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

Collective variable (CV) identification is challenging in complex dynamical systems. To study the 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. RNAP translocation is an essential mechanical step of transcription elongation that dictates gene expression. The translocation generally follows from polymerization product release and proceeds to initial binding or preinsertion 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 mechanochemical coupling mechanisms may be distinct. In the current work, we modeled POLRMT elongation complexes and performed equilibrium MD simulations on the pre- and post-translocation models, with extensive samplings around two 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 coordinate sets to best represent the sampled features from the MD simulations. Our results indicate that POLRMT translocation is likely coupled with NTP binding to enable fingers subdomain opening at post-translocation which would otherwise be nonstabilized, or the translocations may proceed futilely without the fingers opening for incoming NTP initial binding or incorporation. The time scale of the coupled translocation reaches over hundreds of microseconds, as predicted by the VAMPnets analyses. Such a time scale seems to match a last postcatalytic kinetic step suggested for the POLRMT elongation cycle by previous experimental measurements. Our MD simulation studies combining atomic coordinate refinements and dimension reduction analyses on top of extensive conformational samplings thus suggest a variation of Brownian ratcheting in POLRMT translocation, as if the Brownian motions of translocation are coupled with NTP binding, which captures transient fingers subdomain opening to couple the translocation with a sustained fingers opening.