Collective Coordinates and Facilitated Conformational Opening during Translocation of Human Mitochondrial RNA Polymerase (POLRMT) from Atomic Simulations

Collective coordinate (CV) identification is challenging in complex dynamical systems. To study translocation of a single-subunit RNA polymerase (RNAP) during human mitochondrial transcription, we employed all-atom molecular dynamics (MD) as a vehicle to illustrate CV refinement in conformational samplings and dimension reduction analyses. The RNAP translocation is an essential mechanical step of the transcription elongation that dictates gene expression. The translocation generally follows from polymerization product release and proceeds to initial binding or pre-insertion of incoming nucleotides. The human mitochondrial DNA-dependent RNAP (or POLRMT) plays a critical role in cellular metabolism and can be a key molecular off-target in the design of nucleotide analogue antiviral and antitumor drugs due to its structural similarities with many viral RNAPs or RNA dependent RNA polymerases (RdRps). While POLRMT shares particularly high structural similarity with bacteriophage T7 RNAP, previous experimental studies and our current simulations suggest that POLRMT’s mechano-chemical coupling mechanisms may be distinct. In our current work, we modeled POLRMT elongation complexes and performed equilibrium MD simulations on the pre- and post-translocation models, with extensive samplings around different potential translocation paths (with or without coupling to the fingers subdomain conformational change). We then compared time-lagged independent component analysis (tICA) and the neural network implementation of the variational approach for Markov processes (VAMPnets) as dimensional reduction methods on selected atomic CV sets to best represent the sampled data from the MD simulations. Our results indicate that POLRMT translocation is likely coupled with and facilitated by NTP binding to enable fingers subdomain opening at post-translocation which would otherwise be non-stabilized, or the translocations may proceed in futile without leading to the fingers opening for incoming NTP initial binding or incorporation. The timescale of the NTP binding-coupled or facilitated translocation reaches over hundreds of microseconds as predicted by the VAMPnets analyses. Such a timescale seems to match a last post-catalytic kinetic step suggested for the POLRMT elongation cycle by previous experimental detections. By inclusion of NTP binding/catalytic motifs into the analyses, the predicted time scale slightly increases as if additional protein dynamics related to NTP binding activities are considered. Thus, our MD simulation studies combining CV refinements and dimension reduction analyses on top of extensive conformational samplings suggest a distinct mechano-chemical coupling mechanism of POLRMT translocation as if the Brownian motions are slightly assisted by NTP binding.