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 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.
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