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. 2012 Jun 7:5:11.
doi: 10.1186/2046-1682-5-11.

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

Maria L Kireeva  1 , Kristopher Opron, Steve A Seibold, Céline Domecq, Robert I Cukier, Benoit Coulombe, Mikhail Kashlev, Zachary F Burton
Affiliations

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

Maria L Kireeva et al. BMC Biophys. .

Abstract

Background: During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile "trigger loop" of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the "bridge helix" that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing.

Results: All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as "switch" residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop.

Conclusions: Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation.

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Figures

Figure 1
Figure 1
A model for trigger loop hinges, bridge helix hinges and bridge helix bending modes based on all atom molecular dynamics simulations. At the top of the figure, diagrams of the closed TEC, the closed product TEC (after chemistry) and the translocating TEC are shown. DNA is grey; RNA is red; the NTP substrate (or incorporated NMP and pyrophosphate) is blue; the trigger loop (TL) is purple; the bridge helix (BH) is yellow. Interpretations of simulations are shown schematically below. Simulations indicate trigger loop hinges H1 and H2, bridge helix hinges H3 and H4 and bridge helix bend modes B1 (straighter) and B2 (more sharply bent).
Figure 2
Figure 2
The open trigger loop RNAP TEC appears to be the translocating conformation. Vectors describe downstream DNA/DNA and upstream RNA/DNA translocation during simulation. The downstream translocation vector is drawn through DG5 (DNA template dGMP 5) and G37 (RNA GMP 37) 3′ carbons. Translocation is measured as the displacement of an orthogonal projection of DA40 (non-template DNA dAMP 40) 3′ carbon onto the vector relative to the initial structure. The upstream translocation vector is drawn through DT14 (DNA template dTMP 14) and C31 (RNA CMP 31) 3′ carbons. Translocation is measured as the displacement of an orthogonal projection of G33 (RNA GMP 33) 3′ carbon onto the vector relative to the initial structure. Closed-Mg (green) and open (blue) TECs are shown. B) Scatter plot of upstream and downstream translocation (each point represents a 20 ps interval). “X” indicates 10.5 ns time points for the three simulations relative to a (0,0) starting position in the original crystal structures (2O5J for closed; 2PPB for open).
Figure 3
Figure 3
The trigger loop hinges H1 (1230-GEPGTQ-1235) and H2 (1255-GLP-1257). Snapshots of the trigger loop region in open, closed and closed-Mg TECs are compared at about 10 ns simulation. Coloring is in “secondary structure” representation: helix is magenta, 310 helix is blue, coil is white, turn is cyan, β-sheet is yellow. G1230, P1232, H1242, G1255 and P1257 are in stick representation to locate the H1 and H2 hinges and orient the view. ATP and Mg are in space filling representation to locate the active site. Blue arrows indicate differences in secondary structure that develop during simulation.
Figure 4
Figure 4
The bridge α-helix hinges and bending. To the right, 10 ns snapshots of closed, closed-Mg and open bridge helices are compared, colored in secondary structure representation, as in Figure 3. G1076, G1080 and G1081 are in space filling representation so that H3 and H4 hinges can be identified. To the left, proposed switch residues β’ H1075, K1079, D1083, R1087, D1090 and β D429 are shown in stick representation for closed-Mg and open TEC bridge helix structures. Blue arrows indicate differences in secondary structure that develop during simulations.
Figure 5
Figure 5
Transcriptional activities of hinge H1 to H4 amino acid substitutions. A) Elongation and fidelity assays. Blue bars represent transcriptional elongation rate relative to wild type RNAP II at 200 μM NTP concentration. Error bars indicate standard error from exponential curve fitting. Red bars represent transcriptional fidelity, determined by a competition assay (Additional file 1: Figures S9 and S10). Error bars are standard deviation of at least three independent determinations. B) Conserved ionic interactions surrounding the Sc RNAP II H2 hinge.
Figure 6
Figure 6
Exonuclease III mapping of Sc RNAP II TECs. A) Back border (upstream border) 3′-dA11 Sc RNAP II TEC mapping. 3′-dA11 (3′-dATP chain terminated A11 TECs) RNA controls, run in the same reaction, are shown to demonstrate effective chain termination at the A11 position. The strategy for mapping Sc RNAP II TECs in the absence or presence of incoming CTP is indicated at the bottom of the figure. B) Phosphorimager quantification of triplicate determinations done at 30 s exonuclease III incubation. C) Percent increase in the post-translocated state induced with CTP compared to wild type Sc RNAP II, from the data shown in B. Error bars represent standard deviation.
Figure 7
Figure 7
Proposed interactions between bridge helix switch residues β’ H1075 and K1079 and the fork region. Fork residues β R420, D426 and D429 are proposed from simulations to be switch residues. Interacting residues β R142 and R331 also appear to be involved in the switching mechanism.
Figure 8
Figure 8
A conserved conformational switching mechanism centered on β’ D784 about 20 Å from H1. The substrate ATP, Mg-I and Mg-II (green) are in space-filling representation. Proposed switch residues are in stick representation. The two orientations of D784 observed in simulations of closed and open trigger loop RNAP TECs are indicated with blue arrows. Some atomic interactions are indicated with blue lines or emphasized with blue circles.
Figure 9
Figure 9
Transcriptional activities and fidelity of Sc RNAP II substitutions in the active site and proposed switch residues (as in Figure5A).
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