Estimating RNA numbers in single cells by RNA fluorescent tagging and flow cytometry.

Estimating the statistics of single-cell RNA numbers has become a key source of information on gene expression dynamics. One of the most informative methods of in vivo single-RNA detection is MS2d-GFP tagging. So far, it requires microscopy and laborious semi-manual image analysis, which hampers the amount of collectable data. To overcome this limitation, we present a new methodology for quantifying the mean, standard deviation, and skewness of single-cell distributions of RNA numbers, from flow cytometry data on cells expressing RNA tagged with MS2d-GFP. The quantification method, based on scaling flow-cytometry data from microscopy single-cell data on integer-valued RNA numbers, is shown to readily produce precise, big data on in vivo single-cell distributions of RNA numbers and, thus, can assist in studies of transcription dynamics.

Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures.

Temperature shifts trigger genome-wide changes in Escherichia coli's gene expression. We studied if chromosome integration impacts on a gene's sensitivity to these shifts, by comparing the single-RNA production kinetics of a PLacO3O1 promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated PLacO3O1 has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.

Regulation of asymmetries in the kinetics and protein numbers of bacterial gene expression.

Genetic circuits change the status quo of cellular processes when their protein numbers cross thresholds. We investigate the regulation of RNA and protein threshold crossing propensities in Escherichia coli. From in vivo single RNA time-lapse microscopy data from multiple promoters, mutants, induction schemes and media, we study the asymmetry and tailedness (quantified by the skewness and kurtosis, respectively) of the distributions of time intervals between transcription events. We find that higher thresholds can be reached by increasing the skewness and kurtosis, which is shown to be achievable without affecting mean and coefficient of variation, by regulating the rate-limiting steps in transcription initiation. Also, they propagate to the skewness and kurtosis of the distributions of protein expression levels in cell populations. The results suggest that the asymmetry and tailedness of RNA and protein numbers in cell populations, by controlling the propensity for threshold crossing, and due to being sequence dependent and subject to regulation, may be key regulatory variables of decision-making processes in E. coli.

Effects of σ factor competition are promoter initiation kinetics dependent.

In Escherichia coli, the expression of a σ factor is expected to indirectly down-regulate the expression of genes recognized by another σ factor, due to σ factor competition for a limited pool of RNA polymerase core enzymes. Evidence suggests that the sensitivity of genes to indirect down-regulation differs widely. We studied the variability in this sensitivity in promoters primarily recognized by RNAP holoenzymes carrying σ(70). From qPCR and live single-cell, single-RNA measurements of the transcription kinetics of several σ(70)-dependent promoters in various conditions and from the analysis of σ factors population-dependent models of transcription initiation, we find that, the smaller is the time-scale of the closed complex formation relative to the open complex formation, the weaker is a promoter's responsiveness to changes in σ(38) numbers. We conclude that, in E. coli, a promoter's responsiveness to indirect regulation by σ factor competition is determined by the sequence-dependent kinetics of the rate limiting steps of transcription initiation.