title: Dissecting the stochastic transcription initiation process in live Escherichia coli.
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.
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland.
authors.forename: Jason
authors.initials: J
authors.lastname: Lloyd-Price
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland.
authors.forename: Sofia
authors.initials: S
authors.lastname: Startceva
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland.
authors.forename: Vinodh
authors.initials: V
authors.lastname: Kandavalli
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland.
authors.forename: Jerome G
authors.initials: JG
authors.lastname: Chandraseelan
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland.
authors.forename: Nadia
authors.initials: N
authors.lastname: Goncalves
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland.
authors.forename: Samuel M D
authors.initials: SM
authors.lastname: Oliveira
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Häkkinen
authors.affiliation: Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, PO Box 553, Office TC336, 33101 Tampere, Finland andre.ribeiro@tut.fi.
authors.forename: Andre S
authors.initials: AS
authors.lastname: Ribeiro
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D056968:Lac Repressors / Q000378:metabolism; D008957:Models, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013269:Stochastic Processes; D061785:Transcription Initiation, Genetic
chemical_list: D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012321:DNA-Directed RNA Polymerases
keywords: free RNA polymerase; in vivo transcription dynamics; rate-limiting steps; repressor binding dynamics; reversible closed complex formation