Road rules for traffic on DNA-systematic analysis of transcriptional roadblocking in vivo.

TitleRoad rules for traffic on DNA-systematic analysis of transcriptional roadblocking in vivo.
Publication TypeJournal Article
Year of Publication2014
AuthorsHao N, Krishna S, Ahlgren-Berg A, Cutts EE, Shearwin KE, Dodd IB
JournalNucleic Acids Res
Date Published2014 Aug
KeywordsBacterial Proteins, DNA, Bacterial, DNA-Directed RNA Polymerases, Escherichia coli Proteins, Lac Repressors, Promoter Regions, Genetic, Transcription Factors, Transcription, Genetic

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.

Alternate JournalNucleic Acids Res.
PubMed ID25034688
PubMed Central IDPMC4132739
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