Fig. S3.

Conformation of the RNA polymerase clamp in the absence of subunit Rpo6 and Rpo4/7. Our recombinant archaeal model system allowed us to perturb the subunit composition of the labeled RNAPs. We prepared RNAP variants without the stalk subunits Rpo4/7 (RNAPΔRpo4/7) and without subunit Rpo6 that serves as docking site for Rpo4/7. Histograms showing the single-molecule FRET efficiencies determined for the 9-subunit RNAP (RNAP ΔRpo4/6/7; A) and 11-subunit RNAP (RNAP ΔRpo6; C) in comparison with the 10- and 12-subunit RNAP (RNAP ΔRpo4/7 and RNAP wt; B and D). RNAPs were immobilized via a biotin in subunit Rpo11 on a quartz slide for TIRF measurements. The histograms were fitted with a double Gaussian function and the mean FRET efficiencies, E, the percentage distribution of the populations, A, and the coefficient of determination, R², are given with SEs in the histograms. Although addition of subunits Rpo4/7 to a RNAP ΔRpo4/7 enzyme slightly decreases the amount of low FRET population (E = 0.37), no shift was observed in an RNAP ΔRpo6 enzyme. Rpo6 is the docking site for subunits Rpo4/7, and hence, in its absence, Rpo4/7 cannot associate with the RNAP core. The absence of subunit Rpo6 leads to a very broad distribution, suggesting that Rpo6 might play a role in adjusting the position of the clamp. (E) Comparison of free RNAP wt vs. RNAP ΔRpo4/7 datasets: the null hypothesis that the datasets have the same distribution is rejected at the 5% level based on the Pearson χ² test (P = 0.0016704 = 0.17%). In contrast, the null hypothesis that the datasets have the same distribution is not rejected when comparing and analyzing two RNAP wt or RNAP ΔRpo4/7 datasets. The Pearson χ² test for RNAP wt datasets yielded P = 0.134966 (13.5%), and the Pearson χ² test for RNAP ΔRpo4/7 datasets yielded P = 0.679731 (67.9%).