Transcription and precursor processing of normal and mutant human tRNAiMet genes in a homologous cell-free system.

Abstract

Two human tRNAiMet genes were previously cloned from a recombinant library of fetal liver DNA (Santos, T., and Zasloff, M. (1981) Cell 23, 699-709). One gene differed from the common vertebrate sequence by a G to T transversion at position 56, occupied exclusively by a purine in all prokaryotic and eukaryotic tRNAs. In this study, we show that although both tRNAiMet genes are transcribed in an in vitro system from KB cells, normal post-transcriptional processing of the mutant gene transcript is interrupted. While the primary transcript of the normal gene undergoes stepwise excision of its 5' and 3' terminal sequences, 5' preceding 3', only the 5' leader of the primary transcript of the mutant gene is excised, resulting in the accumulation of an intermediate containing an unprocessed 3' trailer. The results suggest that certain eukaryotic tRNA mutations may not appear in mature tRNA species due to the effects of the mutation on precursor processing.