The regulatory role for the ERCC3 helicase of general transcription factor TFIIH during promoter escape in transcriptional activation

Abstract

Eukaryotic transcriptional activators have been proposed to function, for the most part, by promoting the assembly of preinitiation complex through the recruitment of the RNA polymerase II transcriptional machinery to the promoter. Previous studies have shown that transcriptional activation is critically dependent on transcription factor IIH (TFIIH), which functions during promoter opening and promoter escape, the steps following preinitiation complex assembly. Here we have analyzed the role of TFIIH in transcriptional activation and show that the excision repair cross-complementing (ERCC) 3 helicase activity of TFIIH plays a regulatory role to stimulate promoter escape in activated transcription. The stimulatory effect of the ERCC3 helicase is observed until approximately 10-nt RNA is synthesized, and the helicase seems to act throughout the entire course of promoter escape. Analyses of the early phase of transcription show that a majority of the initiated complexes abort transcription and fail to escape the promoter; however, the proportion of productive complexes that escape the promoter apparently increases in response to activation. Our results establish that promoter escape is an important regulatory step stimulated by the ERCC3 helicase activity in response to activation and reveal a possible mechanism of transcriptional synergy.

Figure 1

TFIIH mutants and their transcriptional activities. (A) Mutations of ATP-binding sites in the MO15, ERCC3, and ERCC2 subunits of TFIIH. The lysine residues mutated to alanine are indicated. (B) Silver-stained gel of purified TFIIH mutants. The positions of TFIIH subunits are indicated on the right. WT, wild type. (C) Test (pG5HMC2AT) and control (pMLΔ53C2AT) templates used for in vitro transcription. (D) Activated transcription assays in the presence of TFIIH mutants. The positions of the transcripts from the test (390-nt) and control (290-nt) templates are indicated on the right.

Figure 2

Activated transcription from the premelted templates. (A) Premelted templates used for transcription. The DNA sequences of the premelted regions and the position of the initiation site (+1) are indicated. (B) Basal (−) or activated (+) transcription from the premelted templates in the presence of wild-type TFIIH (WT) or TFIIH with a mutated ERCC3 (K346A), respectively. (C) Quantification of the relative levels of transcription. The data from three independent experiments are represented as means ± SD. The unit for transcriptional levels is arbitrary.

Figure 3

Effect of ERCC3 on promoter escape from premelted templates. Transcription reactions were performed by using the template −9/+2 in the presence of ATP and CTP (lanes 1–4), or ATP, CTP, UTP, and 3′-O-methyl GTP (lanes 5–8). The position of the initiation product (ApC) is indicated on the left, and the short transcripts are indicated on the right. WT, wild type.

Figure 4

Effect of GAL4-VP16 on promoter escape from nonpremelted templates. (A) DNA sequences of pG5HMC2AT+nG templates. The G residue in bold indicates the position at which transcription terminates by 3′-O-methyl GTP. WT, wild type. (B) Short transcripts in the early phase of basal and activated transcription. The positions of transcripts terminated at the G residue are indicated. Asterisks indicate the transcripts from 3 to 11 nt in length that correspond to the same transcripts indicated in Fig. 3. (C) Quantification of the short transcripts. The values are means ± SD of the relative molar amount of each transcript from three independent experiments. (D) Fold activation for each pair of basal and activated transcription. The values are means ± SD from three independent experiments.

Figure 5

Hydrolyzable βγ bond of ATP is required throughout promoter escape in activated transcription. (A) DNA sequences of pG5HMC2AT+nA templates. The first A residue in the G-less cassette is indicated in bold. WT, wild type. (B) Diagram illustrating the experimental design for stalling RNAPII before each A residue in transcription reactions. The templates, transcription factors, and nucleotides were added at the indicated time points. Transcription was performed on each template in the absence (−) or presence (+) of GAL4-VP16 and PC4. (C) Quantification of transcripts. The values are means ± SD from three independent experiments. (D) Fold activation for each template. The values are means ± SD from three independent experiments.

Figure 6

Kinetic analysis of promoter escape in activated transcription. (A) Diagram illustrating the experimental design for in vitro transcription reactions. Ten-fold scales of transcription reactions were performed by using pG5HMC2AT+20G (template 20G), and aliquots were withdrawn at the indicated time points (60 + t). (B) Time course of promoter escape in the presence (activation, Top) or absence (basal, Middle and Bottom) of GAL4-VP16 and PC4. The Bottom (basal, long) indicates the 4-fold longer exposure of the Middle. The position of the 19-nt transcript is indicated. (C) Quantification of the time course of promoter escape. k_a(obs) and k_b(obs) indicate the observed rate constants for activated transcription and basal transcription, respectively. The values (means ± SD) at each time point from three independent experiments are plotted.

Figure 7

Model for transcriptional activation by GAL4-VP16. GAL4-VP16, together with PC4, stimulates the steps before initiation and the promoter escape step. The stimulation of promoter escape occurs through the ERCC3 helicase activity of TFIIH, possibly by directing nonproductive transcription complexes into the productive pathway. The productive complexes enter into elongation and produce the full-length transcript, whereas the nonproductive complexes may only produce abortive transcripts.