title: RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state.
abstract: Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. 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 Mn(2+) 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.
authors.affiliation: National Centre for Biological Sciences (NCBS-TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560065, India and Department of Microbiology and Cell biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Parag
authors.initials: P
authors.lastname: Surana
authors.forename: Vijaya
authors.initials: V
authors.lastname: Satchidanandam
authors.forename: Deepak T
authors.initials: DT
authors.lastname: Nair
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D001665:Binding Sites; D004664:Encephalitis Virus, Japanese / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D012313:RNA / Q000096:biosynthesis* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012313:RNA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012324:RNA-Dependent RNA Polymerase