Structural Basis For Catalysis And Translocation In Viral RNA-dependent RNA Polymerase

RNA viruses,serious threat to human health,are the major causative agents of infectious diseases.Genomic replication and gene transcription of RNA viruses is performed by RNA-dependent RNA polymerase(RdRP).Under the guidance of the template sequence,the polymerase can add N TP or dNTP as the substrate to the product chain.The addition process is also called as the nucleotide add ition cycle(NAC),which includes the process of substrate-induced active site closure,the phosphoryl transfer reaction and translocation which moves the polymerase one position downstream of its template.The molecular mechanism of these processes is an essential content in the field of polymerase research.Poliovirus(PV)Rd RP-RNA-NTP complex crystal structure reveals the unique pattern of Rd RP active site closure.And the hypothesis of Rd RP translocation intermediate is proposed according to these structures.In current study,Rd RP of enteroviruses,the main pathogens of hand-foot-and-mouth disease,have been used to obtain Rd RP complex crystals.By achieving single and multiple nucleotide additions in these crystals,we have solved seven RdRP complex crystal structures at a resolution of 2.5-2.9 ? including the first translocation intermediates.These structures show key processes in the EV71 polymerase catalytic cycle,including the NTP binding,the active site closure of the polymerase,and the phosphoryl transfer reaction.It is also confirmed that RdRP has distinct uniqueness in closure of active site and translocation compared to other major polymerase types.The structure of translocation intermediate in this study shows that the Rd RP template and the product duplex undergoes an asymmetric movement in the translocation,and the movement of the product chain is significantly ahead of the template chain.The intermediate provides the key information for analysis of the translation mechanism.The asymmetric movement of the template and product strands has never been observed or even considered before,and may have great potential for high impact in the nucleic acid polymerase research field.