Promoter escape limits the rate of RNA polymerase II transcription and is enhanced by TFIIE, TFIIH, and ATP on negatively supercoiled DNA.
To measure rate constants for discrete steps of single-round transcription (preinitiation complex formation, promoter escape, and transcript elongation), kinetic studies were performed in a well defined human RNA polymerase II transcription system. These experiments revealed that promoter escape limits the rate of transcription from the adenovirus major late promoter (AdMLP) contained on negatively supercoiled DNA. TFIIE and TFIIH were found to significantly increase fractional template usage during a single round of transcription in an ATP-dependent reaction. The observed rate constant for promoter escape, however, was not greatly affected by TFIIE and TFIIH. Our results are explained by a model in which transcription branches into at least two pathways: one that results in functional promoter escape and full-length RNA synthesis, and another in which preinitiation complexes abort during promoter escape and do not produce full-length RNA transcripts. These results with negatively supercoiled templates agree with our earlier conclusion that TFIIE, TFIIH, and ATP direct promoter escape and support a model in which the TFIIH helicases stimulate promoter escape in an ATP-dependent reaction.
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Campus Box 215, Boulder, CO 80309-0215, USA.
J F
JF
Kugel
J A
JA
Goodrich
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002417:Cattle; D004278:DNA, Superhelical / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000096:biosynthesis; D012319:RNA Polymerase II / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D004278:DNA, Superhelical; D011994:Recombinant Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D051758:Transcription Factor TFIIH; D012313:RNA; D000255:Adenosine Triphosphate; D012319:RNA Polymerase II