Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity

• Published in: FEBS letters Vol. 436; no. 1; pp. 99 - 103
• Main Authors: Fechter, Pierre, Rudinger, Joëlle, Giegé, Richard, Théobald-Dietrich, Anne
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 25.09.1998
• Subjects: Aminoacylation; AspRS, aspartate-tRNA synthetase; Biochemistry - methods; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Kinetics; Promoter; Promoter Regions, Genetic; Ribozyme; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; RNA, Transfer, Tyr - chemistry; RNA, Transfer, Tyr - genetics; RNA, Transfer, Tyr - metabolism; Transcription; Transcription, Genetic; Transzyme; tRNA Tyr; tRNATyr; TyrRS, tyrosyl-tRNA synthetase; Viral Proteins; Promoter; TyrRS, tyrosyl-tRNA synthetase; Aminoacylation; Ribozyme; Transcription; Transzyme; AspRS, aspartate-tRNA synthetase; tRNA Tyr
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)01096-5

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 Tyr, starting with 5′-C 1, and operates as well for yeast tRNA Asp with 5′-U 1. Although the tRNAs produced by the transzyme method are not phosphorylated, they are fully active in aminoacylation with k cat and K m parameters quasi identical to those of their phosphorylated counterparts.