In vivo single-molecule dynamics of transcription of the viral T7 Phi 10 promoter in Escherichia coli

We study the dynamics of transcription initiation of the T7 Phi 10 promoter as a function of temperature, using quantitative polymerase chain reaction (qPCR) and in vivo single-cell, single-ribonucleic acid (RNA) time-lapse microscopy. First, from the mean and squared coefficient of variation of the empirical distribution of intervals between consecutive RNA appearances in individual cells, we find that both the mean rate and noise in RNA production increase with temperature (from 20 °C to 43 °C). Next, the process is shown to be sub-Poissonian in all conditions, suggesting the existence of more than one rate-limiting step and absence of a significant ON-OFF mechanism. Next, from the kinetics of RNA production for varying amounts of T7 RNA polymerases, we find that as temperature increases, the fraction of time that the T7 RNA polymerase spends in open complex formation increases relative to the time to commit to closed complex formation, due to changes in the kinetics of open complex, closed complex, and reversibility of the closed complex formation. We conclude that the initiation kinetics of the T7 Phi 10 promoter changes with temperature due to changes in the kinetics of its rate-limiting steps.