A strategy for dissecting the kinetics of transcription repression mechanisms
Promoters in Escherichia coli include an ‘OFF’ state, during which transcription is halted. Here, we propose a novel empirical method for assessing the time-length spent by promoters in this state. It relies on direct measurements of RNA production kineti
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Laboratory of Biosystem Dynamics, BioMediTech Institute and Faculty of Biomedical Sciences and Engineering, Tampere University of Technology, 33101, Tampere, Finland 2
Computational Intelligence Group of CTS/UNINOVA. Faculdade de Ciências e Tecnologia da Univ. Nova de Lisboa, Portugal
Abstract— Promoters in Escherichia coli include an ‘OFF’ state, during which transcription is halted. Here, we propose a novel empirical method for assessing the time-length spent by promoters in this state. It relies on direct measurements of RNA production kinetics at the single molecule level at different induction levels, followed by an estimation of the RNA production rate under infinite induction, which is then compared to this rate under real, maximum induction. We apply it to the LacO3O1 promoter and infer that, under full induction, on average, 15% of the time between successful transcription events is spent in the OFF state. We verify this result by comparing the kinetics of a mutant strain lacking repressor molecules with that of the inferred rate under infinite induction. We expect this strategy of dissecting the kinetics of transcription repression to be applicable to a wide number of promoters in E. coli. Keywords— Transcription, Induction, Plot, OFF state. I. INTRODUCTION
Novel experimental techniques of microscopy and fluorescent molecular probes have led to the rapid acquisition of invaluable data on the dynamics of gene expression in live cells. One particularly valuable development has been the engineering of the MS2-GFP protein that has the ability to bind specific RNA sequences and, thus, provided multiple binding sites, detecting individual RNA molecules as soon as they are produced in live cells [1]. This technique allows both estimating RNA numbers in individual cells of a population at a given time [1] as well as obtaining RNA production time intervals [2]. This data greatly increased our knowledge on the in vivo dynamics of transcription. Recently, a technique was developed for dissecting the dynamics of active transcription [3]. This method uses measurements of the RNA production dynamics from cells with differing RNA polymerase (RNAP) concentrations, which allows estimating what would be the rate of transcription in cells with infinite RNAP concentration. Here, we propose a novel, similar methodology that uses data from cells with differing intracellular inducer concentrations, to further dissect the kinetics of transcription initiation. In particular, we focus on the promoter OFF state.
© Springer Nature Singapore Pte Ltd. 2018 H. Eskola et al. (eds.), EMBEC & NBC 2017, IFMBE Proceedings 65, DOI: 10.1007/978-981-10-5122-7_274
II. METHODS
A. Cells, Plasmids, and Chemicals We use E. coli strain BW25113 (lacI+ rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) [4], which have the constitutive promoters PlacI+ and ParaC producing, respectively, LacI repressors for the LacO3O1 promoter [5] and AraC repressors for the BAD promoter. We also use the deletion mutant strain JW0336 (BW25113 ΔlacI), lacking th
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