Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II.
Using a defined RNA polymerase II (pol II) transcription system, we have investigated the roles of basal factors at discrete stages during the transcription cycle (e.g., initiation, promoter clearance, and transcript elongation). Abortive initiation assays revealed that TATA-binding protein, transcription factors TFIIB and TFIIF, and pol II were necessary and sufficient to form functional initiation complexes on both linear and supercoiled templates. By contrast, TFIIE, TFIIH, and ATP hydrolysis were additionally required during promoter clearance from linear templates, while negative supercoiling obviated the need for these auxiliary factors. Furthermore, TFIIE, TFIIH, and supercoiling were not required during elongation. Our results suggest a role for TFIIH-associated helicase activity or supercoiling during promoter clearance rather than open complex formation. These results establish abortive initiation as a useful assay for studying functional initiation complex formation in defined eukaryotic transcription systems and provide a framework for investigating regulation at different stages of the eukaryotic transcription cycle.
Goodrich
J A
JA
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Tjian
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D000255:Adenosine Triphosphate; D012319:RNA Polymerase II