A Role for the TFIIH XPB DNA Helicase in Promoter Escape by RNA Polymerase II

Abstract

TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATPdependent activities: a 3 3 5 DNA helicase catalyzed by its XPB subunit, a 5 3 3 DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.TFIIH is a nine-subunit complex that possesses multiple catalytic activities, including DNA-dependent ATPase, DNA helicase, and a protein kinase that is capable of phosphorylating the carboxyl-terminal domain (CTD) 1 of the largest subunit of RNA polymerase II (1). The two largest TFIIH subunits are ATP-dependent DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes. The TFIIH-associated CTD kinase is a three-subunit subassembly, CDK-activating kinase (CAK), which is composed of the kinase/cyclin pair CDK7/cyclin H and the RING-H2 finger protein MAT1. TFIIH subunits are found in a variety of additional subassemblies, including a six-subunit complex (IIH6) containing XPB, XPD, p62, p52, p44, and p34, a five-subunit "core" complex (IIH5) containing XPB, p62, p52, p44, and p34, and a four-subunit XPD/CAK complex (2-6).TFIIH was initially identified by its requirement in transcription initiation by RNA polymerase II (7). Initiation is an ATP-dependent process that requires at minimum the five general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (8,9). Biochemical studies have shown that initiation in this minimal transcription system proceeds through multiple stages beginning with assembly of polymerase and all five general initiation factors into a closed preinitiation complex at the promoter (8, 9) and culminating in ATP-dependent formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts (10 -13). Evidence supporting a role for TFIIH DNA helicase activity in ATP-dependent formation of the open complex was initially suggested by studies indicating that both TFIIH and ATP are dispensible for initiation by RNA polymerase II from artificial promoters containing premelted transcriptional start sites and from promoters on negatively supercoiled DNA templates (14 -19).In addition to its requirement in transcription initiation, TFIIH is also required for efficient promoter escape by RNA polymerase II (18, 20 -22). Mechanistic studies have shown that a fra...

TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA

“…In previous studies (3,4,6,25), efficient promoter escape by RNA polymerase II was shown to depend strongly on the TFIIH XPB DNA helicase, an ATP(dATP) cofactor, and a region of promoter DNA extending Ϸ39-50 bp downstream from the transcriptional start site. In addition, we showed that transcription initiation by RNA polymerase II depends on a region of promoter DNA extending Ϸ23-39 bp downstream from the transcriptional start site when initiation depends on TFIIH and an ATP(dATP) cofactor.…”

“…Previous studies have shown that the TFIIH XPB DNA helicase functions at multiple steps to promote efficient transcription initiation and promoter escape by RNA polymerase II. The TFIIH XPB DNA helicase catalyzes ATP(dATP)-dependent formation of the open complex before synthesis of the first phosphodiester bond of nascent transcripts (2), and it is required to suppress premature arrest of very early RNA polymerase II elongation intermediates at promoterproximal sites Ϸ10-12 bp downstream of the transcriptional start site before their escape from the promoter (3)(4)(5).…”

The DNA replication factor MCM5 is essential for Stat1-mediated transcriptional activation

“…DNA helicases necessary for the initiation of transcription have been well defined. These helicases include the subunits of the transcription factor IIH complex, which specifically interact with RNA Pol II complex on the promoter (34,35). The DNA helicase involved in transcription elongation has remained a mystery.…”

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

“…TFIIH consists of nine subunits including excision repair cross-complementing (ERCC) 3 (XPB), ERCC2 (XPD), and MO15, which have 3Ј-5Ј helicase, 5Ј-3Ј helicase, and C-terminal domain kinase activities, respectively (19,20). Both promoter opening and promoter escape require the ERCC3 helicase activity and (d)ATP hydrolysis (21,22), which can be circumvented by the negative supercoiling of the template DNA (18). In addition to the role in basal transcription, TFIIH is critically important for activated transcription.…”