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. 2014 Oct 21;42(20):12707–12721. doi: 10.1093/nar/gku997

Figure 5.

Figure 5.

Blocking TH1a unfolding prevents hairpin-stabilization of pausing. (A) ScaffoldPEC allows his pause escape measurement by reconstituting ECs upstream of the pause, adding 32P-CTP to advance to C28 (pause-1), and then adding UTP (100 μM) and GTP (10 μM) at time zero to elongate through the pause site. (B) Kinetics of pause behavior for F937–736 RNAP under reduced, oxidized and re-reduced (oxidized and then reduced, rred) conditions, determined from kinetic fits of data included in C, D and E. (C) F937–736 RNAP (reduced) transcription products were resolved through denaturing PAGE. Fraction of C28, U29 and G30/G31 were fit and rates determined by numerical integration algorithm using the reaction scheme in (A) (Materials and Methods). The reduced RNAP state was confirmed through SDS-PAGE (inset). (D) As in (C), but using F937–736 RNAP under oxidized conditions. Transcription products show increased bypass fraction with crosslinking (65% confirmed through SDS-PAGE, inset). (E) As in (C), but using F937–736 RNAP that has been re-reduced (rred). The treatment reverses the pausing effects of crosslinking F937–736. Data are means ± SD of experimental triplicates.