title: Road rules for traffic on DNA-systematic analysis of transcriptional roadblocking in vivo.
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.
authors.affiliation: School of Molecular and Biomedical Sciences (Biochemistry), The University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Nan
authors.initials: N
authors.lastname: Hao
authors.affiliation: Simons Centre for the Study of Living Machines, National Centre for Biological Sciences, Bangalore 560065, India.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Krishna
authors.affiliation: School of Molecular and Biomedical Sciences (Biochemistry), The University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Ahlgren-Berg
authors.affiliation: School of Molecular and Biomedical Sciences (Biochemistry), The University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Cutts
authors.affiliation: School of Molecular and Biomedical Sciences (Biochemistry), The University of Adelaide, Adelaide, SA 5005, Australia.
authors.forename: Keith E
authors.initials: KE
authors.lastname: Shearwin
authors.affiliation: School of Molecular and Biomedical Sciences (Biochemistry), The University of Adelaide, Adelaide, SA 5005, Australia ian.dodd@adelaide.edu.au.
authors.forename: Ian B
authors.initials: IB
authors.lastname: Dodd
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D056968:Lac Repressors / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D014157:Transcription Factors; C072103:transcription repair coupling factor protein, Bacteria; D012321:DNA-Directed RNA Polymerases