Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

Abstract

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the transcription factor Gal4 with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell times sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model where multiple polymerases initiate during a burst as long as the transcription factor is bound to DNA, and a burst terminates upon transcription factor dissociation.

Alternative transcription cycle for bacterial RNA polymerase

“…Antisense transcription, which produces RNAs that have sequences at least in part complementary to ordinary sense gene transcripts, has been observed in organisms from bacteria to humans and is typically initiated from locations throughout the entire genome [9][10][11][12] . While the global biological significance of this pervasive antisense transcription has been questioned 13,14 , antisense RNA production has demonstrated roles in regulating expression of many individual sense genes [15][16][17][18][19][20][21][22] . The origins of antisense transcripts are incompletely understood.…”

Inference and uncertainty quantification of stochastic gene expression via synthetic models

“…The stochasticity of biological processes at the single-cell level is one of the major paradigm shifts of twenty-first-century biology [1][2][3]. Modern experimental methods, ranging from advanced microscopy to single-cell sequencing [4][5][6], have confirmed and detailed the pervasiveness of stochasticity in cellular biology. While these discoveries open new perspectives on the fundamental functioning of living systems, they also create novel challenges towards the development of mathematical models of biological processes, accentuating the role of statistical inference and uncertainty quantification in any modelling effort.…”

Alternative transcription cycle for bacterial RNA polymerase

“…Antisense transcription, which produces RNAs that have sequences at least in part 15 complementary to ordinary "sense" gene transcripts, has been observed in organisms from bacteria to humans and is typically initiated from locations throughout the entire genome (David et al, 2006;Georg and Hess, 2018;He et al, 2008;Irnov et al, 2010). While the global biological significance of this pervasive antisense transcription has been questioned (Lloréns-Rico et al, 2016;Raghavan et al, 2012), antisense RNA production has demonstrated roles in 20 regulating expression of many individual sense genes (Callen et al, 2004;Donovan et al, 2018;Georg and Hess, 2011;Lenstra et al, 2015;Sedlyarova et al, 2016;Shearwin et al, 2005;Tudek et al, 2015;Waters and Storz, 2009). The origins of antisense transcripts are incompletely understood.…”