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

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.

• Publication year

  2014

• Venue

  Nucleic Acids Research

• Publication date

  2014-07-17

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1093/nar/gku627PMID 25034688PMCID 4132739
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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