Release of Human TFIIB from Actively Transcribing Complexes Is Triggered upon Synthesis of 7- and 9-nt RNAs

Abstract

RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription.Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important to maintain the transcriptional activity of Pol II as initiating complexes transition into elongation complexes.

Structural visualization of transcription initiation in action

“…The above result generally agrees with earlier biochemical studies that are not fully consistent with each other. For example, by detecting changes in the transcription bubble, complex stability, or dissociation of GTFs (mainly TFIIB), previous studies showed that the functional transition occurs when RNA reaches 7 ( 35 ), 8 ( 34 ), 9 ( 37 ), 10 ( 33 ), 7 or 9 ( 38 ), 12 or 13 ( 63 ), or more than 9 or 10 nt ( 36 , 57 ). This apparent discrepancy may result from the heterogeneity of transcription initiation, variations in promoters and experimental contexts, and limitations of the detection method (see supplementary text).…”

“…5D). Moreover, TFIIB dissociates from Pol II before RNA reaches 10 nt in length ( 38 , 39 ). Previous studies in yeast Pol II have proposed that steric clashes between RNA and the B-ribbon may lead to TFIIB dissociation ( 44 , 46 ).…”

“…Cryo-EM maps of TC10 to TC17 show similar organization of the EEC (Fig. 2, A, B, and D; (38,39,(63)(64)(65). Thus, Pol II is no longer restrained by the upstream promoter-bound GTFs and is free to move forward and escape the promoter.…”

“…When RNA is 7 to 10 nt long, the transcription bubble expands up to 17 or 18 nt, followed by an abrupt bubble collapse (33)(34)(35)(36)(37). Pol II will eventually break its contact with GTFs, escape from the promoter, and form an early elongation complex (EEC) (38,39). Although structures of reconstituted ITCs have been reported (40)(41)(42)(43)(44)(45)(46), these complexes were not started from a PIC but instead were assembled on artificial RNA-DNA hybrids that were formed by a mismatched transcription bubble in which the template strand paired with a synthetic complementary RNA.…”

“…The remaining GTFs, including TFIID (with TBP), TFIIA, TFIIB, and TFIIH, are either completely dissociated from or flexibly tethered to Pol II through the associations of TFIIF, TFIIE, and/or the exposed promoter elements. The flexibility of GTFs in the EEC may also explain seemingly inconsistent observations of GTFs on transcribing complexes, which vary under different experimental conditions(38,39,(63)(64)(65). Thus, Pol II is no longer restrained by the upstream promoter-bound GTFs and is free to move forward and escape the promoter.…”