Polymerase front channel opening and conservation of RNA conformation.

<p><b>A)</b> Opening of the polymerase front channel to accommodate the exiting RNA shown by the comparison of polymerase structures in their apo and elongation complex forms. All ECs follow the three-color coding scheme while the apo polymerases are shown in green. From left to right: all five PV ECs compared to one apo form (PDB code 1RA6); one CV EC compared to two apo forms (PDB codes 3DDK and 3DCU); two RV ECs compared to two apo forms (PDB code 1XR7 chains A & B). The major conformational differences are highlighted by blue, pink, and grey arrows indicating the movement within the thumb, fingers, and palm domains, respectively. The α-carbon atoms and displacement distances of PV polymerase residues 408 and 440 in the thumb, 102 and 133 in the pinky finger, and 211 in the palm, and their structural equivalents in CV and RV polymerases, are shown as spheres to further highlight the movements. <b>B)</b> Maximum likelihood superimposition of all eight distinct (i.e. not NCS-related) EC dumbbell pairs with the protein backbone Cα atoms of the right side polymerase being used for the alignment. The RNA from the PV_r3 EC pair that contains the most “straight” RNA-RNA junction is shown in black for reference, other complexes are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060272#pone-0060272-g002" target="_blank">Figure 2</a>, and the downstream template RNA is omitted for clarity. Two views are shown to better depict the three dimensional variation of the partner complexes, with the “Top view” looking down into the active site and the “Side view” looking into the NTP entry channel. <b>C)</b> Stereo images of the upstream RNA duplex positions resulting from the protein-based structure alignment shown in panel B. The referencing PV_r3 duplex is in black, other PV duplexes are in yellow, CV duplex is in brown, and RV duplexes are in blue. The RNA helical axes as calculated by nucleic acids analysis program Curves+ are shown in thick sticks using the same coloring scheme. Consistent RNA conformations are observed from position −1 to position −8, with significant lateral variation of the base pairing starting around the −7/−8 base pair step. The middle panel shows the standard deviation of the RNA axis positions at each base step, mathematically suggesting that the upstream duplex conformation is only affected by the variation at the inter-complex junction beyond the −8 position. This corresponds to the point at which the RNA duplex exits from the polymerase and no longer contacts the thumb domain.