Fig. 4: C11 activates C37/53 Pol III transcription factor (TF) complexes with efficient termination for reinitiation. | Nature Communications

Fig. 4: C11 activates C37/53 Pol III transcription factor (TF) complexes with efficient termination for reinitiation.

From: Mechanism of RNA polymerase III termination-associated reinitiation-recycling conferred by the essential function of the N terminal-and-linker domain of the C11 subunit

Fig. 4

a Left: schematic representation of the immobilized SUP4-tRNATyr gene with A-box, B-box promoter elements, and T1 and T2 terminators indicated, that is used to form transcription preinitiation complexes (PICs) described in the text and in Supplementary Fig. 2. Right: schematic description of the PIC assembly and transcription time-course reactions shown in (b); NTPs nucleoside triphosphates. b RNA products of in vitro transcription reactions carried out on a batch of SUP4-tRNATyr transcription complexes reconstituted with either Pol III-holo (lanes 1–5, numbered below), Pol III-core (lanes 6–10), or Pol III-core preincubated with C11 only (core + C11), C37/53 only, or C37/53 + C11 subunits (lanes 11–15, 16–20, and 21–25). All lanes were part of the same transcription experiment and were run on the same gel. Lanes 15 and 16 were separated by empty lanes but were juxtaposed in the image shown as indicated by the vertical line. In this gel system, the T1 and T2 bands correspond to sizes ~107 and ~113 nucleotides (nt), respectively, as illustrated in panel a and annotated on the left side of panel b (see Supplementary Fig. 2b). c The amounts of T1 + T2 RNAs at each time point in b were quantified and calculated with Multigauge V3.2 software39. Two data points for each time point represent independent duplicate biological replicates for the same batch of construct proteins. The means of the two points were used to plot the lines for the sets of proteins as annotated. The blue and red lines were used for visual contrast. The inset shows only the lower three time-course plots on a semi-log scale, base 2. Regression analysis of the quantified data with R2 values is shown in Supplementary Fig. 3. d Release of RNA at the T1 terminator is not sufficient for efficient recycling by Pol III-core. The RNA products of an in vitro transcription reaction as in panel b (30 min.) were separated into released (R) and bound (B), prior to examination by denaturing gel electrophoresis; the T1 and T2 bands are as in panels a and b, corresponding to ~107 and ~113 nucleotides (nt) in length.

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