A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting

Abstract

Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells.

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

“…Consistent with our data, yeast TBP occupies the HSC82 heat shock promoter for 60 s in vivo (58). In human tissue culture cells, RNA polymerase II loads onto promoters approximately every 4 to 9 s to form convoys on an active gene (23). These bursts of RNA polymerase II convoys last for approximately 100 s before the promoter becomes inactive again (23).…”

“…Interestingly, the duration of RNA polymerase II convoys on the mutant TATA promoter is only slightly less than that on a wild-type TATA template (ϳ60 to 80 s) (23). Our study also revealed infrequent p53-mediated binding of TFIID on our mutant core promoter DNA lasting for a significantly long time (ϳ64 s) (Fig.…”

“…In human tissue culture cells, RNA polymerase II loads onto promoters approximately every 4 to 9 s to form convoys on an active gene (23). These bursts of RNA polymerase II convoys last for approximately 100 s before the promoter becomes inactive again (23). Therefore, it is tempting to speculate that the long-lived TFIID residency (ϳ100 s) on our native target promoters could be related to RNA polymerase II convoy duration (ϳ100 s) and transcriptional bursting.…”

“…TFIID can also direct infrequent loading of RNA polymerase II convoys on mutant TATA-containing promoters, leading to bursts of transcription (23). Interestingly, the duration of RNA polymerase II convoys on the mutant TATA promoter is only slightly less than that on a wild-type TATA template (ϳ60 to 80 s) (23).…”

“…Another single-molecule report uncovered TFIIB's dynamic binding to TFIID at the promoter (22). Additional live-cell studies also suggested that the PIC composition is extraordinarily dynamic, with the loading and promoter escape of RNA polymerase II occurring approximately every 4 to 9 s during bursts of transcription (23). An explanation of how activators such as p53 regulate TFIID's dynamic binding to native target DNA remains elusive.…”