title: Chemical synthesis of a biologically active natural tRNA with its minor bases.
abstract: The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.
authors.affiliation: Département de Recherche Fondamentale de la Matière Condensée, CIS Bio International, Centre d'Etudes Nucléaires de Grenoble, France.
authors.forename: D
authors.initials: D
authors.lastname: Gasparutto
authors.forename: T
authors.initials: T
authors.lastname: Livache
authors.forename: H
authors.initials: H
authors.lastname: Bazin
authors.forename: A M
authors.initials: AM
authors.lastname: Duplaa
authors.forename: A
authors.initials: A
authors.lastname: Guy
authors.forename: A
authors.initials: A
authors.lastname: Khorlin
authors.forename: D
authors.initials: D
authors.lastname: Molko
authors.forename: A
authors.initials: A
authors.lastname: Roget
authors.forename: R
authors.initials: R
authors.lastname: Téoule
mesh_terms: D000412:Alanine-tRNA Ligase / Q000378:metabolism*; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009705:Nucleosides / Q000138:chemical synthesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D012344:RNA, Transfer, Ala / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D014158:Transcription, Genetic
chemical_list: D007202:Indicators and Reagents; D009705:Nucleosides; D009838:Oligodeoxyribonucleotides; D012344:RNA, Transfer, Ala; D012194:RNA-Directed DNA Polymerase; D000412:Alanine-tRNA Ligase