Fig. 2: The isolated C-terminal domain (CTD) of C11 can activate RNA 3’-cleavage in backtrack arrested Pol III.

a Diagram of the C11 constructs used in this study; the ones above the dashed horizontal line are used in this figure. b Coomasie blue-stained gel of the C11 proteins used, corresponding to panel a. c Schematic of a DNA scaffold Pol III elongation complex (EC) in which the 3-end of the 12-nt RNA (depected in red) has a 2-nt mismatch with the template, representing an arrested EC with a RNA 3′-overhang in a backtracked state; the asterisk represents the radiolabeled 5′-³²P. d RNA 3′-cleavage assay results. A batch of backtracked arrested EC substrate was aliquoted to different reaction tubes and incubated with either buffer alone or recombinant C11 as indicated above the lanes, for 15 min prior to addition of MgCl₂ as indicated. Total RNA was analyzed. The starting material, backtracked EC, and cleavage products, BEC and CP, are indicated by brackets; the image in lanes 13–14 was from the same assay run on a different gel. Positions of 12-nt and 10-nt RNAs are indicated to the right. e The fraction of CPs, calculated as % cleavage product (CP) = CP/(BEC + CP) × 100, were quantified and calculated with Multigauge V3.2 software³⁹ and plotted against the concentration of C11, CTD, and CTD-L. Two data points for each concentration are representative of independent biological replicates for the same batch of C11 construct proteins, as indicated in the inset. The means of the two points were used to plot the lines for the proteins annotated. Additional assay data are shown in Supplementary Fig. 1a. Linear regression analysis of these quantified data with R² values is shown in Supplementary Fig. 1b.