Uncoupling Promoter Opening from Start-Site Scanning

Abstract

Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.

Figure 1. Role of TFIIK in transcription initiation

(A) SDS polyacrylamide gel electrophoresis 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 panel) or inhibitor-sensitive TFIIK mutant (TFIIK-L83G) (Liu et al., 2004) (right panel). 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 Fig. S1.

Figure 2. Run-off transcription with modified promoters

(A) Schematic illustration of modified promoters (also see Fig. 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.

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.

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 Fig. S4.