The 5'-flanking sequences of Drosophila melanogaster tRNA5Asn genes differentially arrest RNA polymerase III.

Abstract

Three tRNA5Asn genes have been subcloned from a tRNA gene cluster isolated from the cytogenetic locus 42A of Drosophila melanogaster. The three tRNAAsn genes, contained on plasmids pAsn6, pAsn7, and pAsn8, have identical mature tRNA coding regions but have different 5'- and 3'-flanking sequences. In vitro transcription in Drosophila Schneider S3 cell-free extracts showed the tRNAAsn genes had different transcription efficiencies. pAsn8 had a transcription efficiency of approximately 8 transcripts/gene/h, whereas pAsn6 was a less active template at 5 transcripts/gene/h. pAsn7 was the poorest template at 1.5 transcripts/gene/h. Exchanging 5'-flanking regions of the tRNAAsn genes showed that the differences in transcription efficiencies were attributable to the corresponding 5'-flanking region. Transcription of each of the tRNAAsn genes revealed a different optimum for KC1 concentration for each template which also was directly attributable to the corresponding 5'-flanking region. The "salt effect" is not related to the ability of the three tRNAAsn genes to sequester transcription factors as determined using the stable complex competition assay. Rather, this effect appears to be due to the ability of the respective 5'-flanking regions to interact with RNA polymerase III. The poorest transcription template, pAsn7, was a better competitor in the stable complex formation assay than either pAsn8 or pAsn6. We conclude that the pAsn7 stable complex binds and functionally arrests RNA polymerase III in the initiation reaction.