The formation of the RNA polymerase II (Pol II) initiation complex was analyzed using site-specific protein-DNA photo-cross-linking. We show that the RAP74 subunit of transcription factor (TF) IIF, through its RAP30-binding domain and an adjacent region necessary for the formation of homomeric interactions in vitro, dramatically alters the distribution of RAP30, TFIIE, and Pol II along promoter DNA between positions -40 and +26. This isomerization of the complex, which requires both TFIIF and TFIIE, is accompanied by tight wrapping of the promoter DNA for almost a full turn around Pol II. Addition of TFIIH enhances photo-cross-linking of Pol II to a number of promoter positions, suggesting that TFIIH tightens the DNA wrap around the enzyme. We present a general model to describe transcription initiation.

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 (RNA-PII). 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.RNA polymerase II (RNAPII) is the multisubunit enzyme that synthesizes eukaryotic mRNA. The two largest RNAPII subunits, Rpb1 and Rpb2, which are homologous to the ␤Ј and ␤ subunits of prokaryotic RNA polymerases, possess the catalytic activity of the enzyme (5, 71). Rpb1 contains a repeated heptapeptide in its carboxyl-terminal domain (CTD) that becomes highly phosphorylated during the passage from initiation to elongation of transcription (8). Initiation of transcription by RNAPII in vitro requires a set of general transcription initiation factors including TATA-binding protein (TBP), transcription factor IIB (TFIIB), TFIIE, TFIIF, and TFIIH (21,44). TBP binds to the TATA element of promoters and induces an ϳ90°bend in the DNA helix (27,30). TFIIB associates with the TBP-promoter complex (1, 37) and makes promoter contacts on each side of the DNA bend centered on the TATA box (6, 31). TFIIF, which is composed of two subunits called RNAPII-associated proteins 74 and 30 (RAP74 and RAP30), tightly binds to RNAPII and allows the binding of the enzyme to a TBP-TFIIB-promoter complex (4, 16). TFIIE is also composed of two subunits, TFIIE56 and TFIIE34 (25,41), that stabilize the association of RNAPII with the preinitiation complex (50). A complex containing TBP, TFIIB, TFIIE, TFIIF, and RNAPII is capable of initiating transcription on a premelted linear template (23, 45, 61) and on a negatively supercoiled template (46,62,64). Both TFIIE and TFIIF have been shown to play a role in the TFIIH-independent melting of the promoter DNA around the transcription initiation site (TIS) (23,45). TFIIE is also required for the association of TFIIH with the preinitiation complex and the regulation of TFIIH activities (35,39,40). A complex containing TBP, TFIIB, TFIIE, TFIIF, TFIIH, and RNAPII is capable of melting promoter DNA in a region between nucleotides Ϫ9 and ϩ2 of a linear template (22,24,26,69). Open complex formation requires the hydrolysis of the ␤-␥ bond of ATP by the helicaseATPase activity of TFIIH (22,24).Mammalian TFIIH is a nine-subunit complex responsible both for the melting of the template DNA prior to initiation (54, 57) and during prom...

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) functions at multiple stages of transcription and is involved in the coupling of transcription to pre-mRNA processing. We have used site-specific protein-DNA photocross-linking to determine the position of the CTD along promoter DNA in the transcriptional pre-initiation complex. Comparison of the promoter contacts made by RNAP II with or without the CTD indicate that the CTD approaches promoter DNA downstream of the transcriptional initiation site between positions ؉16 and ؉26. Incubation of pre-assembled initiation complexes with antibodies to the CTD prior to UV irradiation led to specific photocross-linking of the IgG heavy chain to nucleotide ؉17, indicating that the CTD is accessible for protein-protein interactions in a complex containing RNAP II and the general initiation factors. In conjunction with previously published observations, our structural data are fully compatible with the notion that DNA wrapping around RNAP II places the CTD and the RNA exit channel into juxtaposition and provide a rationale for contacts between the SRB-mediator complex and core RNAP II observed in the RNAP II holoenzyme.The CTD 1 of Rpb1, the largest subunit of RNAP II, contains a seven-amino acid motif tandemly repeated 52 times in humans and 26 -27 times in yeast (reviewed in Ref. 1). This repeated heptapeptide has the consensus sequence Tyr-SerPro-Thr-Ser-Pro-Ser and is highly conserved among eukaryotic organisms. The CTD is essential for cell viability (2-5), and recent evidence indicates a central role for this domain in the coordination of the various enzymatic activities involved in mRNA formation including transcription, 5Ј-end capping, 3Ј-end formation, and pre-mRNA splicing (reviewed in Ref. 6).Two different forms of RNAP II exist in vivo, namely RNAP IIA and IIO. The IIA form is not phosphorylated on the CTD and preferentially enters the pre-initiation complex, whereas the IIO form is extensively phosphorylated on the CTD and is found in the elongation complex (reviewed in Ref. 7). Conversion of IIA to IIO occurs either concomitant with, or shortly after, the passage from initiation to elongation and involves extensive CTD phosphorylation (8, 9). These observations support the notion that phosphorylation of the CTD regulates the conversion of RNAP II from the form involved in promoter recognition to that associated with the elongation complex. Consistent with a role of the unphosphorylated CTD in preinitiation complex formation, direct protein-protein interactions of the hypophosphorylated CTD with general initiation factors TBP (10), RAP74 (11), and TFIIE34 (11) have been reported. However, a form of RNAP II lacking the CTD (form IIB) is capable of transcriptional initiation at TATA box-containing promoters in vitro, but not at TATA-less promoters (12,13). A number of kinases, including the general initiation factor TFIIH, and one phosphatase that can regulate CTD phosphorylation have been identified (reviewed in Refs. 7 and ...

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 (RNA-PII). 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.

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