Volume 50, Issue 3, Pages 420-429 (May 2013) The Magic Spot: A ppGpp Binding Site on E. coli RNA Polymerase Responsible for Regulation of Transcription Initiation  Wilma Ross, Catherine E. Vrentas, Patricia Sanchez-Vazquez, Tamas Gaal, Richard L. Gourse  Molecular Cell  Volume 50, Issue 3, Pages 420-429 (May 2013) DOI: 10.1016/j.molcel.2013.03.021 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 50, 420-429DOI: (10.1016/j.molcel.2013.03.021) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Mapping 32P-6-thio-ppGpp Crosslinks to the E. coli RNAP β′ Subunit (A) 6-thio-ppGpp; 6-thio group is circled. (B) SDS gel with RNAP holoenzyme or core enzyme after crosslinking to 32P-6-thio-ppGpp without a competitor (0), with 1 mM GTP (GTP), or with 1 mM nonradioactive ppGpp (ppGpp). The position of the comigrating β and β′ subunits is indicated. (C) Wild-type (WT) RNAP or RNAP containing a β′-GFP fusion, crosslinked to ppGpp as in (B). Both panels are from the same gel, but intervening lanes have been deleted for clarity. (D and E) Undigested or thrombin-digested crosslinked RNAP. Wild-type β′ (1,407 residues) contains a thrombin site at position ∼900. Other RNAPs contain an engineered thrombin site at the indicated position (e.g., β′ 675th). Arrows indicate the positions of crosslinked complete-digestion products (fragment endpoints are indicated). Comparison of phosphorimages with stained images of the same gels is shown in Figure S1. (F) A 36-amino-acid crosslinked interval (β′ 612–648; red bar) is common to all crosslinked digestion products. Black bars, thrombin products forming crosslinks; gray bars, hydroxylamine products forming crosslinks. (G) Amino acid sequence of crosslinked interval in β′ (612–648). Positions where substitutions were constructed are underlined. See also Figure S1. Molecular Cell 2013 50, 420-429DOI: (10.1016/j.molcel.2013.03.021) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 RNAP Substitutions that Reduce the Inhibition of Transcription by ppGpp (A) Representative gel showing transcripts produced in vitro by the rRNA promoter rrnB P1 or the vector-derived RNA 1 promoter with wild-type RNAP or β′ K615A RNAP and 0–200 μM ppGpp. (B) Concentration for half-maximal inhibition of rrnB P1 transcription by ppGpp (IC50 values) with wild-type or mutant RNAPs. Values with associated ranges were determined from replicate experiments as illustrated in (A) and (C)–(F). (C–F) Plots of representative transcription experiments, as in (A), quantifying rrnB P1 transcript levels as a function of ppGpp concentration. Transcription with wild-type (WT) and two mutant RNAPs are shown in each panel. Molecular Cell 2013 50, 420-429DOI: (10.1016/j.molcel.2013.03.021) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 RNAP Substitutions Eliminate the Destabilizing Effects of ppGpp on Promoter Complexes, Reduce Crosslinking to 32P-6-thio-ppGpp, and Alter Cell Growth (A) Plot of a representative promoter complex half-life experiment with WT RNAP or K615A, R417A RNAP ± 50 μM ppGpp. Plots indicate the fraction of complexes remaining as a function of time after competitor addition. Half-life values from replicate experiments with associated ranges are shown in (B). (B) Promoter complex half-lives (min) ± ppGpp with indicated RNAPs, quantified as in (A). Error bars represent the range from at least two independent experiments. (C) Representative crosslinking experiment of 32P-6-thio-ppGpp with different RNAPs as in Figure 1B. (D) Relative ppGpp crosslinking to indicated WT or mutant RNAPs, determined as in (C). Relative crosslinking from replicate experiments with associated ranges are shown (see the Experimental Procedures). Error bars represent the range from at least two independent experiments. (E) Growth curves of E. coli wild-type or rpoC R417A, K615A strains grown in LB, washed, and diluted into fresh LB (black, wild-type; green, mutant) or downshifted into MOPS-glucose minimal medium (MM; blue, wild-type; red, mutant). Error ranges for six replicate determinations are shown. Efficiencies of plating (MM/LB) were 1.2 ± 0.5 (wild-type) and 1.2 ± 0.2 (mutant). The time for the mutant strain to achieve maximum exponential growth rate or to reach stationary phase was 4–5 hr longer than for the wild-type strain. Molecular Cell 2013 50, 420-429DOI: (10.1016/j.molcel.2013.03.021) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Location of the ppGpp Binding Site on E. coli RNAP (A) Model of E. coli RNAP, adapted from 3LU0 (Opalka et al., 2010), with residues implicated in ppGpp function shown in blue spacefill. The crosslinked interval (β′ 612–648, yellow) is located between the secondary channel (SC) and α NTDII (green). The location of ppGpp (red spacefill) is predicted from the genetic and biochemical data in Figures 2 and 3. BH, bridge helix; RH, rim helices; Mg2+, active site. (B) Close-up view rotated ∼90° from the view in (A). Residues implicated in binding to ppGpp are indicated. (C) The proposed ppGpp binding site is at the junction of the rigid-body core and shelf modules of RNAP (dark gray, core module; cyan, shelf module; green, clamp) on the T. thermophilus RNAP-Gfh1 cocrystal structure (3AOI, adapted from Tagami et al. [2010]; Gfh1 is not shown). ppGpp (red) is shown at the location corresponding to the proposed binding site on E. coli RNAP (ppGpp does not bind to T. thermophilus RNAP; Figure 3D) (Vrentas et al., 2008). T. thermophilus core module residues corresponding to E. coli α NTDII α E188 (T. thermophilus E182) and R191 (T. thermophilus R185), shown in yellow spacefill, are in a region proposed to form a stabilizing contact with an α helix in β′ in the shelf module (blue; E. coli β′ 408–417; T. thermophilus β′ 685–696) (Tagami et al., 2010). We suggest that substitutions for E. coli residues R191 and E188 would increase complex stability by disfavoring a clamp-open (ratcheted) state. Molecular Cell 2013 50, 420-429DOI: (10.1016/j.molcel.2013.03.021) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Evolutionary Conservation of the Proposed ppGpp Binding Site (A) Three regions comprising the proposed ppGpp binding site in E. coli RNAP. Sixty amino acids from each region are illustrated on the model of E. coli RNAP (3LU0): region 1 (β′ 600–659; yellow), region 2 (β′ 340–399; green), and region 3 (ω 1–60; blue). ppGpp is in red. Residues predicted to contact ppGpp are in spacefill (see Figure 4). (B) Alignments of the E. coli RNAP sequences from the three regions illustrated in (A) with the corresponding regions from Bacillus subtilis subsp subtilis str 168 and Thermus thermophilus HB8. Residues predicted to bind ppGpp in each region of E. coli RNAP are in red. The 36-amino-acid interval in region 1 that crosslinked to 6-thio-ppGpp is in green. Alignments of the three RNAP regions from other bacterial species are provided in Figure S2. Boxes in regions 1 and 3 surround short region containing residues with the largest effects on ppGpp binding. Dots indicate sequence identity. See also Figure S2. Molecular Cell 2013 50, 420-429DOI: (10.1016/j.molcel.2013.03.021) Copyright © 2013 Elsevier Inc. Terms and Conditions