Fig. 3: The isolated C11 NTD stimulates C37/53-mediated Pol III termination from scaffold ECs.

a Left: schematic of Nickel-Nitrilotriacetic acid (Ni-NTA) immobilized Pol III-core elongation complex (EC) assembled on DNA scaffold. The first T:A bp of the T5 terminator, indicated as Term Zone, begins after 12 bp downstream of the 3′ end of the 10-nt RNA primer (depicted in red); an asterisk indicates the 5′-³²P. The nontemplate (NT) and template (T) DNA strands are indicated. Right: schematic description of the Pol III transcription-elongation complex (TEC) assembly and termination reaction. b Gel autoradiogram showing results of 10-min transcription-elongation–termination assay in the presence and absence of C37/53 and the C11 protein constructs indicated above the lanes, M = D91A, E92A (DE-AA) mutant. Transcripts that were released from or remained bound to the immobilized Pol III are indicated above the lanes as R and B, respectively. TZ denotes the positions of the termination products released in the Term Zone, and RT denotes read-through to the end of the DNA. Lanes 25 and 26 were from the same gel, same exposure. The −27 and −25 reflect RNA size markers in nucleotides (nt), these represent transcripts with 3 and 5 uridylates, respectively. c Graphic representation of quantification of transcription efficiency, quantified and calculated with Multigauge V3.2 software³⁹. The bars indicate the means of two data points (black circles) for each condition which represent independent duplicate biological replicates for the same batch of the construct proteins as indicated below.