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.
Highlights
Following assembly of the metazoan RNA polymerase (Pol)2 II transcription preinitiation complex (PIC), productive transcription initiation involves at least three steps [1,2,3,4,5,6,7,8,9]: 1) formation of the open complex state containing an ϳ11-base DNA bubble
We found that transcription start sites (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
We found that transcription initiated normally under these conditions, eliminating a required role for Pol II RNA synthesis in scanning, we surprisingly found that yeast transcription initiated in the absence of hydrolyzable ATP or dATP
Summary
Following assembly of the metazoan RNA polymerase (Pol)2 II transcription preinitiation complex (PIC), productive transcription initiation involves at least three steps [1,2,3,4,5,6,7,8,9]: 1) formation of the open complex state containing an ϳ11-base DNA bubble. 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.
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