title: Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF.
abstract: Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.
authors.affiliation: From the Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, Ohio 45221 balasual@ucmail.uc.edu.
authors.forename: Balasubrahmanyam
authors.identifier: http://orcid.org/0000-0001-6257-7700
authors.initials: B
authors.lastname: Addepalli
authors.affiliation: From the Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, Ohio 45221 Pat.Limbach@uc.edu.
authors.forename: Patrick A
authors.identifier: http://orcid.org/0000-0003-1526-4546
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D011560:Pseudouridine / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012365:RNA, Transfer, Tyr; D011560:Pseudouridine; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase
keywords: CMCT; RNA modification; RluF; acrylonitrile; chemical modification; derivatization; mass spectrometry (MS); pseudouridine; transfer RNA (tRNA); translation