Chemical synthesis of a biologically active natural tRNA with its minor bases.
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.
Gasparutto
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Département de Recherche Fondamentale de la Matière Condensée, CIS Bio International, Centre d'Etudes Nucléaires de Grenoble, France.
Livache
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Bazin
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Duplaa
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AM
Guy
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Khorlin
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Molko
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Roget
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Téoule
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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
D007202:Indicators and Reagents; D009705:Nucleosides; D009838:Oligodeoxyribonucleotides; D012344:RNA, Transfer, Ala; D012194:RNA-Directed DNA Polymerase; D000412:Alanine-tRNA Ligase