Uncoupling Promoter Opening from Start-Site Scanning

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Uncoupling Promoter Opening from Start-Site Scanning Kenji Murakami, Pierre-Jean Mattei, Ralph E. Davis, Huiyan Jin, Craig D. Kaplan, Roger D. Kornberg Molecular Cell Volume 59, Issue 1, Pages 133-138 (July 2015) DOI: 10.1016/j.molcel.2015.05.021 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 59, 133-138DOI: (10.1016/j.molcel.2015.05.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Role of TFIIK in Transcription Initiation (A) SDS-PAGE of purified TFIIH-ΔTFIIK. (B) Run-off transcription with increasing amounts of TFIIK. HIS4 promoter DNA (−96/+112) (Murakami et al., 2013a) was combined with TFIIA, TFIIB, TBP, TFIIE, TFIIH-ΔTFIIK, Pol II-TFIIF complex, and the amounts of TFIIK indicated. Transcripts initiated from upstream and downstream TSSs are indicated by red and black arrows. The asterisk indicates a non-specific product that was not observed by primer extension analysis. (C) Run-off transcription with increasing amounts of TFIIK (left three lanes) or inhibitor-sensitive TFIIK mutant (TFIIK-L83G) (Liu et al., 2004) (right three lanes). The reactions were performed as in (B) except with the addition of 50 μM NA-PP1. (D) Same as (B) with different promoters. See also Figure S1. Molecular Cell 2015 59, 133-138DOI: (10.1016/j.molcel.2015.05.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Run-Off Transcription with Modified Promoters (A) Schematic illustration of modified promoters (also see Figure S2). The wild-type SNR20 promoter, the SNR20 promoter with a C+4G mutation introduced in the TSS (SNR20 91W), and the SNR20 promoter with deletions (indicated by dashed lines) bringing the +1 TSS to positions 29, 31, and 33 bp from the TATA box (SNR20 29D, SNR20 31D, SNR20 33D). (B) Run-off transcription with the modified SNR20 promoters. For reactions with the inhibitor-sensitive mutant TFIIK-L83G, 1.1 mM NA-PP1 was added. Molecular Cell 2015 59, 133-138DOI: (10.1016/j.molcel.2015.05.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Exonuclease III Footprints of the PIC and PIC-ΔTFIIK 32P-labeled HIS4 (−96/+112) DNA was incubated with TFIIA, TFIIB, TBP, TFIIE, TFIIH-ΔTFIIK, and Pol II-TFIIF complex with or without nucleotides as indicated over the lanes, followed by treatment with exonuclease III and gel electrophoresis. Positions of protected fragments are indicated by black (PIC) and red (PIC-ΔTFIIK) arrows. Molecular Cell 2015 59, 133-138DOI: (10.1016/j.molcel.2015.05.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Schematic Representation of Promoter Opening and TSS Scanning Schematic representation of the PIC (left panel) and proposed changes upon promoter opening (middle panel) and TSS scanning (right panel). DNA is blue and green. TFIIK is yellow. Upstream and downstream TSSs are indicated by red and black arrows. See also Figure S4. Molecular Cell 2015 59, 133-138DOI: (10.1016/j.molcel.2015.05.021) Copyright © 2015 Elsevier Inc. Terms and Conditions