Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/89372
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Type: Journal article
Title: Road rules for traffic on DNA - systematic analysis of transcriptional roadblocking in vivo
Author: Hao, N.
Krishna, S.
Ahlgren-Berg, A.
Cutts, E.
Shearwin, K.
Dodd, I.
Citation: Nucleic Acids Research, 2014; 42(14):8861-8872
Publisher: Oxford University Press (OUP)
Issue Date: 2014
ISSN: 1362-4962
0305-1048
Statement of
Responsibility: 
Nan Hao, Sandeep Krishna, Alexandra Ahlgren-Berg, Erin E. Cutts, Keith E. Shearwin, and Ian B. Dodd
Abstract: Genomic DNA is bound by many proteins that could potentially impede elongation of RNA polymerase (RNAP), but the factors determining the magnitude of transcriptional roadblocking in vivo are poorly understood. Through systematic experiments and modeling, we analyse how roadblocking by the lac repressor (LacI) in Escherichia coli cells is controlled by promoter firing rate, the concentration and affinity of the roadblocker protein, the transcription-coupled repair protein Mfd, and promoter-roadblock spacing. Increased readthrough of the roadblock at higher RNAP fluxes requires active dislodgement of LacI by multiple RNAPs. However, this RNAP cooperation effect occurs only for strong promoters because roadblock-paused RNAP is quickly terminated by Mfd. The results are most consistent with a single RNAP also sometimes dislodging LacI, though we cannot exclude the possibility that a single RNAP reads through by waiting for spontaneous LacI dissociation. Reducing the occupancy of the roadblock site by increasing the LacI off-rate (weakening the operator) increased dislodgement strongly, giving a stronger effect on readthrough than decreasing the LacI on-rate (decreasing LacI concentration). Thus, protein binding kinetics can be tuned to maintain site occupation while reducing detrimental roadblocking.
Keywords: Transcription Factors; DNA, Bacterial; Bacterial Proteins; Escherichia coli Proteins; Transcription, Genetic; Lac Repressors; Promoter Regions, Genetic; DNA-Directed RNA Polymerases
Rights: © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
RMID: 0030008661
DOI: 10.1093/nar/gku627
Grant ID: http://purl.org/au-research/grants/arc/DP110101470
Appears in Collections:Molecular and Biomedical Science publications

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