PROMOTER ESCAPE BY RNA-POLYMERASE-II - A ROLE FOR AN ATP COFACTOR IN SUPPRESSION OF ARREST BY POLYMERASE AT PROMOTER-PROXIMAL SITES

It is well established that TFIIH-dependent transcription by RNA polym erase II requires a hydrolyzable ATP cofactor for synthesis of the fir st phosphodiester bond of nascent transcripts. Whether an ATP cofactor is also required after initiation for escape of RNA polymerase II fro m the promoter has, however, been controversial. We have now addressed this question directly by investigating the ability of RNA polymerase II transcription complexes containing short, similar to 5-8-nucleotid e transcripts synthesized in the presence of limiting nucleotides to e scape the promoter in the absence of an ATP cofactor in a basal transc ription system reconstituted with purified RNA polymerase II and gener al initiation factors. Depletion of ATP had a profound effect on the a bility of initiated complexes to progress into the elongation phase: w hereas in the presence of ATP, the majority of transcription complexes could be chased away from the promoter proximal region, most complexe s deprived of ATP catalyzed synthesis of only a few phosphodiester bon ds and then ceased elongation after synthesizing transcripts less than 10-14 nucleotides in length. A significant fraction of these transcri pts could be extended following addition of ATP, indicating that they were contained in arrested, but potentially active elongation complexe s. Like the ATP-requiring step in initiation, ATP-dependent suppressio n of arrest by RNA polymerase II at promoter proximal sites is inhibit ed by adenosine 5'-O-(thio)triphosphate. Transcription complexes conta ining transcripts longer than 9-10 nucleotides are insensitive to inhi bition by ATP gamma S, indicating that susceptibility to ATP sensitive arrest is a property of very early elongation complexes. Taken togeth er, our findings reveal a novel role for an ATP cofactor in transcript ion by RNA polymerase II.