1. Volume 50, Issue 6, Pages 882-893 (June 2013)
Cys-Pair Reporters Detect a Constrained Trigger Loop in a Paused RNA Polymerase 
Dhananjaya Nayak, Michael Voss, Tricia Windgassen, Rachel Anne Mooney, Robert Landick 
Molecular Cell 
Volume 50, Issue 6, Pages (June 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 50, 882-893DOI: (10.1016/j.molcel.2013.05.015)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 TL Conformations and Nucleotide Addition by RNAP
(A) Models for TL conformations during the nucleotide addition cycle and pausing. The TL is unfolded and mobile during translocation and NTP binding but must fold into a helical hairpin (TH) with side chain contacts to the NTP to promote rapid nucleotide addition (Vassylyev et al., 2007b; Wang et al., 2006). PECs arise by sequence-induced rearrangements via an elemental pause intermediate. Escape from the hairpin-stabilized PEC, in which the RNA 3′ nucleotide can fray, appears to be inhibited by two factors: slow translocation and slow TL folding (Toulokhonov et al., 2007). The relative contributions of these two factors are not known and may vary among pause sites.
(B) Structure of the EC and location of select CPR residues. Closed-clamp (yellow) TthEC bound to NTP (pdb 2o5j; Vassylyev et al., 2007b) plus unfolded TL (green) from σA-bound RNAP (1iw7; Vassylyev et al., 2002). Clamp opening (purple; 4gzy) is favored by pause sequences and nascent RNA hairpins and may inhibit translocation or TL folding (Tagami et al., 2010; Toulokhonov et al., 2007; Weixlbaumer et al., 2013). The enlarged region shows the folded (TH; orange) and unfolded TL (green) conformations together with four intermediate TL conformations observed in RNAP crystal structures: TthRNAP bound to streptolydigin (1zyr; pale green; Tuske et al., 2005), SceRNAPII bound to TFIIS (1y1v, purple; Kettenberger et al., 2004), SceRNAPII bound to dUTP (2nvq, red; Wang et al., 2006), and SceRNAPII in −1 backtracked EC (3gtg, yellow; Wang et al., 2009). TL Cα positions for amino acid 937 in EcoRNAP (colored spheres) and the Cα positions of key CPR partner residues (blue spheres) are shown. In EcoRNAP, a 188 aa insertion occurs in the active-site distal arm of the TL (connection points indicated as small gray spheres); the insertion, which accommodates rapid TL folding, is omitted for clarity. Additional CPRs used in this study are shown in Figure S1.
(C) Diagrammatic depiction of three TL CPRs involving β′I937C and able to crosslink from folded (F , orange), partially folded (P , red), or unfolded (U , green) TL conformations. The small gray spheres indicate the location of the 188 aa insertion in E. coli RNAP; the blue spheres are as in (B). The cap consists of several RNAP folds (see Discussion), including the F-loop in which β′736 is located (Miropolskaya et al., 2010). See also Figure S1.
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4. Figure 2 Formation of Crosslinks by CPRs
(A) Crosslink formation by CPR F in free RNAP detected by SDS-PAGE. Different redox potentials were created using mixtures containing different concentrations of CSSC and DTT. After quenching reactions with iodoacetamide, RNAP subunits were separated by SDS-PAGE and stained with Coomassie blue R. The extent of crosslinking was quantified as the fraction of the more slowly migrating, crosslinked species, S-Sβ′, relative to the total (β′ + S-Sβ′).
(B) Time courses of crosslinking by CPR F at different CSSC concentrations. The fast-rate component (appKCSSC = 2.9 ± 0.4 mM; appVmax = 4.1 ± 0.2 min−1) affected 48%, 48%, 30%, and 17% of the RNAP, and the slow component (0.063 ± 0.007 min−1) affected ∼25% of the RNAP at 0.1, 1, 2.5, and 10 mM CSSC, respectively. Data are means ± SD of experimental triplicates.
(C) Reaction scheme for CPR oxidation with CSSC to form interconverting single- and double-mixed disulfides (SCSH, SHSC, SCSC) and disulfide crosslinks. Geometry of the transition state for crosslink requires Cα distances of ∼6 Å and in-line S atoms separated by ∼2.39 Å (Wiita et al., 2006).
(D) Formation of crosslinks by CPRs F , P , and U in free RNAP as a function of redox potential generated with 2.5 mM CSSC and varying DTT. Data are means ± SD of experimental triplicates. See also Figure S2.
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5. Figure 3 Transcription Elongation Properties of the TL CPRs
(A) EC scaffold (assembled with nontemplate, template, and RNA oligos NT-5069, T-4903, and RNA-7636, respectively; Table S1). 5′-[32P]RNA13 (red) used for EC reconstitution could extend 8 nt upon addition of ATP and GTP (lower case italics; see Experimental Procedures). The inset shows the variant scaffold used to incorporate 3′-deoxyG and enable measurement of NTP binding (NT-7571, T-7572, RNA-7573; Table S1).
