-machi, Tochigi 329-04 Received for publication, December 1, 1995 Bacillus subtilis, which belongs to Gram-positive eubacteria, has been predicted to have a minor isoleucine tRNA transcribed from the gene possessing the CAT anticodon, which corresponds to methionine. We isolated this tRNA and determined its sequence including modified nucleotides. Modified nucleotide analyses using TLC, UV, and FAB mass spectros copy revealed that the first letter of the anticodon is modified to lysidine [4-amino-2-(N6 - lysino)-1-ƒÀ-D-ribofuranosyl pyrimidine]. As a result, this tRNA agrees with the minor one predicted from the DNA sequence and is thought to decode the isoleucine codon AUA. Key words: Bacillus subtilis, FAB mass spectroscopy, lysidine, modified nucleotide, tRNA. In the codon table, isoleucine is assigned to three codons, lysidine in the wobble position can form a base pair only AUU, AUC, and AUA, but theoretically two kinds of with adenosine, not with guanosine, and consequently the anticodon (GAU and UAU) are sufficient to decode these minor tRNA11e can read only the AUA codon. At the same three codons. To decode the AUA codon, some organisms time, lysidine contributes to a change in charging specificity utilize a minor species of tRNA11e whose first letter of the from methionine to isoleucine (7). anticodon is a unique or an unknown modified nucleotide. In B.
    [Show full text]
  • WO 2017/112943 Al 29 June 2017 (29.06.2017) W P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/112943 Al 29 June 2017 (29.06.2017) W P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C07K 14/705 (2006.01) A61K 31/7088 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, C12N 15/12 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, (21) International Application Number: KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, PCT/US2016/068552 MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, (22) International Filing Date: NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, 23 December 2016 (23. 12.2016) RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, (25) Filing Language: English ZA, ZM, ZW. (26) Publication Language: English 4 Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 62/387,168 23 December 201 5 (23. 12.2015) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 62/290,413 2 February 2016 (02.02.2016) US TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (71) Applicant: MODERNATX, INC.
    [Show full text]
  • Discovery and Characterization of Trnaile Lysidine Synthetase (Tils)
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 584 (2010) 272–277 journal homepage: www.FEBSLetters.org Review Discovery and characterization of tRNAIle lysidine synthetase (TilS) Tsutomu Suzuki *, Kenjyo Miyauchi Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan article info abstract Article history: In the bacterial decoding system, the AUA codon is deciphered as isoleucine by tRNAIle bearing lysi- Received 12 November 2009 dine (L, 2-lysyl-cytidine) at the wobble position. Lysidine is an essential modification that deter- Revised 21 November 2009 mines both the codon and amino acid specificities of tRNAIle. We identified an enzyme named Accepted 24 November 2009 tRNAIle lysidine synthetase (TilS) that catalyzes lysidine formation by using lysine and ATP as sub- Available online 26 November 2009 strates. Biochemical studies revealed a molecular mechanism of lysidine formation that consists Edited by Manuel Santos of two consecutive reactions involving the adenylated tRNA intermediate. In addition, we deci- phered how Escherichia coli TilS specifically discriminates between tRNAIle and the structurally sim- ilar tRNAMet, which bears the same anticodon loop. Recent structural studies unveiled tRNA Keywords: tRNA recognition by TilS, and a molecular basis of lysidine formation at atomic resolution. Lysidine Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. TilS AUA codon Wobble modification Anticodon 1. Lysidine plays a critical role in decoding the AUA codon as Ile tRNAIle bearing the CAU anticodon was switched to Met, and this tRNA translates the AUG codon as Met [4].
    [Show full text]
  • Agmatidine, a Modified Cytidine in the Anticodon of Archaeal Trna , Base
    Agmatidine, a modified cytidine in the anticodon of archaeal tRNAIle, base pairs with adenosine but not with guanosine Debabrata Mandala,1, Caroline Köhrera,1, Dan Sub, Susan P. Russellc, Kady Krivosc, Colette M. Castleberryc, Paul Blumd, Patrick A. Limbachc, Dieter Söllb, and Uttam L. RajBhandarya,2 aDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520; cDepartment of Chemistry, University of Cincinnati, Cincinnati, OH 45221; dGeorge Beadle Center for Genetics, University of Nebraska, Lincoln, NE 68588 Contributed by Dieter Söll, December 24, 2009 (sent for review December 2, 2009) Modification of the cytidine in the first anticodon position of the Eukaryotes, on the other hand, contain a tRNAIle with the anti- Ile Ile AUA decoding tRNA (tRNA2 ) of bacteria and archaea is essential codon IAU (I ¼ inosine), which can read all three isoleucine co- for this tRNA to read the isoleucine codon AUA and to differentiate dons using the wobble pairing rules of Crick. They also contain a between AUA and the methionine codon AUG. To identify the tRNAIle with the anticodon ΨAΨ, which is thought to read only modified cytidine in archaea, we have purified this tRNA species the isoleucine codon AUA but not the methionine codon AUG from Haloarcula marismortui, established its codon reading proper- (8). Given these two distinct mechanisms in bacteria and ties, used liquid chromatography–mass spectrometry (LC-MS) to eukaryotes, a question of much interest is how the archaeal map RNase A and T1 digestion products onto the tRNA, and used tRNAIle accomplishes the task of reading the AUA codon.
