Transcription of eukaryotic tRNA genes in vitro. II. Formation of stable complexes.

Abstract

Drosophila tRNA genes form stable transcription complexes in vitro, as we have demonstrated by kinetic analyses of transcription experiments in Drosophila Kc cell extracts. tRNA genes added to transcriptionally active cell-free extracts rapidly and stably sequester a transcription factor, inhibiting transcription of a tRNA gene added later. We describe a simplified competition assay dependent on the ability of tRNA genes to form stable complexes. Through the use of this assay with deletion mutations of a Drosophila tRNAArg gene, we demonstrate that stable transcription complex formation is dependent on the DNA region extending from the 5' end of the sequence encoding the T-stem of the tRNA to more than 10 base pairs downstream from the transcription termination sequence. Stable transcription complex formation involves an initial, rapid factor binding followed by rearrangement of the gene-factor complex to a transcriptionally active state. Factor binding to form the stable transcription complex is kinetically dependent on the sequence 5' to the gene region encoding the D-stem, and thermodynamically dependent on the gene region encoding the D-stem and -loop.