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Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRpGTP structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRpGTP structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a checkpoint to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.<\/jats:p>","DOI":"10.1093\/nar\/gkt1106","type":"journal-article","created":{"date-parts":[[2013,11,30]],"date-time":"2013-11-30T01:45:52Z","timestamp":1385775952000},"page":"2758-2773","source":"Crossref","is-referenced-by-count":72,"title":["RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state"],"prefix":"10.1093","volume":"42","author":[{"given":"Parag","family":"Surana","sequence":"first","affiliation":[{"name":"National Centre for Biological Sciences (NCBS-TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560065, India and 2Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vijaya","family":"Satchidanandam","sequence":"additional","affiliation":[{"name":"National Centre for Biological Sciences (NCBS-TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560065, India and 2Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deepak T.","family":"Nair","sequence":"additional","affiliation":[{"name":"National Centre for Biological Sciences (NCBS-TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560065, India and 2Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore 560012, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2013,11,28]]},"reference":[{"key":"2020112407563946100_gkt1106-B1","volume-title":"Flaviviridae: The Viruses and Their Replication","author":"Lindenbach","year":"2007","edition":"5th edn"},{"key":"2020112407563946100_gkt1106-B2","doi-asserted-by":"crossref","first-page":"e437","DOI":"10.1371\/journal.pntd.0000437","article-title":"Japanese encephalitis-a pathological and clinical perspective","volume":"3","author":"Ghosh","year":"2009","journal-title":"PLoS Negl. 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