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

    Tutkimustuotos: ArtikkeliScientificvertaisarvioitu

    32 Sitaatiot (Scopus)

    Abstrakti

    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.
    AlkuperäiskieliEnglanti
    Sivut203-214
    JulkaisuDNA Research
    Vuosikerta23
    Numero3
    DOI - pysyväislinkit
    TilaJulkaistu - 29 maalisk. 2016
    OKM-julkaisutyyppiA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä

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