FEBS Letters | |
Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity | |
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 tRNATyr, starting with 5′-C1, and operates as well for yeast tRNAAsp 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
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
RO201912020306577ZK.pdf | 213KB | download |