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

• Published in: Journal of molecular biology 2020-06, Vol.432 (14), p.4049-4060
• Main Authors: Ly, Elina, Powell, Abigail E., Goodrich, James A., Kugel, Jennifer F.
• Format: Article
• Language: English
• Subjects: DNA - genetics; Fluorescence; general transcription factor; Humans; Kinetics; Promoter Regions, Genetic - genetics; RNA - biosynthesis; RNA - genetics; RNA polymerase II; RNA Polymerase II - genetics; single molecule; Single Molecule Imaging - methods; TIRF; transcription; Transcription Factor TFIIB - genetics; Transcription, Genetic - genetics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2020.05.005

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 for Pol II to successfully escape the promoter as initiating complexes transition into elongation complexes. [Display omitted] •Single-molecule studies reveal heterogeneity in transcription complexes.•There is a tight correlation between release of TFIIB and active complexes.•The release of TFIIB is independent of the BRE promoter element.•TFIIB release from active complexes is triggered at two distinct points.•TFIIB dissociates with a defined rate as Pol II continues transcribing the template.