5'-flanking sequences that inhibit in vitro transcription of a xenopus laevis tRNA gene

Abstract

Two series of deletion mutants have been constructed in the 5'-flanking DNA of a variant methionine tRNA gene of X. laevis. When assayed in homologous cell-free extracts, mutants of both series are expressed at very different levels and they initiate transcription at either or both of the two major sites used by the wild-type gene. No similarities are evident in the 5'-flanking regions of well expressed mutants, but two DNA segments are identified that inhibit transcription. The most inhibitory one, between residues -20 and -12, consists of 9 bp of alternating purines and pyrimidines: TGCGCGTGC. The second has only a weak inhibitory effect when in its normal location from -43 to -32, but it can reduce transcription severely when brought closer to the gene; it is composed of 12 bp of alternating purines and pyrimidines with one residue out of alternation: ATGCACAGCGCA. The composition of these tracts has some possible implications for their conformation, which may account for their inhibition of gene expression.