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

Abstract

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.