Transcription of eukaryotic tRNA genes in vitro. I. Analysis of control regions using a competition assay

Abstract

The regions of a Drosophila tRNAArg gene responsible for the "wild type" in vitro transcription level were determined by a transcription-competition assay. Cell-free transcription extracts programmed with 5' and 3' deletion mutants of the tRNAArg gene were used to quantitate the efficiency of transcription and to measure the ability of these DNAs to compete for transcription factors compared to the wild type tRNAArg gene. The results show that those portions of the gene which code for the D-stem/D-loop and T-stem/T-loop of the tRNA product are the regions responsible for competitive ability. These regions were previously shown to contain the intragenic control sequences for eukaryotic tRNA gene transcription and are respectively referred to as the D- and T-control regions. The presence of both the D- and T-control regions is essential for maximum competitive strength. The 5'-flanking and 5' stem regions adjacent to the D-control region have a function in the competitive ability of the D-control region while the 3'-flanking and the 3' stem regions adjacent to the T-control region have a function in the competitive ability of the T-control region. These results are consistent with a model for promotion of tRNA gene transcription that involves recognition by transcription factors of the two control regions. Optimal binding of the transcription factors is dependent upon sequences adjacent to and flanking the intragenic control regions.