Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

Abstract

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Keywords: DNA helicase; DNA unwinding; transcription initiation.

Fig. 1.

ATP-dependent helicase and translocase activities of TFIIH. (A) Helicase and triple helix substrates. Helicase substrates are designed to distinguish 5′ → 3′ and 3′ → 5′ enzyme polarity. The short displaced oligonucleotide is fluorescently labeled for visualization. The triplex substrate is formed via Hoogsteen base pairs between the 22 nucleotide (nt) ³²P-labeled triplex forming oligo (TFO) and the purine rich strand of the PCR generated duplex. (B) Helicase assay testing ATP-dependence and polarity of WT TFIIH or TFIIH containing the ATPase mutant Rad3-E236Q. (C) Triplex disruption assay testing ATPase requirements for triplex displacement with either WT TFIIH or TFIIH containing the ATPase defective mutants Rad3-E236Q or Ssl2-E489Q.

Fig. 2.

Template requirements for triplex disruption by TFIIH. (A) Triple helix substrates used to monitor TFIIH translocation. TFO22 was generated by annealing the 22-nt TFO to the 22-bp triplex target sequence. This smaller triplex was somewhat less stable than the 142-bp triplex DNA, likely due to no additional dsDNA. To make the 142-bp and circular triplex templates, the TFO was annealed to a PCR-generated 142-bp duplex or a 3.2-kb plasmid containing the triplex target sequence. (B) Triplex disruption assay using the TFO22 triple helix template and the TFIIH derivative Rad3 E236Q. (C) Time course of triplex disruption comparing the circular 3.2-kb plasmid triplex and the linear 142-bp triplex templates. Intact triplex was measured at each time point and quantified by comparison with a standard curve. The percent disruption of each triplex is indicated. ATP and dATP both function in the TFO displacement assay (compare with Fig. 1C) and in OC formation (7).

Fig. 3.

Polarity of Ssl2 catalyzed TFIIH translocation. (A) Triple helix substrates designed to test the polarity of Ssl2 translocation. Top and bottom strand triplexes are annealed as shown using PCR-generated duplex DNA and the TFO. PAGE-purified oligonucleotides were annealed with the TFO to generate the biotin-containing triplexes where a 5′-biotin labeled oligonucleotide is positioned immediately upstream of the triplex on the top or bottom duplex strand. (B) Triplex disruption assay comparing templates with the TFO annealed to either the top or bottom strand of the duplex. Either WT or Rad3 E236Q TFIIH was used as indicated. (C) Time course of triplex disruption from biotin templates by TFIIH (Rad3-E236Q). Reactions were incubated for the indicated times with 1 mM dATP at 26 °C and quantified for intact triplex remaining. (D) Quantitation of results in C.

Fig. 4.

In vitro transcription from promoters with DNA backbone blocks on the nontemplate DNA strand. (A) HIS4 promoter derivatives with Cy3 DNA backbone insertions. DNAs were constructed from synthetic oligonucleotides and contained Cy3 positioned 37, 41, or 46 bp downstream from HIS4 TATA. Transcription was assayed by primer extension using the lacI oligonucleotide as shown. (B) In vitro transcription using the reconstituted yeast Pol II system and the promoters in A. Lanes 1–4 contain the indicated amounts of a transcription reaction used to generate a standard for quantitation of transcription signals relative to the unmodified template. Lanes 5–7 are transcription reactions using the indicated Cy3 templates. Percent transcription relative to the unblocked template is indicated. No transcription is observed when TFIIH is omitted (lanes 8–11). (C) HIS4 promoter derivatives identical to those in A except that they contain the 12 nucleotide single-stranded DNA bubble as shown. This bubble allows transcription initiation by Pol II in the absence of other general factors (33). (D) In vitro transcription using purified yeast Pol II on the bubble templates and assayed by primer extension. Lanes 1–4 contain the indicated amounts of a transcription reaction using the non-Cy3 bubble template. Lanes 5–7 are transcription reactions using the Cy3-modified bubble templates. Lanes 8–9 are mock transcription reactions lacking Pol II and assayed by primer extension. The products marked by * are due to primer extension of the DNA template, which is blocked by Cy3. These blocked products are not visible in B, lanes 9–11, as they are significantly longer than the RNA products initiated at the HIS4 TSS.

Fig. 5.

Dependence of the ATP hydrolysis rate on DNA length. In all reactions, TFIIH (Rad3 E236Q) was preincubated with DNA for 40 min before ATP addition. (A) The phosphate released (P_i) as a function of time for a range of DNA lengths shows a linear dependence that can be fit to give a steady-state rate of ATP hydrolysis. (B) The steady-state rate of ATPase hydrolysis as a function of DNA length and DNA concentration. Concentration is plotted as micromolar-base pairs (µMbp), and the data are fit to Michaelis–Menten curves, allowing for the extraction of K_M and V_max parameters. The V_max of the plasmid data and its associated error are shown by two dashed horizontal lines. (C) K_M as a function of DNA length. (D) V_max as a function of DNA length. Fit shown using analytical expression for the dependence of V_max on DNA length for the model described in the text with a step size of 1 bp and a processivity of 10 bp (SI Materials and Methods).

Fig. 6.

dsDNA translocase model for open complex formation. The Ssl2 subunit of TFIIH tracks in the 5′ → 3′ direction on the nontemplate promoter DNA strand (red). Because TFIIH movement is constrained due to interaction with other PIC components, translocation results in insertion and rotation of promoter DNA into the Pol II cleft, leading to DNA unwinding (right arrows indicate rotation and direction of dsDNA movement). The short persistence length of Ssl2/TFIIH predicts that the OC state is unstable, in agreement with experimental observations (9, 10).