SARS-CoV-2 RdRp Follows Asynchronous Translocation Pathway for Viral Transcription and Replication

RNA-dependent RNA polymerase (RdRp) is the replicase machinery for SARS-CoV-2 and thereby it has become one of the most promising drug targets to combat the pandemic as well as the healthy threat posed by the novel coronavirus. Translocation is one essential step for RdRp to exert the viral replication and transcription, and it describes the dynamic process in which the double-stranded RNA moves upstream by one base pair position to empty the active site for the continuous substrate incorporation. However, the molecular mechanisms underlying the dynamic translocation of SARS-CoV-2 RdRp remain elusive. In the current study, we have elucidated the molecular insights into the translocation dynamics of SARS- CoV-2 RdRp by constructing a Markov State Model based on extensive molecular dynamics simulations. We have identified two previously uncharacterized intermediates which pinpoint an asynchronous and rate-limiting translocation of the nascent-template duplex. The movement of the 3’-terminal nucleotide in the nascent strand lags behind its upstream nucleotides due to the uneven protein environment while the translocation of template strand is delayed by the hurdle residue K500. Although the motions of the two strands are not synchronous, they share the same “ratchet” to stabilize the system in the post-translocation state, suggesting a coupled Brownian-ratchet model. Overall, our study has provided the intriguing insights into the translocation dynamics with unprecedented molecular details, which would significantly deepen our understanding about the transcriptional mechanisms of SARS-CoV-2.