Figure 4.

Functional analysis of the U136/U145/A146 mutant 6S-1 RNA unable to form a hairpin in the 3′-portion of the central bulge. (A) 5′-Endlabelled 6S-1 RNA was incubated with σ^A-RNAP in the presence of NTPs for different time periods as in Figure 2A. Samples were then either loaded on a 7.5% native PAA gel using 1 × TBE as electrophoresis buffer (left panel) or 0.5 × TBE, 160 mM KOAc, 5 mM Mg(OAc)₂, 1 mM DTT, pH 8.6 (right panel) as electrophoresis buffer. Lanes C1: samples incubated for 180 min at 37°C in the absence of NTPs; lanes C2: as lanes C1 but without RNAP. For further details, see legend to Figure 2. (B) In vitro transcription was conducted as in Figure 1A, comparing wt (wt 6S-1) and U136/U145/A146 mutant 6S-1 RNA (mut 6S-1) as template; C/G/UTP, NTP mixture lacking ATP. For further details, see legend to Figure 1A. (C) Binding of 5′-endlabelled wt 6S-1 RNA (open squares) and U136/U145/A146 mutant 6S-1 RNA (filled squares) to the σ^A-RNAP holoenzyme, based on experiments as shown in (A) (right), but in the absence of NTPs (see also Beckmann et al, 2011). Apparent K_d values, based on two independent experiments, were 0.24±0.06 μM for wt 6S-1 RNA and 0.56±0.17 μM for the mutant 6S RNA, as derived from non-linear regression analysis using the equation for a single ligand binding site. Errors, standard errors of the mean (s.e.m.); some error bars are small such that they are masked by the symbol. Similar binding curves were obtained by 7.5% native PAGE in 1 × TBE buffer. The experimental end point for complex formation is lower for the mutant versus wt RNA, suggesting that the fraction of 6S-1 mutant conformers that are unable to bind to RNAP is increased relative to wt 6S-1 RNA. (D) Model of the mutant 6S-1 RNA structure before and after the pRNA-induced structural rearrangement. The three base exchanges are highlighted.