Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity.
A limitation for a universal use of T7 RNA polymerase for in vitro tRNA transcription lies in the nature of the often unfavorable 5'-terminal sequence of the gene to be transcribed. To overcome this drawback, a hammerhead ribozyme sequence was introduced between a strong T7 RNA polymerase promoter and the tDNA sequence. Transcription of this construct gives rise to a 'transzyme' molecule, the autocatalytic activity of which liberates a 5'-OH tRNA transcript starting with the proper nucleotide. The method was optimized for transcription of yeast tRNA(Tgammar), starting with 5'-C1, and operates as well for yeast tRNA(Asp) with 5'-U1. Although the tRNAs produced by the transzyme method are not phosphorylated, they are fully active in aminoacylation with k(cat) and Km parameters quasi identical to those of their phosphorylated counterparts.
UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
P
P
Fechter
J
J
Rudinger
R
R
Giegé
A
A
Théobald-Dietrich
D001671:Biochemistry / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D016337:RNA, Catalytic; D012365:RNA, Transfer, Tyr; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases