Fig. 6: C11 cleavage-defective CTD mutants interfere with termination during Pol III recycling. | Nature Communications

Fig. 6: C11 cleavage-defective CTD mutants interfere with termination during Pol III recycling.

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. 6

a In vitro transcription recycling reactions using the SUP4-tRNATyr transcription complexes reconstituted with Pol III-core (lane 1), or aliquots of a batch of Pol III-core preincubated with C37/53 alone (lane 2) or plus increasing amounts of C11-Δhp (deleted hairpin) or C11-(DE-AA) at 100, 200, and 500 nM, as indicated. The T1 and T2 bands are as described for Fig. 4a, b, d. b Schematic representation of the suppressor-tRNA gene in S. pombe strains ySHA24 and yRS6A used in c and d. The complementary read-through (cRT) region can form extensive base pairing with the tRNA region, therefore if it is transcribed will interfere with the formation of a functional tRNA, making suppression dependent on termination at T156. c tRNA-mediated suppression (TMS) phenotypes in the red–white ade6-704 assay as described in the text. Lighter color indicates increased termination at T1, a darker color indicates decreased termination at T1. d Graphic representation of quantification of relative activities in the dual nanoluciferase/firefly-luciferase TMS assay described in the text; nLuc and fflyLuc relative light units (RLU) are arbitrary. Bars indicate the means of two data points (circles) for the test constructs indicated representing independent biological replicates.

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