Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters

Abstract

SummaryTranscription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters—an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.

Timing polymerase pausing with TV-PRO-seq: dissecting the interplay of pausing duration and location, and gene expression

“…2D). Our finding of surprisingly short promoter proximal pausing appears to agree with recent studies hinting at rapid Pol II turnover in this region 23,24 . This suggests that, instead of pausing, this region rather features a high rate of abortive transcription, which might be a standard feature of metazoan Pol II transcription.…”

Global analyses of the dynamics of mammalian microRNA metabolism

“…Even at a production rate of 24 copies/cell per min (two standard deviations below the calculated rate), the rate of miR-21a production would be more than 2-fold faster than that of the most rapidly produced mRNA in NIH3T3 fibroblasts and comparable to that of pre-rRNA production from a single pre-rRNA locus in HeLa cells (Schwanhäusser et al 2011;Turowski and Tollervey 2015). When considering that miR-21 is transcribed from two alleles of a single locus and that RNA polymerase II (PolII) elongates with a rate constant of ~4.3 kb/min and has a footprint of ~40-50 nucleotides (Darzacq et al 2007;Darst et al 1991;Rice et al 1993;Krebs et al 2017), our results imply that at the calculated rate of production, the Mir21 locus is coated in elongating PolII at approximately 50% of its maximum density. Such efficient PolII recruitment is presumably challenging and greater then 2-fold more efficient recruitment would be impossible, which helps explain why some highly expressed miRNAs are transcribed from multiple genes-the extreme being miR-430, which makes up 99% of the miRNA in the early zebrafish embryo and is transcribed from an array of >90 genes (Wei et al 2012;Giraldez et al 2005).…”

Continuous transcription initiation guarantees robust repair of transcribed genes and regulatory regions in response to DNA damage

“…Recent advances in the field of transcription regulation point to the fact that activation of paused genes is mediated through switching from a premature termination state of Pol II at PPP sites to a processive elongation state 13,16,17 , implying that continuous initiation is required for fast transcriptional induction 16 . Our results, showing that persistent initiation guarantees a prolonged transcription-coupled NER, are functionally linked to the fact that DNA damage-triggered widespread PPP release of a given Pol II is sufficient to drive immediate initiation of the next Pol II (see Fig.…”

“…Signal-regulated phosphorylation of these factors and of serine 2 residue (S2P) of Pol II CTD by P-TEFb is required for productive elongation [13][14][15] . It recently emerged that, if this step does not occur rapidly, start-RNAs are terminated 14,16 , implying that Pol II turnover at PPP sites is high and that replenishment of Pol II engaged in early transcription is achieved by the continuous re-entry of pre-initiating Pol II into PICs 16,17 .…”