    [Show full text]
  • Designing Minimal Genomes Using Whole-Cell Models
    ARTICLE https://doi.org/10.1038/s41467-020-14545-0 OPEN Designing minimal genomes using whole-cell models ✉ Joshua Rees-Garbutt1,2,7, Oliver Chalkley1,3,4,7, Sophie Landon1,3, Oliver Purcell5, Lucia Marucci1,3,6,8 & ✉ Claire Grierson1,2,8 In the future, entire genomes tailored to specific functions and environments could be designed using computational tools. However, computational tools for genome design are 1234567890():,; currently scarce. Here we present algorithms that enable the use of design-simulate-test cycles for genome design, using genome minimisation as a proof-of-concept. Minimal gen- omes are ideal for this purpose as they have a simple functional assay whether the cell replicates or not. We used the first (and currently only published) whole-cell model for the bacterium Mycoplasma genitalium. Our computational design-simulate-test cycles discovered novel in silico minimal genomes which, if biologically correct, predict in vivo genomes smaller than JCVI-Syn3.0; a bacterium with, currently, the smallest genome that can be grown in pure culture. In the process, we identified 10 low essential genes and produced evidence for at least two Mycoplasma genitalium in silico minimal genomes. This work brings combined computational and laboratory genome engineering a step closer. 1 BrisSynBio, University of Bristol, Bristol BS8 1TQ, UK. 2 School of Biological Sciences, University of Bristol, Bristol Life Sciences Building, 24 Tyndall Avenue, Bristol BS8 1TQ, UK. 3 Department of Engineering Mathematics, University of Bristol, Bristol BS8 1UB, UK. 4 Bristol Centre for Complexity Science, Department of Engineering Mathematics, University of Bristol, Bristol BS8 1UB, UK.
    [Show full text]
  • Mapping Post-Transcriptional Modifications Onto Transfer
    Review Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry Robert L. Ross, Xiaoyu Cao and Patrick A. Limbach * Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, OH 45221‐0172, USA; [email protected] (R.L.R.); [email protected] (X.C.) * Correspondence: [email protected]; Tel.: +1‐513‐556‐1871 Academic Editor: Jürg Bähler Received: 30 December 2016; Accepted: 15 February 2017; Published: 22 February 2017 Abstract: Liquid chromatography, coupled with tandem mass spectrometry, has become one of the most popular methods for the analysis of post‐transcriptionally modified transfer ribonucleic acids (tRNAs). Given that the information collected using this platform is entirely determined by the mass of the analyte, it has proven to be the gold standard for accurately assigning nucleobases to the sequence. For the past few decades many labs have worked to improve the analysis, contiguous to instrumentation manufacturers developing faster and more sensitive instruments. With biological discoveries relating to ribonucleic acid happening more frequently, mass spectrometry has been invaluable in helping to understand what is happening at the molecular level. Here we present a brief overview of the methods that have been developed and refined for the analysis of modified tRNAs by liquid chromatography tandem mass spectrometry. Keywords: modified nucleosides; RNA sequencing; tRNA; tandem mass spectrometry; LC‐MS/MS 1. Transfer Ribonucleic Acids Transfer ribonucleic acids (tRNAs) are the smallest of the three main types of RNAs. They are an adapter molecule, which bridges the divide between the genetic code stored in DNA to functional proteins that are necessary for cellular viability.
    [Show full text]
  • Characterization of UVA-Induced Alterations to Transfer RNA Sequences
    biomolecules Article Characterization of UVA-Induced Alterations to Transfer RNA Sequences Congliang Sun, Patrick A. Limbach and Balasubrahmanyam Addepalli * Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA; [email protected] (C.S.); [email protected] (P.A.L.) * Correspondence: [email protected] Received: 15 September 2020; Accepted: 5 November 2020; Published: 8 November 2020 Abstract: Ultraviolet radiation (UVR) adversely affects the integrity of DNA, RNA, and their nucleoside modifications. By employing liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based RNA modification mapping approaches, we identified the transfer RNA (tRNA) regions most vulnerable to photooxidation. Photooxidative damage to the anticodon and variable loop regions was consistently observed in both modified and unmodified sequences of tRNA upon UVA (λ 370 nm) exposure. The extent of oxidative damage measured in terms of oxidized guanosine, however, was higher in unmodified RNA compared to its modified version, suggesting an auxiliary role for nucleoside modifications. The type of oxidation product formed in the anticodon stem–loop region varied with the modification type, status, and whether the tRNA was inside or outside the cell during exposure. Oligonucleotide-based characterization of tRNA following UVA exposure also revealed the presence of novel photoproducts and stable intermediates not observed by nucleoside analysis alone. This approach provides sequence-specific information revealing potential hotspots for UVA-induced damage in tRNAs. Keywords: UVR; photooxidation; tRNA; post-transcriptional nucleoside modifications; cusativin; RNA modification mapping; RNA oxidation 1. Introduction Transfer RNAs (tRNAs) deliver amino acids to the site of ribosome-mediated protein synthesis while decoding the messenger RNA (mRNA).
