Modulation of transcriptional activity and stable complex formation by 5'-flanking regions of mouse tRNAHis genes.

We determined the nucleotide sequences of three mouse tRNAHis genes and a tRNAGly gene present in two different lambda clones. One lambda clone contained two tRNAHis genes 600 base pairs (bp) apart in opposite orientations. The other clone contained a tRNAHis and a tRNAGly gene 569 bp apart in the same orientation. The coding regions of the three tRNAHis genes were identical to sequenced mammalian tRNAHis if posttranscriptional modifications are not considered. Notably, the three tRNAHis genes and a fourth gene previously sequenced by us contained within the flanking regions, various amounts of short, conserved 5' leader sequences and 3' trailer sequences directly abutting the coding regions. Otherwise the flanking regions were not homologous. Deletion mutants of one of the tRNAHis genes were constructed which contained 228, 99, 9, and 3 bp of the wild-type 5'-flanking region, respectively. Deletion of 5'-flanking sequences from positions -9 to -4 reduced transcriptional activity substantially (ca. fivefold) in a HeLa cell S-100 lysate. This effect was independent of the vector sequences in the deletion clone, implying that the region from -4 to -9 of the intact gene contains a positive modulatory element for transcription in vitro. The deletion mutant containing 3 bp of wild-type 5'-flanking sequence also had a greatly reduced ability to inhibit the transcription of a second tRNA gene in a competition assay. Thus, the normal 5'-flanking region influences the ability of the gene to form stable complexes with transcription factors. These data further indicate that a mammalian transcription extract is sensitive to 5'-flanking-region effects if a suitable tRNA gene is assayed.