A detailed mutational analysis of the eucaryotic tRNAmet1 gene promoter

We have isolated phage M13 clones containing the X. laevis trnamet1 gene, each having one or a few C leads to T transitions in the tRNA coding sequence. Nearly every G-C and C-G base pair in the tDNA has been mutagenized. The importance of these altered nucleotides in transcription by RNA polymerase III has been assessed by injecting the cloned DNAs into frog oocyte nuclei together with alpha-32P-GTP and measuring the synthesis of labeled tRNAmet1. Several G-C and C-G base pairs in the structural gene appear to be major promoter determinants, because when mutated, transcription is reduced 3-fold to 20-fold. Most of these determinants occur between nucleotides 7 to 19 and 49 to 61 in sequence regions highly conserved among eucaryotic as well as procaryotic tRNAs. Several additional G-C and C-G base pairs between these two regions also contribute to promoter activity; their location suggests that a stem-loop structure in the DNA encoding the tRNA's anticodon arm is important for RNA polymerase III promoter function.