RNA polymerase efficiently transcribes through DNA-scaffolded, cooperative bacteriophage repressor complexes

Abstract

DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA with specific binding sites, while phage λ CI repressor dimers bind to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by Escherichia coli RNA polymerase (RNAP) through these protein complexes. 186 CI, or λ CI, bound along unlooped DNA negligibly interfered with transcription by RNAP. Wrapped and looped topologies induced by these scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with high affinity, these repressors are not effective roadblocks to transcription.

Keywords: atomic force microscopy; bacteriophage repressors; roadblock efficiency; topology; transcription.

Figure 1.

(A) A diagram of the DNA template shows the main features of DNA molecules used in transcription measurements: promoter (green, hooked arrow), RNAP (yellow arrow), associated transcript (blue arrow), terminator (red cross) and streptavidin marker (purple arrow). (B) AFM images of control experiments in the absence of repressor. Particles and DNA features are marked according to the color scheme in (A). Nascent RNA associated with all RNAP particles is visible as dark blue structures emanating from the RNAP particles.

Figure 2.

The progress observed for RNAP in the presence of the 186 CI repressor is summarized. (A) A diagram of the DNA template shows the spacings between relevant DNA features. (B) A gallery of AFM images shows transcription elongation complexes and DNA templates wrapped around 186 CI bound at pR. 186 CI particles are marked by cyan arrows and all particles and DNA features are marked according to the color scheme in (A). (C) A gallery of transcription elongation complexes shows DNA templates wrapped around 186 CI bound at pR as well as a repressor bound at FR. (D) The probability of progress by RNAP was plotted for different experimental conditions with the measured positions of 186 CI indicated as a bar histogram. The N values are numbers of molecules with associated RNAP for which RNAP or 186 CI positions were measured. The colored dots indicate the fraction of elongation complexes found along the DNA molecule at distances greater than or equal to x from the promoter. Color-coded heights in AFM images rise from low, orange values through higher dark blue and cyan and the highest features appear yellow. [186 CI] was 150 nM (without NTPs) or 500 nM otherwise.

Figure 3.

The 186 CI heptameric wheel is disrupted by transcription. Measurements of the diameters (A) and volumes (B) of 186 CI particles measured in AFM transcription assays are shown in whisker and violin plots. All 186 CI particles anywhere on DNA templates with bound RNAP were counted. Without transcription there are large and small particles. Larger particles disappear with transcription while the smaller ones remain.

Figure 4.

The progress observed for RNAP in the presence of the λ CI repressor is summarized. (Left) (A) A diagram of the DNA template shows the spacings between relevant DNA features. (B) A gallery of transcription elongation complexes shows DNA templates with λ CI bound to OL and/or OR operators. The bottom AFM image includes a second RNAP that had started elongation on the DNA template, following a first RNAP that had just bypassed λ CI bound at the OR region before stalling. (C) A gallery of transcription elongation complexes along a looped DNA template are shown. Colored arrows marking features follow color code in (A). No looped molecules were found with RNAP inside or after the loop. Heights in AFM images rise from dark blue to cyan and the highest features appear yellow. (D) The probabilities of RNAP progress in the presence of the λ CI repressor in unlooped or (E) looped DNA templates are shown. The N values are numbers of molecules with associated RNAP for which RNAP or 186 CI positions were measured. The bar histogram indicates the measured locations of bound λ CI. The colored dots indicate the fraction of elongation complexes found at distances greater than or equal to x from the promoter along the DNA molecule.

Figure 5.

Transcription evicts repressors to reduce the number of repressor molecules bound to DNA as shown by the number of different configurations of DNA molecules bound by 186 CI (A) or λ CI (B) particles without (red) or with (blue) transcription. (A) In conditions of active transcription, a higher fraction of DNA molecules with no 186 CI bound, and a lower proportion of DNA molecules with both pR and FR bound by protein were observed. (B) Actively transcribing RNAP also decreased the number of λ CI-mediated looped complexes substantially, while the number of DNA molecules with no repressor molecules bound increased. All traceable molecules were counted regardless of whether an RNAP was bound.

Figure 6.

Schematic of RNA polymerase disrupting the 186 CI (A) and λ CI (B) repressors. RNA polymerase likely disrupts the 186 CI complex causing the wheel-like structure to dissociate, but 186 CI may reassemble on the high affinity PR sites after RNAP passage. For λ CI, disruption of the first bound dimer encountered by advancing RNAP, and loss of the cooperative interactions between dimers, may weaken binding by subsequent dimers, allowing them to be more easily dislodged. After RNAP passage, the dimers may slowly reassemble on the DNA.

Figure 7.

Roadblocks were ranked in order of strength (Table 1) according to the calculation described in Materials and Methods. Lac repressor forming a loop was most effective. Cooperative λ CI and 186 CI oligomers did not impede RNAP significantly.