The extent of a eukaryotic tRNA gene. 5'- and 3'-flanking sequence dependence for transcription and stable complex formation.

We have examined the 5'-and 3'-flanking sequence requirements for the "wild type transcription properties" of a Drosophila tRNA Arg gene through the use of transcription assays in cell-free extracts. Thirty-three base pairs of the 5' flank immediately adjacent to the sequence encoding the mature tRNA are necessary for efficient transcription in Drosophila Kc cell extract. Sequences affecting factor binding to form stable transcription complexes extend more than 60 base pairs into the 5' flank, and approximately 35 base pairs into the 3' flank. HeLa cell extract exhibits dependence, albeit reduced, on the same 5'-flanking sequence; it also has 3'-flanking sequence requirements for maximal stable complex formation. This requirement of in vitro transcription for flanking sequence is not dependent on the use of a homologous system, but is dependent on the cellular source of the extract.