Road rules for traffic on DNA—systematic analysis of transcriptional roadblockingin 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.

Proteins mediating DNA loops effectively block transcription

“…it suggests that rather than dislodging obstacles, RNA polymerase simply waits for obstacles to disappear. This is compatible with the in vivo results in which promoter firing rate and the multiplicity of paused TECs affected read through 18. Such extended pauses indicate that during a transcription interval of 60 s, LacI can resist an advancing RNA polymerase particularly when bound to a high affinity operator like O1.…”

“…Indeed, it is well known that LacI bound to the high affinity Os operator potently interferes with transcriptional elongation 16, 17, 18. TEC observations on unlooped DNA further illustrate this point.…”

“…Pauses in tether shortening (transcription elongation) were observed at the extension corresponding to O1 (dashed line at −0.2 µm). Pauses at the roadblock were measured and are reported in Figure 5 together with the mean lifetimes of LacI‐operator complexes estimated as the inverse of the measured off‐rates 18. Notice that the mean pause time is similar to the average dwell time of LacI on O1 in vitro.…”

“…In addition, LacI tetramers exhibit higher affinity for operators in looped as compared to unlooped DNA 15. LacI bound to an operator can block transcription initiation with up to 99.5% efficiency in vivo16, 17 that depends on the promoter firing rate and whether multiple RNA polymerases act cooperatively 18. Lac repressor can also obstruct eukaryotic RNA polymerase II19 to a greater or lesser degree depending on accessory factors 20…”

Regulatable and Modulable Background Expression Control in Prokaryotic Synthetic Circuits by Auxiliary Repressor Binding Sites

“…23 These LacI−lacO models further suggest that single RNA polymerases at low promoter firing rates can actively dislodge LacI at its lacO position but that at high promoter firing rates, multiple individual RNA polymerases transcribing the 5′-leader region may become clogged upstream of the bound LacI to lacO or may occlude the binding site from access to LacI. However, a clear distance effect of lacO positioning to the promoter on the road-blocking magnitude was not detected, and double lacO sites were not considered in this study.…”