(B) Wild-type and CPR ECs were formed, crosslinked, and extended in steps 1–5 (Experimental Procedures). For each RNAP, samples were prepared prior to reaction with CSSC (−CSSC), after crosslinking with 0.5 mM CSSC for 30 min at RT (+CSSC; estimated 75%, 80%, and 35% crosslinked for U , P , and F , respectively), and after re-reduction with 5 mM DTT (+CSSC→DTT). For each sample, portions were removed and separated by denaturing PAGE (Experimental Procedures) before addition of NTPs (lane 1) and after incubation at 37°C with GTP and ATP (10 μM each) for 5, 10, 30, 120, or 240 s (lanes 2–6) or after further incubation at 37°C with GTP and ATP elevated to 0.5 mM each for 5 min (lane 7).
(C) Kinetics of G14 addition by ECs assembled with wild-type RNAP (Experimental Procedures). Data are means ± SD of experimental triplicates.
(D) G14 addition rates by reduced (red) and oxidized (ox) wild-type and CPR ECs. ECs were reduced or oxidized as described for (B), except that F EC was oxidized with 20 mM oxidized DTT for 2 hr at RT. CPR ECs U , P and F exhibited 75%, 80%, and 65% crosslinking, respectively. G14 addition by oxidized CPR ECs was biphasic and, in each case, gave a rapid G14 addition rate for a minority fraction consistent with the noncrosslinked EC and a majority rate shown in the figure that could be assigned to the crosslinked CPR EC. Assay of EC F at 10 mM GTP confirmed a saturating G14 addition rate of ∼25 s−1. Data are means ± SD of experimental triplicates.
(E) Effect of NTP binding on formation of CPR crosslinks. ATP (0.5 mM), noncognate NTPs, or 2′dATP (+, 1 mM; ++, 10 mM) were incubated with 3′-deoxyG-containing ECs (A and Experimental Procedures) at Eh = −0.3 V (0.5 mM CSSC, 0.5 mM DTT). ATP had the same effects whether added before or 60 min after CSSC/DTT. Data are means ± SD of experimental triplicates. See also Figure S3.
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6. Figure 4 GreB and DksA Inhibit TL Folding
(A) GreB and DksA bind in the RNAP secondary channel and preclude TL folding. TL CPRs are depicted as in Figure 1C.
(B–G) CPR crosslinking in core RNAP was measured as a function of DksA (black) or GreB (gray) concentration (0.01–10 μM) at Eh = −0.14 V (2.5 mM CSSC, 0.05 mM DTT; Experimental Procedures) for CPRs targeting the unfolded, partially folded, and folded TL conformations (Figure S1). (B) U ; (C) U ; (D) P ; (E) P ; (F) F ; (G) F The crosslink may be possible from either the folded or partially folded conformations. Data are means ± SD of experimental triplicates. See also Figure S4.
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7. Figure 5
CPR Crosslinking Profiles a Constrained TL Conformation in PECs
(A) PEC scaffold (NT-5069, T-5420, RNA-4865; Table S1). For dwell-time measurements, PECs were reconstituted with a 27 nt RNA lacking the 3′ C and U (RNA-4867; Table S1).
(B) Pause dwell times for wild-type, oxidized wild-type (ox), and reduced CPR PECs (Experimental Procedures). Data are means ± SD of experimental triplicates.
(C–H) CPR crosslinking as a function of redox potential for RNAP (blue), EC (gray), and PEC (black). EC was reconstituted with scaffold shown in Figure 3A. Varying Eh was generated by mixing 2.5 mM CSSC with 0.05–20 mM DTT. The integrity of ECs and PECs was verified by native gel electrophoresis (Figure S3). (C) U ; (D) U ; (E) U ; (F) P ; (G) F ; (H) F Data are means ± SD of experimental triplicates. See also Figure S5 and Table S2.
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8. Figure 6 NTP-Induced TL Folding Is Inhibited in the PEC
(A) NTP binding to EC CPRs was assayed by initially reacting n-1 ECs (NT-7571, T-7572 and RNA-7573; Table S1) with 3′-dGTP, followed by incubation with 0.001–10 mM ATP. NTP binding to PEC CPRs was assayed by initially reacting n-1 PECs (NT-5069, T-5420 and RNA-4867; Table S1) with 3′-dUTP, followed by incubation with 0.001–10 mM GTP.
(B–G) Fraction of CPR crosslinked versus [NTP] at Eh = –0.3 V (0.5 mM CSSC, 0.5 mM DTT) for ECs or PECs. (B) U (EC); (C) U (PEC); (D) P (EC); (E) P (PEC); (F) F (EC); (G) F (PEC). Data are means ± SD of experimental triplicates.
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9. Figure 7 RfaH NTD Stimulates upon TL Folding in the PEC
(A) Model of E. coli RNAP EC and hairpin-stabilized PEC. RfaH NTD has been proposed to bind the β′ clamp helices and the β gate loop to stabilize a closed-clamp conformation (Sevostyanova et al., 2011).
(B and C) Crosslinking in PECs as a function of RfaH NTD concentration for P (B) and F (C) at Eh = −0.3 V (0.5 mM CSSC, 0.5 mM DTT). The effect of RfaH NTD on 3′-dNMP-containing PECs and ECs was also evaluated in the presence or absence of 10 mM GTP or ATP, respectively, as depicted in the bar graph. ∗, indistinguishable results for these assays were obtained whether RfaH was added before or after PEC formation. Data are means ± SD of experimental triplicates. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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