Fig. 5
From: Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo
The G-dC base pair at the 5′ end of the RNA-DNA hybrid interferes with RNAP translocation in vivo and in vitro. (a) PIEs generated by the single-length RNET-seq analysis for 14-, 15- or 16-nt reads from WT and ΔgreAB cells. DNA positions −9, −10 and −11, where −1 corresponds to the 3′ RNA base, are shown. Pausing was defined by P(0.9, 50). The full-length PIEs are shown in Figs. S9 and S10 in Additional file 1. For 14-nt reads, the pause sties of mapqmean >10 are used (n = 286 and 258 for WT and ΔgreAB, respectively; Fig. S11 in Additional file 1). (b) Model for robust transcription pausing in the post- (14 nt), pre-translocated (15 nt) or 1-bp backtracked (16 nt) state according to the −9, −10 or −11 position of the riboG-dC. (c) Ten-nucleotide RNA strands (top) and the template DNA strands (TDS) in the ECs used for the biochemical assay. The RNA and template DNA bases, carrying sequence different from the original G−10 scaffold, are indicated in red. (d) Effects of different −10 and +1 bases on the elongation (upper) and pyrophosphorolysis (lower) of an EC carrying a 10-nt transcript (EC10). Reaction scheme is shown at the top. The apparent rate constants (k) for these two reactions were obtained by fitting the data to single-exponential curves. The mean values of two or three independent experiments ± standard deviations are shown. PPi pyrophosphate