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 polymerase II requires a hydrolyzable ATP cofactor for synthesis of the first phosphodiester bond of nascent transcripts. Whether an ATP cofactor is also required after initiation for escape of RNA polymerase II from 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, approximately 5-8-nucleotide transcripts synthesized in the presence of limiting nucleotides to escape the promoter in the absence of an ATP cofactor in a basal transcription system reconstituted with purified RNA polymerase II and general initiation factors. Depletion of ATP had a profound effect on the ability of initiated complexes to progress into the elongation phase: whereas in the presence of ATP, the majority of transcription complexes could be chased away from the promoter-proximal region, most complexes deprived of ATP catalyzed synthesis of only a few phosphodiester bonds and then ceased elongation after synthesizing transcripts less than 10-14 nucleotides in length. A significant fraction of these transcripts could be extended following addition of ATP, indicating that they were contained in arrested, but potentially active elongation complexes. Like the ATP-requiring step in initiation, ATP-dependent suppression of arrest by RNA polymerase II at promoter-proximal sites is inhibited by adenosine 5'-O-(thio)triphosphate. Transcription complexes containing transcripts longer than 9-10 nucleotides are insensitive to inhibition by ATPgammaS, indicating that susceptibility to ATP-sensitive arrest is a property of very early elongation complexes. Taken together, our findings reveal a novel role for an ATP cofactor in transcription by RNA polymerase II.
Dvir
A
A
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
Conaway
R C
RC
Conaway
J W
JW
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D009843:Oligoribonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic / Q000187:drug effects*
D009843:Oligoribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D012319:RNA Polymerase II