1. Volume 57, Issue 3, Pages 408-421 (February 2015)
The Ratcheted and Ratchetable Structural States of RNA Polymerase Underlie Multiple Transcriptional Functions 
Shun-ichi Sekine, Yuko Murayama, Vladimir Svetlov, Evgeny Nudler, Shigeyuki Yokoyama 
Molecular Cell 
Volume 57, Issue 3, Pages (February 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 57, 408-421DOI: (10.1016/j.molcel.2014.12.014)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
Cys-Pair Crosslinking Analyses of RNAP Structural Forms and Effects of Nucleic Acids, Gfh1, and GreA
(A) Schematics of two RNAP structural forms: the tight and ratcheted forms.
(B) Schemes of the nucleic acid scaffolds. EC15, the posttranslocation state of the elongation complex (EC); EC16, the pretranslocation state of EC; BC17, the one-nucleotide backtracked state; BC24, the eight-nucleotide backtracked state.
(C) The two Cys sites (α R185C and β′ E692C) introduced into T. thermophilus RNAP. The RNAP coordinates in the ratcheted form (PDB 3AOH, colored) are superimposed on those in the tight form (PDB 2O5I, gray) by the α subunit. The Cα–Cβ bonds are shown as orange sticks.
(D) Cys-pair crosslinking (CPX) formation. The variant was incubated at 25°C for 2 hr in pH 6.5 buffer, in the presence or absence of the EC15 scaffold or Gfh1. After the incubation, the proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE).
(E) Quantitation of CPX formed in (D). The amounts of α–β′ crosslinks (normalized to EC15) are depicted as bars. Data represent mean ± SD (n = 4). The absolute values are provided in Table S1. Unpaired t test, ∗∗∗p <
(F) The effects of the nucleic acid scaffolds and GreA. The variant was preassembled with the EC15, EC16, BC17, or BC24 scaffold, and CPX was measured in the absence or presence of the GreA variant (D42A/E45A). The reaction was performed at 25°C for 90 min in pH 8.0 buffer.
(G) Quantitation of CPX formed in (F). ∗∗p < 0.01.
See also Figure S1 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Enzymatic Activities by RNAP in the Ratcheted Form
(A) Experimental setup for the RNA cleavage reaction. Cleavage of the 5′-fluorescently labeled 17-mer RNA in BC17 by RNAP yields a 15-mer RNA.
(B) Experimental setup for the nucleotide addition and its reversal. Upon the addition of a substrate NTP, the 15-mer RNA in EC15 is extended by one nucleotide to yield a 16-mer RNA. Conversely, the 16-mer RNA in EC16 is trimmed at its 3′ end to yield a 15-mer RNA upon the addition of pyrophosphate.
(C) Intrinsic RNA cleavage. The wild-type RNAP, the maximally (∼74%) oxidized CPX variant (CPXOX), and that rereduced with DTT (CPXRED) were assembled with the BC17 scaffold. The reaction was performed at 40°C, in 50 mM Tris-HCl (pH 7.8), 50 mM KCl, and 10 mM MgCl2. The graph depicts the time course of the reaction. The dashed line shows the curve of CPXRED multiplied by 0.26, to approximate the contribution of the uncrosslinked fraction (∼26%) in CPXOX. Data represent mean ± SD (n = 3) (for all graphs in this figure). For many data points, the errors were very small and are obscured by the graph symbols.
(D) GreA-dependent RNA cleavage (15°C). The inset shows a magnification of the earlier time points.
(E) Single nucleotide (UMP) addition to EC15 (20°C).
(F) Pyrophosphorolysis of EC16 (30°C).
See also Figure S2.
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5. Figure 3 Structure of the BC
(A) Overall structure of the BC in two orientations. The structural modules of RNAP are colored (core, gray; shelf, cyan; clamp, yellow-green; jaw-lobe and β′-NCD, light blue; BH, purple; TL, green). The nucleic acids are colored as in Figure 1B.
(B) Structural comparison with EC. The structures of BC [colored as in (A)] and EC (light orange, PDB 2O5I) are superimposed by their core modules.
(C) The BC catalytic site. The 2Fo–Fc electron density maps for DNA/RNA, FL, and TL are shown as blue meshes.
(D) The proofreading site. The active site of RNAP is viewed from the secondary channel.
(E) Comparison of the TL conformation with the backtracked and frayed RNAP II.
(F) The NTP-insertion complex (slate, PDB 2O5J), superimposed on BC via the shelf module. In the straight conformation, TL would clash with the extruded RNA 3ʹ end (A16) and FL.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Structure of the Gre-C1-Bound Complex
(A) Overall structure of the Gre-C1-bound RNAP in two orientations. Gre-C1 is shown in magenta, with a transparent surface. RNAP is colored as in Figure 3A. The catalytic magnesium (Mg I) is shown as an orange sphere.
(B) Structural comparison with the EC. The Gre-C1-bound RNAP (colored as in A) and EC (PDB 2O5I, brown) are superimposed by their core modules.
(C) Comparison of the TL conformation with the TFIIS-bound RNAP II.
(D) The CPX efficiencies in the CPX_ΔTL variant (normalized to EC16). The reaction was performed at 25°C for 90 min in pH 8.0 buffer. Data represent mean ± SD (n = 4). Unpaired t test, ∗∗∗p <
(E) Comparison of the active sites. The active sites in the NTP-insertion complex (PDB 2O5J), the BC, and the Gre-C1-bound RNAP are shown. The 2Fo–Fc electron density map for Gre-C1 is shown as a blue mesh (contoured at 1.0σ). The Cα atoms of Asp42 and Glu45 are shown as spheres.
(F) Comparison of the secondary channels.
See also Figures S4 and S5.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 The RNA-Binding Mode Switches upon Gre Binding
(A) The path of the RNA 3ʹ end switches upon Gre binding. The Gre-C1-bound complex is superimposed on the BC by their core modules. The RNA in BC is colored red, except for the +2 residue (A16), which is yellow. As no density was observed for RNA in the Gre-C1-bound complex, it was modeled and shown in green. The β Met560 residue is shown as a stick model.
(B) Intrinsic and GreA-dependent RNA cleavage activities by the wild-type and β M560W mutant RNAPs (40°C). An image of the denaturing gel and a graph of the time courses of RNA cleavage in BC17 are shown.
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8. Figure 6 Effects of Mismatch, his-Pause Scaffold, and Upstream Hairpin
(A) CPX efficiencies in the mismatch-paused complexes. The CPX variant was preassembled with the indicated scaffolds, and the S-S bond formation was measured in the absence or presence of the GreA variant (D42A/E45A). The reaction was performed at 25°C for 80 min in pH 8.0 buffer. Data represent mean ± SD (normalized to EC16, n = 4). Unpaired t test, ∗p < 0.05, ∗∗∗p <
(B) CPX efficiencies in the his-pause complex.
(C) CPX efficiencies in the terminator-hairpin-containing complex.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Structural States of RNAP for Distinct Transcription Functions
Schematic representations of the three structural states of RNAP and their relevance in transcriptional events.
Molecular Cell  , DOI: ( /j.molcel )
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