Figure 5. RfaH inhibits PH stimulation of pausing and RNAP swiveling more effectively and binds ECs more tightly than NusG.
A. Scaffold used to test asRNA stimulation of pausing. The sequence is identical to that used to determine the hisPEC structure (Kang et al., 2018), except that the PH is replaced with an asRNA binding target (Kolb et al., 2014).
B. Experimental scheme to measure PH stimulation of pausing using asRNA.
C. Effect of RfaH-NGN and NusG on stimulation of pausing by an RNA duplex. Following UTP and GTP addition to the radiolabeled C18 EC, the fraction of U19 present as a function of time was determined in the presence of indicated ligands. EC fractions corresponding to bypass, elemental pause, and swiveled states are indicated on the y-axis. Data shown are means and s.d. from at least three replicates.
D. Effect of NusG on stimulation of pausing by 0.5 μM asRNA. NusG decreased the slower, apparently swiveled fraction of U19 only modestly, from 0.57 ± 0.02 to 0.49 ± 0.02. Data shown are means and s.d. from at least three replicates.
E. Depiction of RNAP swiveling and location of CTR Cys residues. PH-induced swiveling changes distance from β′C258i to its potential disulfide partners, βC1045i or β′C843 from 3 Å and 10 Å, respectively, to 4 Å and 7 Å (Kang et al., 2018).
F. RfaH-NGN but not NusG decreased the ratio of CTR reporter disulfides on the hisPEC scaffold (containing PH; Kang et al., 2018), indicating that RfaH-NGN but not NusG shifts the equilibrium from the swiveled to the unswiveled RNAP conformation. Data shown are means and s.d. from at least three replicates.
G. RfaH and NusG retention on the EC. A linear DNA template with a T7A1 promoter, the ops element, and an EcoRI site 128 nt downstream from the opsP site was immobilized on streptavidin beads via a biotin on the nt-DNA (top). A cleavage-deficient EcoRIQ111 protein (RB) was bound to roadblock the transcribing RNAP. Halted ops10 (G42) ECs were formed by step-wise transcription with NTP subsets and incubated with radiolabeled RfaH or NusG. After washing away the unbound RfaH/NusG, transcription was resumed by addition of all NTPs with or without an excess of unlabeled NusG, which is expected to bind to RNAP upon dissociation of the pre-bound factor. G42 and roadblocked ECs were washed to remove unbound RfaH/NusG and analyzed by scintillation counting.
H. RfaH (magenta bars) and NusG (green bars) binding to the immobilized ECs. The residual factor binding to the roadblocked ECs is expressed relative to that observed with the G42 EC, which is defined as 100%. Data shown are means and s.d. from three biological replicates.