Real-Time Observation of Transcription Initiation and Elongation on an Endogenous Yeast Gene

Abstract

Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur in vivo. Here, we describe a method of fluctuation analysis of fluorescently labeled RNA to measure dynamics of nascent RNA—including initiation, elongation, and termination—at an active yeast locus. We find no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle. By measuring the abundance and intranuclear mobility of an upstream transcription factor, we observe that the gene firing rate is directly determined by trans-activating factor search times.

Two DNA-encoded strategies for increasing expression with opposing effects on promoter dynamics and transcriptional noise

“…This suggestion is supported by a study showing that promoter firing rate is dictated by the time required for a transcription factor to find its gene within the nucleus (Larson et al 2011). In contrast, when strengthening a transcription factor binding site, our results suggest that the increase in expression is primarily achieved by an increase in the average number of transcripts produced during the active state.…”

“…Consequently, unraveling the sources that underlie expression variability has been a topic of considerable interest. Several studies demonstrated that genes are transcribed in bursts (Ross et al 1994;Blake et al 2003;Golding et al 2005;Larson et al 2011;Suter et al 2011), such that the expression variability due to transcription is determined by the frequency with which the bursts occur (burst frequency) and the number of transcripts produced per burst (burst size) . These parameters are determined in part by fluctuations in trans factors that are extrinsic to the promoter, such as the concentrations of the regulating transcription factors and RNA polymerases (Elowitz et al 2002;Kaern et al 2005).…”

Imaging Translation in Single Cells Using Fluorescent Microscopy

“…An alternate strategy that measures translation-dependent changes to reporter mRNAs themselves may allow translation to be monitored with the single molecule resolution in living cells. Besides the well-established fluorescent labeling method based on the MS2 bacteriophage coat protein, fluorescent RNA labeling has also been shown using the PP7 bacteriophage coat protein, the lN peptide and the U1A spliceosomal protein (Brodsky and Silver 2000;Takizawa and Vale 2000;Daigle and Ellenberg 2007;Larson et al 2011). Because all four RNA-protein complexes are orthogonal, it should be possible, in principle, to specifically label a reporter mRNA in both the coding region as well as the 3 0 UTR with spectrally distinct fluorescent proteins.…”

Tuning gene expression variability and multi-gene regulation by dynamic transcription factor control

“…The rate at which a nascent RNA escapes from the transcription site (k esc ) is given by the RNA dwell-time which includes elongation and termination, leading to k esc = (elongation time) -1 + (termination time) -1 . The termination time was set to the literature value of 70 seconds 9 and the elongation time was set to 100 seconds based on the transcript length of 2000 bases and an average elongation rate of 20 bases per second 9 . We found that the predicted dynamics of nascent RNA accumulation closely resemble the experimental data ( Supplementary Fig.…”

“…Eukaryotic genes are usually 103 monocistronic and thus, promoter libraries are typically used to adjust relative expression 104 levels 9 . Hence, we built a set of light-responsive promoters differing in the promoter 105 backbone and EL222 binding site number.…”