1. Dynamic Error Correction and Regulation of Downstream Bubble Opening by Human RNA Polymerase II 
Xue Q. Gong, Chunfen Zhang, Michael Feig, Zachary F. Burton 
Molecular Cell 
Volume 18, Issue 4, Pages (May 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 NTP-Driven Translocation
(A) Pretranslocated RNAP II EC, n + 1 GTP (n = RNA length) (blue, space filling; phosphates in white) substrate loaded in the RNAP II main channel i + 2 site.
(B) Incoming GTP passing over the bridge α helix (green).
(C) Incoming n + 1 GTP in the i + 1 active site, oriented toward the secondary pore. Relevant protein density is indicated. Invariant or highly conserved amino acids Rpb1 Ala828 (yellow, space filling), Rpb2 Arg512, and Rpb2 Glu529 that show the greatest displacement during GTP loading are indicated (S. cerevisiae) (Gnatt et al., 2001; Westover et al., 2004a). RNA is red; DNA is orange. The n GMP is red (space filling); the 3′-oxygen is silver. The active site Mg2+-A is purple. An animation of the NTP-driven translocation model is in Movie S1 and
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
By Using the 40-CAAAGGCCTTT-50 Template, i + 2 GTP Drives *A43·GTP→A43 Isomerization Reversal
(A) Model to describe isomerization reversal (IR). DNA is brown. RNA is red. NTP substrates are blue. α-amanitin is orange. Mg2+-A is magenta.
(B) Representative gel data. Times are in seconds (s). The isomerized *A43·GTP EC reverts to A43 with exposure to 2.5 mM GTP and 0.5 mM α-amanitin (EDTA quench). A41 results from TFIIS-induced dinucleotide cleavage from A43 (Zhang et al., 2003).
(C) Isomerization reversal. Filled symbols indicate EDTA quench (isomerization). Open symbols indicate HCl quench (bond synthesis). Red symbols indicate G44+ % (G44 plus all longer transcripts), + α-amanitin. Black symbols indicate G45+ %, + α-amanitin. Gray symbols indicate control reactions with no α-amanitin added (G44+ % [squares] and G45+ % [circles]) (Zhang and Burton, 2004). Data were calculated from A43, neglecting A41 and shorter transcripts, which are slow to advance. Post and Pre indicate fractions of post- and pretranslocated A43 ECs, respectively. Error bars indicate SD of three or four independent experiments. For some data points, symbols obsure error bars.
(D) Isomerization reversal is not observed at 500 and 100 μM GTP + α-amanitin.
(E) NTP dependence of isomerization reversal with the 40-CAAAGGCCTTT-50 template (EDTA quench). Where shown, error bars indicate SD of three or four independent experiments.
The reaction protocol and RNA sequence are shown (B–E).
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4. Figure 3 The i + 2 and i + 3 NTPs Drive Isomerization Reversal
Protocols and sequences are indicated.
(A) i + 2 and/or i + 3 CTP drive *A43·GTP →A43 isomerization reversal with the 40-CAAAGCCTTT-49 template. The gray line indicates the reaction with no α-amanitin added (EDTA quench). GC indicates 2.5 mM GTP and CTP were added. Error bars represent SD of three independent experiments.
(B) 2.5 mM dCTP fails to drive *A43·GTP→A43 isomerization reversal. Failure of the EDTA and HCl quench curves to converge indicates that pyrophosphate release is not complete at 1 s. Error bars represent SD of three independent experiments.
(C) Only CTP drives *A43·GTP→A43 isomerization reversal. *A43·GTP IR % is calculated from comparison of the 0.01 and 0.2 s time points (EDTA quench). A negative IR % indicates that the reaction proceeds in the forward direction after 0.01 s. Error bars represent SD of three independent experiments.
Experiments with the 40-CAAAGCCTTT-49 template (A–C).
(D) Experiments with the 40-CAAAGACTTT-49 template. By using the 40-CAAAGACTTT-49 template, i + 2 ATP and i + 3 CTP drive *A43·GTP→A43 isomerization reversal. Indicated chase NTP additions are at 2.5 mM (EDTA quench). The minimum working concentrations of ATP, CTP, and UTP are 5 μM during the chase because of prior additions.
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5. Figure 4 NTP-Driven Dynamic Error Correction by Human RNAP II
Protocol and RNA sequence are shown. No TFIIS was added. CTP suppresses misincorporation of AMP for GMP at the G44 position with the 40-CAAAGCCTTT-49 template. Misincorporation with ATP alone (lanes 2–6), ATP + CTP (lanes 7–11), and ATP + UTP (lanes 12–16). A44* misincorporation percentage is indicated for lanes 6, 11, and 16 (ovals). This is misincorporation rather than scavenging of trace GTP, because A44* ECs do not rapidly advance in the presence of 2.5 mM CTP (lanes 10 and 11). G44 ECs would advance to stall at C46. No α-amanitin was added.
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6. Figure 5
By Using the 40-CAAAGTCTTT-49 Template, i + 2 UTP and i + 3 CTP Stimulate Maintenance of the Posttranslocated EC Stalled at A43
(A) Model describing regulation of downstream bubble opening in the presence of i + 2 and i + 3 NTPs. Colors are as shown in Figure 2A.
(B) Maintenance of the open bubble. Indicated UTP and CTP additions were made during the 10 μM ATP pulse. 2.5 mM GTP was added, and reactions were quenched (EDTA) after s. Only posttranslocated A43 ECs isomerize within s so that these ECs can advance to G44 (Gong et al., 2004; Zhang and Burton, 2004; Zhang et al., 2005). Error bars indicate SD of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Main Channel NTP Loading, Regulation of the Open Downstream Bubble, and α-amanitin Binding
The template DNA strand is orange. RNA is red. Nontemplate DNA is blue-gray. NTPs are blue (i + 1), red (i + 2), and silver (i + 3). Other features are labeled. The image is based on yeast RNAP II structures (Bushnell et al., 2002; Gnatt et al., 2001; Westover et al., 2004b).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions