In Vivo Transcription Kinetics of a Synthetic Gene Uninvolved in Stress-Response Pathways in Stressed Escherichia coli Cells

Abstract

The fast adaptation of Escherichia coli to stressful environments includes the regulation of gene expression rates, mainly of transcription, by specific and global stress-response mechanisms. To study the effects of mechanisms acting on a global level, we observed with single molecule sensitivity the effects of mild acidic shift and oxidative stress on the in vivo transcription dynamics of a probe gene encoding an RNA target for MS2d-GFP, under the control of a synthetic promoter. After showing that this promoter is uninvolved in fast stress-response pathways, we compared its kinetics of transcript production under stress and in optimal conditions. We find that, following the application of either stress, the mean rates of transcription activation and of subsequent RNA production of the probe gene are reduced, particularly under oxidative stress. Meanwhile, the noise in RNA production decreases under oxidative stress, but not under acidic shift. From distributions of intervals between consecutive RNA productions, we infer that the number and duration of the rate-limiting steps in transcription initiation change, following the application of stress. These changes differ in the two stress conditions and are consistent with the changes in noise in RNA production. Overall, our measurements of the transcription initiation kinetics of the probe gene indicate that, following sub-lethal stresses, there are stress-specific changes in the dynamics of transcription initiation of the probe gene that affect its mean rate and noise of transcript production. Given the non-involvement of the probe gene in stress-response pathways, we suggest that these changes are caused by global response mechanisms of E. coli to stress.

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

“…Then, we observed cells with the chromosome integrated gene at 30 °C, when subject to Topotecan, an inhibitor of Topoisomerase I activity 58 , 59 (Methods). Topoisomerase I releases negative, but not positive supercoiling 60 . We expect these cells to exhibit a similar behavior as when at 10 °C, which Fig.…”

“…It took, on average, ~3 minutes to move cells from the incubator to the microscope, assemble the imaging chamber with slides and cells, and start the observation. Images were then analyzed by the software iCellFusion 83 and CellAging 60 , 84 , 85 (example Figs S3 and S4 ).…”

Kinetics of the cellular intake of a gene expression inducer at high concentrations

“…5,6,15,23,24 This may be because, under these conditions, the recently reported phenomenon of buildup of positive supercoiling with transcription events, which may lead to short-length transcriptional bursts, 27 is too weak due to the 'lack of topological barriers' on the plasmid. Namely no significant 'bursts' in transcription were reported.…”

“…Namely no significant 'bursts' in transcription were reported. 5,6,15,23,24 This may be because, under these conditions, the recently reported phenomenon of buildup of positive supercoiling with transcription events, which may lead to short-length transcriptional bursts, 27 is too weak due to the 'lack of topological barriers' on the plasmid. 27 Further, this phenomenon is expected to affect tangibly only highly expressed genes, while in our measurements we recorded mean intervals between transcription events longer than 1000 s under full induction.…”

Sequence-dependent model of genes with dual σ factor preference

“…To reduce the model, we assume that the numbers of RNAP.σ 70 and RNAP.σ 38 in the cells are significantly larger than 1, which is expected. Also, we assume that the ratelimiting steps in transcription initiation (during which a promoter is occupied and thus new events cannot start [39,40]) are shorter in time-length than the intervals between transcription events. If so, for any σ 70+38 gene:…”