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. 1998 Jun 9;95(12):6722-7.
doi: 10.1073/pnas.95.12.6722.

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription

K Yokomori  1 , C P Verrijzer, R Tjian
Affiliations

An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription

K Yokomori et al. Proc Natl Acad Sci U S A. .

Abstract

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

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Figures

Figure 1
Figure 1
The TFIIH-independent function of TFIIE. (A) The effect of TFIIE in basal transcription was determined in the absence of TFIIH as described in Materials and Methods, by using the templates indicated. Transcription was in the absence (lanes 1 and 3) or presence of recombinant TFIIE (lane 2; 2 ng, and lane 4; 4 ng each of E56/E34) or mutant (mt) TFIIE (lane 5; 4 ng each of E34 and mt E56; ref. 13). Primer extension products of the transcripts are indicated by an arrowhead (G6TI) and a bracket (E4). (B) The effect of TFIIE on TBP binding to AdML 2 and E4 promoter DNA in DNase I-footprinting analyses on the nontranscribed DNA strand (lanes 1–4 and 11–13) and the transcribed DNA strand (lanes 5–10). Proteins in the binding reactions are indicated on the top, including “no protein” (lanes 4, 5, and 11). Ten nanograms each of TBP and TFIIA and 8 ng each of E56 and E34 were used. Nucleotide positions on the promoter relative to the start site of transcription of the AdML 2 promoter are numbered, and protected regions against DNase I digestion over the TATA box are indicated by brackets. The protection indicated by an asterisk in lane 8 corresponds to the cryptic TATA sequence further upstream of the TATA box. TATA box (“TATAAAA”) is indicated by a bar.
Figure 2
Figure 2
E56 promotes cooperative binding of TBP to the TATA element. (A) Analyses of the effect of the individual subunits of TFIIE on TBP binding to the AdML promoter by DNase I-footprinting assays. TBP (10 ng), E56 (8 ng), and E34 (8 ng) were used in the binding reactions as indicated on the top. The protection over TATA box is indicated by a bracket. The transcription start site is +1. (B) DNase I-footprinting analysis of the effect of a zinc-finger mutant of E56 on TBP binding to the promoter region. The assays were carried out in the presence of a constant amount of TBP (6 ng) (lanes 2–6) with 8 ng and 4 ng of either the wild-type E56 (lanes 3 and 4) or the zinc-finger mutant E56 (lanes 5 and 6). Lane 1 is in the absence of any protein. The protected region is indicated by a bracket. (C) Analysis of E56 and TBP binding to the AdML promoter in hydroxyl radical-footprinting assays. DNA was incubated with no protein (lane 1) or TBP (10 ng, lanes 2–4) in the absence of other proteins (lane 2), or in the presence of E34 (8 ng, lane 3) or E56 (8 ng, lane 4) before treatment with hydroxyl radicals. The area over the TATA box protected by TBP is indicated by a bracket (nucleotide position −23 to −32), and further protection caused by the presence of E56 is indicated by a bar (lane 4, −25 to −28). [These experiments were repeated four times.] Corresponding sequences are shown underneath, indicating the TBP protection (a bracket) and the change of protection by E56 (arrows). The hypersensitive band created by TBP also is indicated by an arrowhead (nucleotide −26). The TATA box is underlined.
Figure 3
Figure 3
The effect of E34 on TBP/E56 and TBP/TFIIA binding to the promoter. (A) Analysis of the effect of E34 on promoter binding by E56 and TBP by DNase I-footprinting assays. DNA was incubated with either no protein (lane 1) or 6 ng of TBP alone (lane 2–4) in the presence of 3 ng of E56 (lane 3) or 3 ng each of E56 and E34 (lane 4). The protection is indicated by a bracket. (B) The effect of TFIIE and E34 on TBP/TFIIA and TBP/TFIIB binding to the promoter in DNase I-footprinting analysis in the presence of limiting amounts of TBP (5 ng), TFIIA (3 ng), TFIIB (3 ng), and TFIIE (2 ng of E56 and E34). Lane 1 is no protein. Proteins in the binding reactions are indicated on the top. Open arrowheads indicate two bands that are present in the presence of TBP/TFIIA/E34 (lane 5; also see Fig. 1B, lane 2) but disappeared upon the addition of E56 (lane 4). The TFIIA/TBP-dependent protection of a cryptic TATA sequence is indicated by a bracket with an asterisk (lanes 4 and 5).
Figure 4
Figure 4
Detection of E34 interactions with DNA and TFIIA. (A) Detection of E34 and DNA interactions by UV-crosslinking experiments by using AdML 2-promoter region (−72 to +8) as a template. E34 protein alone (lane 1) and TBP/E56 (lane 2) were subjected to crosslinking DNA and immunoprecipitated by anti-E34 antibodies. (B) Detection of the direct interaction of E34 with the large subunit of TFIIA in vitro protein interaction assays. Five percent of the input 35S-labeled proteins were shown. In lanes 1–9, proteins indicated on top of the lanes were mixed with GST-hTFIIAα β [(lanes 3, 4, and 8) or GST protein (lanes 5, 6, and 9)] on glutathione Sepharose beads. In lanes 10–18, in vitro translated 35S-labeled proteins indicated on top of the lanes were incubated with either flag-tagged E34 protein bound to M2 beads (lanes 12, 13, and 17) or M2 beads alone (lanes 14, 15, and 18). Full-length proteins are indicated by arrowheads. The 14-kDa subunit of TFIIA (indicated by a closed arrowhead) migrates aberrantly (indicated by an open arrowhead) in the input because of the comigrating hemoglobin in the rabbit reticulocyte lysate.
Figure 5
Figure 5
An alternative model for the assembly of a PIC. These models are different from the conventional stepwise assembly pathway in which TFIIE together with TFIIH are the last basal factors to enter the PIC. (A) TFIIE participates in the initial binding of TBP/TFIID to the promoter before the recruitment of the RNA pol II/TFIIF complex and TFIIH. It is pertinent to note that a role of TFIIE during the first steps of PIC assembly does not preclude a role during TFIIH recruitment (see Discussion). (B) To recruit TFIIH, TFIIE may either shift to alternate positions within the PIC or may contact both TBP/TFIIA and TFIIH at the same time, possibly accompanied by conformational changes of DNA upon PIC assembly (e.g., DNA bending by TBP/TFIID). This model illustrates the potential dual role of TFIIE in PIC formation and does not attempt to accommodate distinct RNA pol II complexes that have been described.
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