Fig. 6.
Recycling of RfaH. a 2D [1H, 13C] methyl-TROSY spectra of [I,L,V]-RfaH alone (200 µM), in the presence of equimolar concentration of opsEC (23 µM), and upon titration of RfaH:opsEC with NusG-NTD (concentration of stock solutions 240 µM and 486 µM); molar ratio [I,L,V]-RfaH:opsEC:NusG-NTD is indicated in color. α and β indicate the all-α or all-β state of the RfaH-CTD. b Experimental set-up to follow RfaH state using in vitro transcription assay. c Determination of RfaH effect on single-round RNA synthesis. The relevant RNA region is shown on the left, with the ops element highlighted in green. Prominent pause sites (U38, G39, and C40) are indicated. Halted α32P-labeled A24 ECs were chased in the presence of RfaH-NTD, RfaHFL, or supernatants from roadblocked (RB) or free (SN) first-round reactions on the WT or G35C (corresponds to G8C in the ops element) template. Reactions were quenched at the indicated times (in seconds) and analyzed on 10% denaturing acrylamide gels; a representative gel is shown. d The fractions of RNA species indicated were determined from 360-s time points. The ratios of RNA in the presence and in the absence of the RfaH variant indicated were determined from three independent biological replicates and are shown as mean ± standard deviation. Source data are provided as a Source Data file