Direct observation of frequency modulated transcription in single cells using light activation

Abstract

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.DOI: http://dx.doi.org/10.7554/eLife.00750.001

The nonlinear dynamics and fluctuations of mRNA levels in cross-talking pathway activated transcription

“…However, there has been few studies observing the correlation between an upstream transcription activation and a downstream transcription rate (Larson, 2011). In a recent study, Larson et al (2013) developed a single molecule approach for measuring the dynamical transcription activity of genes responsive to the activation of steroid-receptors. They demonstrated how the ensemble steroid dose response arises from the stochastic behavior of individual genes.…”

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

“…However, the timing and coordination of these interactions remained elusive. Initial biochemical characterization suggested that transcription initiation may take 30 min to 1 h in vitro (Hawley and Roeder 1987), and similar or longer time scales hold true for transcription initiation in the cell as measured by the latest single-molecule imaging-based technologies (Larson et al 2013). On the other hand, much shorter residence times have been reported for gene-specific activators (Chen et al 2014) and the GTF TFIIB in living cells (Chen et al 2002).…”

Transcriptional refractoriness is dependent on core promoter architecture

“…Burst size, that is, the number of transcripts per burst, and burst frequency, that is, the number of transcriptional bursts per time unit are gene specific and appear to depend on the promoter architecture, such as the presence of a CAAT box, a TATA box, the size of the nucleosome-free region as well as the location and number of transcription factor binding sites [7][8][9][10][11][12] . Burst sizes of between 1 and 450 transcripts per burst have been observed followed by periods of inactivity of up to several hours 7,8,[12][13][14][15][16][17][18][19] .…”