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. 2021 Jul 19;10:e68829. doi: 10.7554/eLife.68829

Figure 2. The PcrA-CTD binds to a conserved motif in the SI1 domain of RNAP.

(A) Relative HDX measured for a region of the RNAP β subunit (residue numbers on x axis) within the CTD-RNAP complex (blue) compared to RNAP alone (red). A small region of RpoB (at amino-acid positions around ~300) becomes significantly protected by interaction with the PcrA CTD as the exchange time becomes longer. (B) The protected region maps to a conserved motif in the SI1 domain of B. subtilis RpoB. This region is organised differently in E. coli RpoB, but the same conserved amino acid motif appears in a slightly different position in the structure (black arrow). (C) Structure of the B.subtilis (upper panel) and E. coli (lower panel) β2 (red) - SI1(green) domains indicating the beta-loop structure containing a putative interaction motif at the tip (black arrows). This sequence is well-conserved in bacterial RNA polymerases and the consensus sequence is shown in weblogo format beneath each structure. (D) In vitro pulldown of RpoB using PcrA as a bait (see Materials and methods for details). Mutation of the conserved glutamate (E301) in the putative helicase interaction motif dramatically reduces RpoB pulldown.

Figure 2.

Figure 2—figure supplement 1. HDX protection plots for the remaining RNAP subunits in the CTD-RNAP experiment.

Figure 2—figure supplement 1.

(A) Complete data for the β subunit shown in Paved format as used for Figure 2A. The green box indicates the protected area. (B–F) DYNAMX HDX butterfly plots for the β, β’, α, δ, and ε subunits of RNAP. Negative values represent protected regions and positive values represent regions that are exposed upon binding of the CTD. The green box in panel B indicates the region of β that is protected by the PcrA CTD. Note that there are no significant protection signals anywhere else in the entire RNAP complex. For difference plots, four replicates were performed for each of the four independent colour-coded time points (orange dots 0.5 min, red dots 2 min, blue dots 5 min, black dots 10 min). Grey shading indicates the standard deviation of all charge states and replicates per peptide.
Figure 2—figure supplement 2. The CTD-interacting motif is located in different regions of the SI1 domain among landmark organisms.

Figure 2—figure supplement 2.

A multiple sequence alignment for the β2 (red line) and SI1 (green line) domains of the RNAP β subunit for the organisms indicated. Pink shading indicates the putative helicase interaction motif which is positioned differently in E. coli-like compared to B. subtilis-like RNAP. Note that the motif was not clearly identified in all bacterial RNAPs.
Figure 2—figure supplement 3. Mutations to E301 do not alter the overall structure of the β subunit of RNAP.

Figure 2—figure supplement 3.

CD spectra for the proteins indicated were obtained at 0.25 mg/ml as described in the Materials and methods. The spectra are all similar and show a high α helical content as expected based on the cryo-EM structure (predicted spectrum shown as red line), suggesting that the wild type and mutant proteins are globally folded.