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. Author manuscript; available in PMC: 2017 Jun 16.
Published in final edited form as: Mol Cell. 2016 May 26;62(6):811–823. doi: 10.1016/j.molcel.2016.04.029

Figure 1. DksA Restores a Response to ppGpp to RNAPs Lacking the Previously-Identified ppGpp Binding Site.

Figure 1

(A) Inhibition of rrnB P1 transcription by ppGpp in vitro using WT RNAP or two different RNAPs lacking Site 1, Δω RNAP and M8 RNAP (ωΔ 2–5, β’R362A, R417A, K615A, Y626A). Means and ranges shown here and in (C), (D), and (F) are from two independent experiments. See Figure S1A and Supplemental Experimental Procedures.

(B) Representative gel showing in vitro transcripts from rrnB P1 and the vector-encoded RNA I promoter in reactions with RNAP lacking Site 1 (Δω RNAP) and either 2 μM DksA and 0–160 μM ppGpp or ppGpp without DksA.

(C) Inhibition of rrnB P1 transcription by ppGpp as in (A) but with 2 μM DksA. 2 μM DksA alone, with no ppGpp, inhibited transcription similarly for each RNAP (transcript levels ~0.6 relative to no DksA).

(D) Relative rrnB P1 transcript levels in vitro, normalized to levels without DksA or ppGpp for each RNAP.

(E) IC50s for ppGpp for inhibition of rrnB P1 with WT or RNAP lacking Site 1 (Δω RNAP) and the indicated DksA concentrations. Values with 95% confidence intervals are shown. For WT RNAP without DksA (Site 1 only), n=10; WT RNAP with 2 μM DksA (Sites 1 and 2), n=8; Δω RNAP with 0.5 μM DksA (Site 2 only), n=2; Δω RNAP with 2 μM DksA (Site 2 only), n=16; Δω RNAP with 6 μM DksA (Site 2 only), n=2.

(F) Inhibition of rrnB P1 transcription by ppGpp using RNAP lacking Site 1 (Δω RNAP) and 0, 0.5, 2, or 6 μM DksA. Inhibition by DksA alone, with no ppGpp, varied with the DksA concentration; transcript levels relative to no DksA, were 1.07 ± 0.18 at 0.5 μM DksA; 0.60 ± 0.04 at 2 μM DksA; 0.29 ± 0.02 at 6 μM DksA. See Figure S1B for plot without normalization.

(G) Crosslinking of 32P-6-thio-ppGpp to DksA in reactions with DksA (2.5, 5 or 10 μM WT or 1.7, 3.3 or 6.6 μM N88I) and 0.6 μM RNAP lacking ppGpp Site 1 (Δω RNAP, lanes 1–8), or DksA alone (lanes 11–16). Reactions in lanes 1, 2, 9, 10, 17, and 18 used RNAP lacking Site 1 (Δω or M8) or WT RNAP but no DksA. The crosslink with WT RNAP is to β’. Both gels (4–12% SDS-PAGE) were from the same experiment.

(H) Crosslinking as in (G) with 2.5 μM N88I-DksA and 0.75 μM WT RNAP or RNAP lacking Site 1 (Δω) ± 1 mM unlabeled ppGpp competitor.

(I) Crosslinking as in (G) with 0.75 μM WT RNAP and 6 μM WT, N88I, or L15F-DksA, ± 1 mM unlabeled ppGpp competitor. See also Figure S1B.