Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites ‘dTSS’, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS ≤ 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.
Escherichia coli uses the ability of σ factors to recognize specific DNA sequences in order to quickly control large gene cohorts. While most genes respond to only one σ factor, approximately 5% have dual σ factor preference. The ones in significant numbers are ‘σ70+38 genes’, responsive to σ70, which controls housekeeping genes, as well as to σ38, which activates genes during stationary growth and stresses. We show that σ70+38 genes are almost as upregulated in stationary growth as genes responsive to σ38 alone. Also, their response strengths to σ38 are predictable from their promoter sequences. Next, we propose a sequence- and σ38 level-dependent, analytical model of σ70+38 genes applicable in the exponential, stationary, and in the transition period between the two growth phases. Finally, we propose a general model, applicable to other σ factors as well. This model can guide the design of synthetic circuits with sequence-dependent sensitivity and plasticity to transitions between the exponential and stationary growth phases.Author SummaryPresent challenges in Synthetic Biology include the design of genetic circuits that are robust to growth phase transitions and whose responsiveness is sequence-dependent, and, thus predictable prior to design. We present and validate an empirical-based, sequence-dependent analytical model of E. coli genes with dual responsiveness to the regulators σ70 and σ38. These genes, supported by our sequence-dependent model, could become building blocks for synthetic genetic circuits functional in both the exponential and the stationary growth phases.
Adaptation to cold shock (CS) is a key survival skill of gut bacteria of warm-blooded animals. In E. coli, this skill emerges from a complex transcriptional program of multiple, timely-ordered shifts in gene expression. We identified short-term, cold shock repressed (CSR) genes by RNA-seq and provide evidence that their variability in evolutionary fitness is low and that their responsiveness to cold emanates from intrinsic features. Given that their single-cell variability in protein numbers increases after CS, we hypothesized that the responsiveness of a large portion of CSR genes is triggered by the high propensity for transcription locking due to positive supercoiling buildup (PSB). We then proposed a model of this phenomenon and, in support, show that nearly half of CSR genes are highly responsive to Gyrase inhibition. Also, their response strengths to CS and Gyrase inhibition correlate and most CSR genes increase their single-cell variability in protein numbers. Further, during CS, the cells' nucleoid density increases (in agreement with increased numbers of positive supercoils), their energy levels become depleted (while the resolving of positive supercoils is ATP dependent), and the colocalization of Gyrases and the nucleoid increases (in agreement with increased time length for resolving supercoils). We conclude that high sensitivity to PSB is at the core of the short-term, cold shock responsive transcriptional program of E. coli and propose that this gene feature may be useful for providing temperature sensitivity to chromosome-integrated synthetic circuits.
The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the gene’s numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.
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