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

FEBS Letters

Rudinger, Joëlle¹  Fechter, Pierre¹  Théobald-Dietrich, Anne¹  Giegé, Richard¹ 

[1] UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 Rue René Descartes, 67084 Strasbourg Cedex, France

关键词: Aminoacylation;    Promoter;    Ribozyme;    Transcription;    Transzyme;    tRNATyr;    AspRS;    aspartate-tRNA synthetase;    TyrRS;    tyrosyl-tRNA synthetase;   

DOI  :  10.1016/S0014-5793(98)01096-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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′-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 K _m parameters quasi identical to those of their phosphorylated counterparts.

【 授权许可】

Unknown