title: Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II.
abstract: Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.
authors.affiliation: From the Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and.
authors.forename: James
authors.initials: J
authors.lastname: Fishburn
authors.affiliation: the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Eric
authors.initials: E
authors.lastname: Galburt
authors.affiliation: From the Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and shahn@fredhutch.org.
authors.forename: Steven
authors.initials: S
authors.lastname: Hahn
mesh_terms: D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000502:physiology; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000737:chemistry / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology; D051758:Transcription Factor TFIIH / Q000737:chemistry / Q000502:physiology; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: C489575:SSL2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051758:Transcription Factor TFIIH; D012319:RNA Polymerase II; D004265:DNA Helicases
keywords: ATP; DNA translocase; RNA polymerase II; gene transcription; general transcription factor (GTF); transcription start site (TSS); yeast transcription