A DNA-tethered cleavage probe reveals the path for promoter DNA in the yeast preinitiation complex

Abstract

To directly map the position of promoter DNA within the RNA Polymerase II (Pol II) transcription Preinitiation Complex (PIC), FeBABE was tethered to specific sites within the HIS4 promoter and used to map exposed surfaces of Pol II and the general transcription factors in proximity to DNA. Our results distinguish between previously proposed models for PIC structure and demonstrate that downstream promoter DNA is positioned over the central cleft of Pol II with DNA upstream of TATA extending toward the Pol II subunit Rpb3. Also mapped were segments of TFIIB, IIE, IIF, and IIH in proximity to promoter DNA. DNA downstream of the transcription bubble maps to a path between the two helicase subdomains of the TFIIH subunit Rad25 (XPB). Together, our results show how the general factors and Pol II converge on promoter DNA within the PIC. KeywordsRNA Pol II; preinitiation complex; hydroxyl radical; general transcription factor Transcription of protein-coding genes by Pol II requires the recruitment of Pol II and the general transcription factors (GTFs) to promoters to form a PIC 1-3 , analogous to the Closed Complex state of bacterial RNAP. Prior to transcription initiation, the PIC transitions into the Open Complex state, where ATP and the TFIIH helicase subunit XPB promote melting of DNA surrounding the transcription start site and positioning of the template strand of the promoter within the active site cleft of Pol II 1,4-7 . In metazoans, ∼12 base pairs (bp) of DNA are melted in the Open Complex, and transcription typically initiates at a single start site ∼30-bp downstream from the TATA element. However, transcription start site selection in S. cerevisiae is more complex since initiation occurs between 40−120-bp from TATA 8 . Despite these differences in start site position, the upstream edge of the transcription bubble maps to ∼20-bp downstream of TATA both in metazoan systems and S. cerevisiae 7,9-11 . These observations led to the suggestion that yeast Pol II "scans" downstream DNA for a suitable initiation site 9 .Much insight into the mechanism of Pol II transcription has been gained from crystal structures of yeast . Structural studies have shown that for elongating Pol II, the template DNA strand lies inside the deep central cleft located between Rpb1 and Rpb2, the two largest Email: shahn@fhcrc.org, Telephone: 206 667 5261, Fax: 206 667 6497. Publisher's Disclaimer: This PDF receipt will only be used as the basis for generating PubMed Central (PMC) documents. PMC documents will be made available for review after conversion (approx. 2−3 weeks time). Any corrections that need to be made will be done at that time. No materials will be released to PMC without the approval of an author. Only the PMC documents will appear on PubMed Central --this PDF Receipt will not appear on PubMed Central. More recently, separate studies examining TFIIB interaction with Pol II have provided two additional models of PIC structure 23-25 . X-ray structure analysis of a complex of Pol II and TFIIB 23 showed that the TFI...

Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex*

“…The barriers to exonuclease III digestion of the PIC around positions Ϫ70 to Ϫ9 are in good agreement with the observed size and location of the 68-bp psoralen bubble at the end of the DNA fragment and are consistent with previous protein-DNA cross-linking studies performed with yeast nuclear extract (24) and with a reconstituted human system (25). The TATA sequence, between positions Ϫ63 and Ϫ56, would be located off-center within the PIC, with an additional 40 bp lying within the PIC on the downstream side.…”

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

“…TFIIB binding to this location in association with the TBP-DNA complex has been proposed to bend promoter DNA around Pol II and to position the promoter above the central cleft. In agreement with this model, hydroxyl radical-generating probes linked to promoter DNA suggest that the path of DNA lies above the central cleft 19 . Additionally, this study, along with earlier protein-DNA crosslinking studies in the human transcription system 20-23 , suggested that TFIIF, TFIIE, and TFIIH are positioned near the central cleft.…”

“…The localization of TFIIF and TFIIE to opposite sides of the Pol II central cleft shows how these factors are in position to affect post-recruitment steps in the mechanism of transcription initiation. Previously, we proposed a model for the binding of TFIIA/TFIIB/TBP/DNA on Pol II based on photocrosslinkers attached to TFIIB, hydroxyl radical generating probes attached to TFIIB and promoter DNA, and structural studies of these factors [8][9][10]19,31,32,35,36 (Fig. 7a).…”

Inactivated RNA Polymerase II Open Complexes Can Be Reactivated with TFIIE

“…NTPs and TFIIE presumably facilitate the additional melting necessary to reveal the unpaired template strand at the initiation site, in agreement with the location proposed for TFIIE within the PIC (36,38). If NTPs for transcription and TFIIE are both absent when the Ϫ9 to Ϫ2 bubble is formed, TFIIH may be unable to begin translocation.…”