Fig 1.
The modeled PPi-bound T7 RNA elongation complex.
(A) The overall structure of the complex (protein in white; DNA non-template strand in purple; DNA template strand in green; RNA strand in yellow). (B) The zoomed in view of the active site, with essential amino acids shown in the upper panel (blue licorice: positively charged; red licorice: negatively charged). PPi is shown in red spheres. The Mg2+ in the active site is in green sphere. The O-helix is shown in cyan and the PPi release channel is highlighted in wheat color. The lower panel provides a surface representation.
Fig 2.
The population density profile for conformations sampled along the simulated PPi release pathways.
The profile is shown on a potential-like surface along the PPi RMSD coordinate. Three population states are identified: S1a, S1b, and S2, with representative snapshots shown for each state. The color scheme in the structure is the same as that in Fig 1. The convergence tests of the profile is provided in SI S2 Fig.
Fig 3.
The MSM construction for the PPi release in the T7 RNAP elongation.
(A) A structural view of the PPi distribution (shown in spheres) sampled from the MD simulations. Each sphere represents the center of the mass of the PPi group. The distribution was generated from randomly chosen MD snapshots for states S1a (in purple sphere), S1b (in green), and S2 (in orange). (B) The three-state macrostate representation generated from the MSM construction. The equilibrium populations from the MSM are indicated. (C) Distance analyses between several key residues and the PPi group in the three-state representation.
Fig 4.
The O-helix rotation detected in microsecond MD simulations.
(A) The rotation angles of the O-helix were measured from simulation (i), (ii) and (iii), and are shown in blue, green, and red, respectively. (B) The molecular snapshot view of the O-helix (green) taken from the unperturbed simulation (i). The initial closed form of the O-helix is also shown (cyan). (C and D) The molecular snapshots taken from the off-charge simulation (ii) and the no PPi simulation (iii), respectively, with the O-helix shown as in (B).
Fig 5.
The side chain swing of Lys472 assists the PPi release.
(A) The molecular view of Lys472 side chain that swings from pointing inside toward PPi in the active site (S1) to pointing outside to the solvent (S2). The two major configurations of the side chain were sampled from the long simulation (i and iii) and from the SMD simulations. (B) The Lys472-MgA distance measured from the long simulation (i) and (iii), with and without PPi, respectively. (C) The 2D density map (-ln P) generated from many short simulations used for the MSM construction. The map is depicted along the distance between Lys472 (NZ) and MgA and along the PPi RMSD. (D) The Lys472-MgA distance change along with the PPi-MgA distance change, obtained from three SMD simulations for nanoseconds, ran from different initial conditions/directions to pull PPi out.
Fig 6.
Comparing T7 RNAP with other RNAPs or DNAPs on the key PPi interactions around the active site. (A) T7 RNAP (PDB:1S77); (B) yeast Pol II [26]; (C) bacterial RNAP [25]; (D) human mitochondrial RNAP (PDB:3SPA); (E) T7 DNAP (PDB:1T7P); (F) E. coli DNAP I (PDB:1KLN).
(G) A schematic of the jump from the cavity model, depicting the key lysine/arginine module that assists the PPi release from the active site (the colored disks represent the three meta-stable states as identified for T7 RNAP); (H) The sequence alignment of the single-subunit polymerases based on similarities of special sequence motifs and molecular structures. The essential charged residues for the PPi release are labeled. The conservations of corresponding residues in the sequence alignment are highlighted.