Dissecting the stochastic transcription initiation process in live Escherichia coli

Jason Lloyd-Price, Sofia Startceva, Vinodh Kandavalli, Jerome G. Chandraseelan, Nadia Goncalves, Samuel M.D. Oliveira, Antti Häkkinen, Andre Sanches Ribeiro

    Research output: Contribution to journalArticleScientificpeer-review

    22 Citations (Scopus)

    Abstract

    We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.
    Original languageEnglish
    Pages (from-to)203-214
    JournalDNA Research
    Volume23
    Issue number3
    DOIs
    Publication statusPublished - 29 Mar 2016
    Publication typeA1 Journal article-refereed

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    • Publication forum level 1

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