Energy requirement for specific transcription initiation by the human RNA polymerase II system

Abstract

The energy requirement for specific transcription initiation and elongation by the human RNA polymerase II system was studied in vitro using partially purified transcription factors from HeLa cell nuclear extracts. The synthesis of the 536-nucleotide long run-off transcript resulting from initiation at the adenovirus major late promoter was found to be dependent upon the presence of either ATP or dATP (with the imido derivative adenyl-5'-yl imidodiphosphate being used as the substrate for the RNA polymerase elongation reaction). An identical requirement for hydrolysis of the phosphate bond in an adenosine nucleotide was observed for the synthesis of the decanucleotide transcribed from the major late promoter in the absence of the GTP substrate. In contrast, the nonhydrolyzable analog adenyl-5'-yl imidodiphosphate fully substitutes for ATP during the subsequent elongation of these short transcripts, which demonstrates that the energy requirement occurs at an earlier step of the transcription reaction. Thus the particular transcription factor that requires ATP (or dATP) hydrolysis for its function must act prior to, or concomitant with, formation of the first few phosphodiester linkages by the RNA polymerase II.