Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein-DNA photo-cross-linking

Abstract

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions -9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions -5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.

FIG. 1

Cross-linking of TBP, TFIIB, TFIIF (RAP74 and RAP30), TFIIE (TFIIE56 and TFIIE34), RNAPII (Rpb1, Rpb2, and Rpb5) and TFIIH (p89/XPB, p80/XPD, and p62) along promoter DNA. (A) Photoprobes derived from the adenovirus major late promoter. For each photoprobe, positions of the photoreactive (U) and radiolabeled (*) nucleotides are indicated. (B) Summary of cross-linking data. Specific cross-links that do (gray boxes) and do not (open boxes) require the presence of TFIIH are indicated. Black boxes indicate the new cross-links induced in the presence of ATP. A cross-linking signal was considered specific when its intensity was significantly higher in reactions containing TBP than in those lacking TBP (see text). A number of photoprobes that place the photonucleotide either upstream of position −62/−64 or downstream of position +35/+38 have also been used, but no significant cross-linking signals were obtained.

FIG. 2

Cross-linking of TFIIH subunits along promoter DNA. (A) Cross-linking of TFIIH subunits upstream of the TATA element, downstream of the TIS, and to position −5. Photo-cross-linking experiments using photoprobes −39/−40, −5, and +13 were performed with TFIIB, TFIIE, TFIIF, TFIIH, and RNAPII in either the presence (+) or absence (−) of TBP. In these experiments, a truncated form of RAP74 [RAP74(1-217)], which migrates at ∼35 kDa, was used to facilitate visualization of the cross-linking signals in the 50- to 100-kDa region of the gels. (B) Identification of affinity-labeled TFIIH subunits. Cross-linked polypeptides were immunoprecipitated (IP) with specific antibodies directed against recombinant p89/XPB and p80/XPD and analyzed by SDS-PAGE. The immunoprecipitated polypeptides comigrated with p89/XPB and p80/XPD controls that were electrophoresed on the gel and revealed by silver staining. p62 was identified according to its mobility in the cross-linking gels, which is indistinguishable from that of the silver-stained subunit but slightly different from that of TFIIE56. (C) Cross-linking of recombinant TFIIH. TFIIH reconstituted from its cloned subunits (rIIH9) was included in cross-linking experiments using photoprobe +13. p89/XPB carries a His tag (His-p89), and its mobility is slightly slower than that of the natural subunit.

FIG. 3

Cross-linking of p89/XPB using various RAP74 deletion mutants. Cross-linking experiments using photoprobes −39/−40, −5, and +13 were performed with TFIIB, TFIIE, TFIIH, RNAPII, RAP30, and RAP74(1-517), RAP74(1-217), and RAP74(1-172)] in the presence (+) or the absence (−) of TBP. RAP74(1-172) does not contain the homomeric interaction region 1 (HIR1), while RAP74(1-217) and RAP74(1-517) do. A summary of p89/XPB cross-linking using the RAP74 deletion mutants is shown in Table 1.

FIG. 4

Cross-linking of p89/XPB using various TFIIE34 deletion mutants. (A) Linear representation of TFIIE34 wild type (wt) and deletion mutants. The ability to support basal transcription (TX) in vitro is indicated by a plus sign. (B) Cross-linking experiments using photoprobe +13 were performed with TBP, TFIIB, TFIIE, TFIIF, and RNAPII in the presence of the various TFIIE34 deletion mutants (mut). Positions of the cross-linked TFIIE34 fragments are indicated by arrows on the gel. A summary of TFIIE34 deletion mutant cross-linking is provided in Table 2. TFIIE34(Δ4-75) cross-links have been confirmed by immunoprecipitation with an antibody raised against recombinant TFIIE34. (C) Cross-linking reactions using photoprobes +13 and +26 were performed as for panel B except that TFIIH was included in the reactions. A summary of p89/XPB cross-linking using the TFIIE34 deletion mutants is provided in Table 3.

FIG. 5

Nucleotide requirement for the new promoter contacts by RNAPII in the −9/+2 region. The cross-linking experiments using photoprobes −5 and +1 were performed in the presence of ATP, ATPγS, or GTP or in the absence of ribonucleoside triphosphate (NTP).

FIG. 6

Cross-linking of TFIIH with a mutation in p89/XPB associated with XP. (A) Cross-linking experiments using photoprobes −5 and +13 were performed in the absence or presence of TFIIH isolated either from HeLa cells or from patient cells with either a wild-type (IIH-XPBwt) or mutated [IIH-XPB(C-A)] TFIIH. (B) Runoff transcription activity of the various TFIIHs. Immunopurified wild-type (IIH-XPBwt) and mutated [IIH-XPB(C-A)] TFIIH were analyzed using Western blotting (WB) and in vitro transcription assays (Transcription). An SDS-polyacrylamide gel containing highly purified HeLa TFIIH is included for comparison. Positions of the TFIIH subunits and runoff transcript (309 nucleotides [nt]) are indicated. (C) DNA helicase activity of the various XPBs. rXPB, either wild type (rXPBwt), with a mutated ATP-binding site [rXPB(GKT)], or carrying the C-A mutation [rXPB(C-A)], was analyzed in a standard helicase assay. The positions of the heteroduplex substrate and the products of both 5′-3′ and 3′-5′ unwinding reactions are shown. −, reaction performed in the absence of XPB; Δ, reaction heated to serve as a positive control. rXPBs were analyzed by Western blotting (WB).

FIG. 7

(A) Proposed structure for the preinitiation complex containing TBP, TFIIB, TFIIF (F74 and F30), TFIIE (E56 and E34), TFIIH, and RNAPII. The relative positions of the various factors and RNAPII are as predicted from our cross-linking data and published observations from a number of laboratories (see Discussion). In the front view, the complex is shown with the promoter DNA between the TATA element and the TIS being placed in the direction of the view. (B) A model for the mechanism of promoter melting by TFIIH. TFIIH is in yellow, and its p89/XPB subunit is in orange. For a detailed description, see Discussion.