Computational protocol: T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking

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Protocol publication

[…] Based on the modeled pre- and post-trans states of the T7 RNAP elongation complexes (ECs), we first performed a Climber simulation () to obtain an initial low-energy translocation pathway. The conformations generated by the above-mentioned Climber simulation were then used as the initial seeds for the subsequent two rounds of equilibrium MD simulations. Finally, we used the dataset from the second round of MD simulations to construct the MSM and reveal the key intermediate states involved in the translocation process along with the associated thermodynamic and kinetic properties. The main procedures are summarized in Figure . We additionally employed an isomap analysis () to determine whether the initial pathway dictates the actual translocation captured by the MSM. [...] Based on the modeled pre- and post-trans structures, we first obtained an initial low-energy translocation pathway using a modified version of the Climber algorithm (). We selected the fingers domain (residues from 554 to 812) and all DNA/RNA chains as the morphed regions since these regions undergo substantial conformational changes before and after the translocation as observed from the crystal structures (,). During the Climber simulation, the external forces were applied only to the atoms in the morphed region, whereas the remainder of the system could respond to the structural changes in the morphed region during each morphing cycle. The target number of morphing cycles was set to 1200, and each morphing cycle consisted of 100 iterations of morphing with 10 steps of conjugate gradient-energy minimization for each 10 morphing iterations. The minimum distance (root mean square deviation, RMSD = 0.5 Å) to the target structure was reached after 600 morphing cycles. Finally, to generate the starting conformations for the subsequent MD simulations, we chose 20 representative structures from the above-mentioned Climber simulation using the k-centers clustering algorithm implemented in the MSMbuilder software (–). The chosen distance metric consisted of the RMSD values of the heavy atoms of the TN and the two adjacent nucleotides (nts), the heavy atoms of the RNA 3΄-end nt, and the Cα atoms of 64 residues in the fingers domain (residues 627–690). Then, we chose a central conformation for each cluster as the representative structure. [...] We performed two rounds of MD simulations. The first round of MD simulations was initiated from the 20 representative conformations obtained from the Climbing simulation (20 × 20 ns NPT simulations at 1 bar, 310 K). To eliminate the biases introduced by the Climber simulations, we selected 100 representative conformations from the first round of MD simulations (see upper right in ). To ensure that we included enough transitions between the different states, we randomly selected 30 additional conformations from the first round of MD simulations (see upper right in ). We then used these 130 conformations as the starting structures for the second round of MD simulations (NPT simulations at 1 bar, 310 K). Finally, we only used the dataset from the second round of simulations to construct the MSM. However, one particular trajectory generated from one of the 30 additional MD conformations had no direct transitions to other MD trajectories, which prohibited the construction of a connected transition network. Thus, we removed this particular trajectory from our model construction. Therefore, in total, we used 129 MD trajectories (129 × 80 ns ∼10.3 μs) for the MSM construction.All MD simulations were performed using the GROMACS-5.1 software package (–). The AMBER99sb force field with PARMBSC0 nucleic acid parameters (–) was used to describe the system. The glycosidic angles of most of the RNA nucleotides adopted an anti-form in the simulation (), except for the unstably paired i-site nucleotide, which demonstrated that the PARMBSC0 force field can describe the DNA/RNA hybrid system well. The whole cubic simulation box contained 29,300 Tip3p water molecules. To neutralize the electrostatic charges of the system and maintain the ionic concentration at 0.15 M, 139 Na+ ions and 94 Cl– ions were added. The final system contained a total of 103,146 atoms. The cut-off values for the van der Waals (vdW) and short-range electrostatic interactions were set to 10 Å. Long-range electrostatic interactions were treated using the Particle-Mesh Ewald method (). The neighbor list was updated after every five steps, and the time step was 2 fs.For the first round of MD simulations, we adopted the following procedure for each of the 20 starting conformations: (i) 2000 steps of energy minimization using the steepest descent algorithm; (ii) 500 ps of NPT equilibration by position restraining the heavy atoms of the system with a force constant of 1000 kJ mol−1 nm2. The temperature was maintained at 310 K using a velocity rescaling thermostat () with a coupling constant of 0.1 ps−1; and (iii) production runs of 20-ns NPT MD simulations were conducted at 310 K and 1 atm using the velocity rescaling thermostat and the Parrinello-Rahman Barostat (,), respectively.We then applied the k-centers clustering algorithm to cluster the MD conformations obtained during the first round of MD simulations into 100 groups. Before the clustering, the first 5-ns simulation data of each 20-ns MD trajectory were discarded to eliminate the bias from the Climber simulation. We then chose the central conformation of each of the 100 clusters as the representative conformation and added another 29 conformations to increase the transition counts between certain regions. Finally, these 129 conformations were used to perform the second round of MD simulations. The MD setup was the same as that used during the first round, and the production runs were set to 80 ns each.Finally, we performed an isomap analysis by projecting the representative conformations obtained by the Climber simulation and those obtained by the unbiased MD simulations onto the three top isomap vectors (). The MD conformations substantially deviated from the initial translocation pathway obtained by the Climber simulation, and the 2D isomap projections for the five sub-datasets obtained by the MD simulations became similar (see ). Thus, the analysis indicated that our samplings reached convergence reasonably well, and the unbiased MD simulations had successively eliminated the bias introduced by the Climber simulation. […]

Pipeline specifications

Software tools Climber, MSMBuilder, GROMACS
Application Protein structure analysis
Diseases Carcinoma, Transitional Cell, Genetic Diseases, Inborn
Chemicals Amino Acids, Nucleotides