Dual Roles for Transcription Factor IIF in Promoter Escape by RNA Polymerase II

Abstract

Transcription factor (TF) IIF is a multifunctional RNA polymerase II transcription factor that has well established roles in both transcription initiation, where it functions as a component of the preinitiation complex and is required for formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts, and in transcription elongation, where it is capable of interacting directly with the ternary elongation complex and stimulating the rate of transcription. In this report, we present evidence that TFIIF is also required for efficient promoter escape by RNA polymerase II. Our findings argue that TFIIF performs dual roles in this process. We observe (i) that TFIIF suppresses the frequency of abortive transcription by very early RNA polymerase II elongation intermediates by increasing their processivity and (ii) that TFIIF cooperates with TFIIH to prevent premature arrest of early elongation intermediates. In addition, our findings argue that two TFIIF functional domains mediate TFIIF action in promoter escape. First, we observe that a TFIIF mutant selectively lacking elongation activity supports TFIIH action in promoter escape, but is defective in suppressing the frequency of abortive transcription by very early RNA polymerase II elongation intermediates. Second, a TFIIF mutant selectively lacking initiation activity is more active than wild type TFIIF in increasing the processivity of very early elongation intermediates, but is defective in supporting TFIIH action in promoter escape. Taken together, our findings bring to light a function for TFIIF in promoter escape and support a role for TFIIF elongation activity in this process. TFIIF1 was originally identified by its requirement in promoter-specific transcription initiation by RNA polymerase II. Mammalian TFIIF is a heterodimer composed of ϳ30 kDa (RAP30) and ϳ74 kDa (RAP74) subunits (1). Substantial evidence suggests that TFIIF performs multiple functions in transcription initiation. Although TFIIF is not essential for selective binding of RNA polymerase II to promoters, TFIIF functions as an integral component of the preinitiation complex and strongly stabilizes binding of polymerase to TFIID and TFIIB at the promoter (2-4). In addition, TFIIF is required for entry of TFIIE and TFIIH into the preinitiation complex (2, 4 -6), for subsequent open complex formation catalyzed by the TFIIH DNA helicase, and for synthesis of the first phosphodiester bond of nascent transcripts (7-9). Although it is presently not known whether TFIIF merely functions as a scaffold for binding of TFIIH to the preinitiation complex or whether TFIIF actively participates in formation of the open complex, evidence suggests that TFIIF interacts with promoter DNA in the preinitiation complex and induces a dramatic conformational change that results in wrapping of DNA for nearly a full turn around RNA polymerase II and may facilitate formation of the open complex (10 -12).In addition to its role in transcription initiation, TFIIF is also capable of potentl...

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

“…Previous studies have shown (i) that TFIIH and an ATP(dATP) cofactor are dispensable for initiation by RNA polymerase II from the Ad(Ϫ9͞Ϫ1) promoter but are required for efficient promoter escape (3), and (ii) that TFIIH and an ATP(dATP) cofactor are not required for either transcription initiation or promoter escape by RNA polymerase II from the Ad(Ϫ9͞ϩ9) promoter (5). As illustrated in Fig.…”

“…Results of previous experiments suggest that a TFIIF activity required for transcription initiation presents an impediment to efficient promoter escape and is at least partly responsible for inducing the premature arrest of very early RNA polymerase II elongation intermediates (5). Together with results of cross-linking experiments suggesting that TFIIF and TFIIE promote tight wrapping of DNA around RNA polymerase II by making contacts in the initiation complex with promoter DNA upstream of the TATA box and downstream of the transcriptional start site (10,11,41), this observation raises the possibility that protein-DNA contacts between promoter DNA and TFIIF and TFIIE might present an impediment to promoter escape.…”

“…The resulting DNA fragment, which contains sequences that extend 77 bp upstream and 367 bp downstream from the transcriptional start site, was gel purified before use in transcription reactions. The Ad(Ϫ9͞Ϫ1) and Ad(Ϫ9͞ϩ9) templates were prepared as described (3,5).…”

“…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).…”

Conserved architecture of the core RNA polymerase II initiation complex

“…Indeed, this domain binds DNA and is required for initiation 21 , and TFIIF suppresses abortive initial transcription 22 . The proximity of the Tfg2 WH domain to TFIIB indicates how TFIIF could stabilize TFIIB on Pol II during initial transcription 23 .…”