title: Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position.
abstract: The 2-thiouridine (s(2)U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon-anticodon base pairing at the ribosome A-site, and prevents frameshifting during translation. By mass spectrometry of affinity-purified native Escherichia coli tRNA1(Gln)UUG, we show that the complete modification at the wobble position 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm(5)s(2)U). The crystal structure of E. coli glutaminyl-tRNA synthetase (GlnRS) bound to native tRNA1(Gln) and ATP demonstrates that cmnm(5)s(2)U34 improves the order of a previously unobserved 11-amino-acid surface loop in the distal β-barrel domain of the enzyme and imparts other local rearrangements of nearby amino acids that create a binding pocket for the 2-thio moiety. Together with previously solved structures, these observations explain the degenerate recognition of C34 and modified U34 by GlnRS. Comparative pre-steady-state aminoacylation kinetics of native tRNA1(Gln), synthetic tRNA1(Gln) containing s(2)U34 as sole modification, and unmodified wild-type and mutant tRNA1(Gln) and tRNA2(Gln) transcripts demonstrates that the exocyclic sulfur moiety improves tRNA binding affinity to GlnRS 10-fold compared with the unmodified transcript and that an additional fourfold improvement arises from the presence of the cmnm(5) moiety. Measurements of Gln-tRNA(Gln) interactions at the ribosome A-site show that the s(2)U modification enhances binding affinity to the glutamine codons CAA and CAG and increases the rate of GTP hydrolysis by E. coli EF-Tu by fivefold.
authors.affiliation: Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA; Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239, USA.
authors.forename: Annia
authors.initials: A
authors.lastname: Rodriguez-Hernandez
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Spears
authors.forename: Kirk W
authors.initials: KW
authors.lastname: Gaston
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
authors.forename: Howard
authors.initials: H
authors.lastname: Gamper
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
authors.forename: Rob
authors.initials: R
authors.lastname: Kaiser
authors.forename: Paul F
authors.initials: PF
authors.lastname: Agris
authors.forename: John J
authors.initials: JJ
authors.lastname: Perona
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000926:Anticodon / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D009690:Nucleic Acid Conformation; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000502:physiology*; D011487:Protein Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: C105273:2-thiouridine; D000926:Anticodon; D009705:Nucleosides; D012351:RNA, Transfer, Gln; D013891:Thiouridine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase
keywords: HPLC; LC–MS/MS; WT; XIC; extracted ion chromatogram; fMet; formylmethionine; glutaminyl-tRNA synthetase; high-pressure liquid chromatography; liquid chromatography–tandem mass spectrometry; peptidyl transfer; pre-steady-state kinetics; ribosome; transfer RNA; wild-type; β-mercaptoethanol; βME