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

posted on 30.09.2014, 02:57 by Anantha-Barathi Muthukrishnan, Antti Martikainen, Ramakanth Neeli-Venkata, Andre S. Ribeiro

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.