    [Show full text]
  • The Evolution and Functions of Trna Modifications
    life Review From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications Katherine M. McKenney 1,2 and Juan D. Alfonzo 1,2,3,* 1 The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA; [email protected] 2 The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA 3 Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA * Correspondence: [email protected]; Tel.: +1-614-292-0004 Academic Editors: Adrian Gabriel Torres and David Deamer Received: 11 January 2016; Accepted: 7 March 2016; Published: 14 March 2016 Abstract: All nucleic acids in cells are subject to post-transcriptional chemical modifications. These are catalyzed by a myriad of enzymes with exquisite specificity and that utilize an often-exotic array of chemical substrates. In no molecule are modifications more prevalent than in transfer RNAs. In the present document, we will attempt to take a chemical rollercoaster ride from prebiotic times to the present, with nucleoside modifications as key players and tRNA as the centerpiece that drove the evolution of biological systems to where we are today. These ideas will be put forth while touching on several examples of tRNA modification enzymes and their modus operandi in cells. In passing, we submit that the choice of tRNA is not a whimsical one but rather highlights its critical function as an essential invention for the evolution of protein enzymes. Keywords: tRNA modification; evolution; genetic code 1. Before There Was RNA, There Were Modifications Through the years there has been a seemingly limitless and constant supply of ideas and discussions about the origin of life; all envision numerous scenarios with the common theme that, based on modern day biological systems, replication and information storage were needed for a self-sustaining system to survive.
    [Show full text]
  • Combining Trna Sequencing Methods to Characterize Plant Trna Expression and Post- Transcriptional Modification
    bioRxiv preprint doi: https://doi.org/10.1101/790451; this version posted October 2, 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 4.0 International license. Combining tRNA sequencing methods to characterize plant tRNA expression and post- transcriptional modification Jessica M. Warren1, Thalia Salinas-Giegé2, Guillaume Hummel2, Nicole L. Coots1, Joshua M. Svendsen1, Kristen C. Brown1, Laurence Maréchal-Drouard2, Daniel B. Sloan1 1Department of Biology, Colorado State University, Fort Collins, CO 80523-1878, USA 2Institut de biologie moléculaire des plantes-CNRS, Université de Strasbourg, F-67084 Strasbourg, France Corresponding author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/790451; this version posted October 2, 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 4.0 International license. ABSTRACT Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high- throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications.
    [Show full text]
  • WO 2015/051214 Al 9 April 2015 (09.04.2015) W P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/051214 Al 9 April 2015 (09.04.2015) W P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07K 14/705 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/US20 14/058967 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 3 October 2014 (03. 10.2014) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (25) Filing Language: English PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (26) Publication Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/886,137 3 October 201 3 (03. 10.2013) US (84) Designated States (unless otherwise indicated, for every 61/903,485 13 November 2013 (13. 11.2013) US kind of regional protection available): ARIPO (BW, GH, 61/952,906 14 March 2014 (14.03.2014) us GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 62/052,139 18 September 2014 (18.09.2014) us TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (71) Applicant: MODERNA THERAPEUTICS, INC.
    [Show full text]
  • Novel Base-Pairing Interactions at the Trna Wobble Position Crucial for Accurate Reading of the Genetic Code
    ARTICLE Received 8 Oct 2015 | Accepted 13 Dec 2015 | Published 21 Jan 2016 DOI: 10.1038/ncomms10457 OPEN Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code Alexey Rozov1,*, Natalia Demeshkina1,*, Iskander Khusainov1,2, Eric Westhof3, Marat Yusupov1 & Gulnara Yusupova1 Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex Lys X-ray structures, we demonstrate how Escherichia coli tRNA UUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism. 1 Department of Integrated Structural Biology, Institute of Genetics and Molecular and Cellular Biology, INSERM, U964; CNRS/University of Strasbourg, UMR7104, 1 rue Laurent Fries, BP 10142, Illkirch 67404, France. 2 Institute of Fundamental Medicine and Biology, Kazan Federal University, Karl Marx 18, Kazan 420012, Russia. 3 Architecture and Reactivity of RNA, Institute of Molecular and Cellular Biology of the CNRS, University of Strasbourg, UPR9002, 15 rue Rene Descartes, Strasbourg 67084, France.
    [Show full text]