The 5' flanking sequence negatively modulates the in vivo expression and in vitro transcription of a human tRNA gene

The consequences of altering the 5' flanking region of a human amber suppressor tRNA(ser) gene on phenotypic expression in vivo and transcription in vitro was examined by constructing a series of upstream deletion and substitution mutants. The resulting tDNA variants were examined for functional tRNA expression in vivo, by measuring suppression of a nonsense mutation in the Escherichia coli chloramphenicol acetyltransferase (cat) gene in co-transfection assays, and for transcriptional activity in vitro using HeLa cell nuclear extracts. Mutant genes in which the 18 nucleotides 5' proximal to the coding region were deleted and replaced with heterologous sequences were 2 to 5 fold more active in vivo in comparison to the wild type gene. There was a strong, but not exclusive, correlation between the levels of nonsense suppression observed in vivo and transcriptional activity in vitro. In certain cases, introduction of an oligonucleotide encompassing this 18 nucleotide element upstream of more active tRNA genes reduced both the levels of suppression and template activity. These results indicate that the immediate 5' contiguous sequence of this tRNA gene negatively modulates expression both in vivo and in vitro.