Literature summary for 2.7.7.48 extracted from

Surana, P.; Satchidanandam, V.; Nair, D.T.
RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state (2014), Nucleic Acids Res., 42, 2758-2773.

Activating Compound

Activating Compound	Comment	Organism	Structure
GTP	flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus	Japanese encephalitis virus

Cloned(Commentary)

Cloned (Comment)	Organism
the gene segment corresponding to the RdRp module corresponds to amino acid 272905 of the NS5 protein, recombinant expression of the enzyme as N-terminally GST-tagged protein in Escherichia coli strain C41(DE3)	Japanese encephalitis virus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme in apo-form or in complex with ATP or GTP, jRdRp (0.08 mM) is incubated with GTP/ATP (5 mM) for 30 min before setting crystallization trials, screening and method optimization, hanging drop vapour diffusion method, mixing of protein-ATP/GTP with 10-18% w/v PEG 4000, 100 mM Tris-Cl buffer, pH 8.0, and 7% v/v 1-propanol, 25°C, for the apo-enzyme mixing of 0.096 mM protein with 10-15% PEG 5000 mono methyl ether, 100 mM Tris-Cl, pH 8.0, and 200mM NaSCN, 25°C, X-ray diffraction structure determination and analysis at 2.28-3.65 A resolution, molecular replacement using the apo-enzyme structures of WNV and DENV, PDB IDs 2HFZ and 2J7W, as search models	Japanese encephalitis virus

Crystallization (Comment)

Organism

purified recombinant enzyme in apo-form or in complex with ATP or GTP, jRdRp (0.08 mM) is incubated with GTP/ATP (5 mM) for 30 min before setting crystallization trials, screening and method optimization, hanging drop vapour diffusion method, mixing of protein-ATP/GTP with 10-18% w/v PEG 4000, 100 mM Tris-Cl buffer, pH 8.0, and 7% v/v 1-propanol, 25°C, for the apo-enzyme mixing of 0.096 mM protein with 10-15% PEG 5000 mono methyl ether, 100 mM Tris-Cl, pH 8.0, and 200mM NaSCN, 25°C, X-ray diffraction structure determination and analysis at 2.28-3.65 A resolution, molecular replacement using the apo-enzyme structures of WNV and DENV, PDB IDs 2HFZ and 2J7W, as search models

Japanese encephalitis virus

Protein Variants

Protein Variants	Comment	Organism
D541A	site-directed mutagenesis, the mutant's elongation activity is marginally reduced compared to the wild-type enzyme	Japanese encephalitis virus
R734A	site-directed mutagenesis, the mutant catalyzes elongation almost identically to the wild-type enzyme	Japanese encephalitis virus
R742A	site-directed mutagenesis, the mutant catalyzes elongation almost identically to the wild-type enzyme	Japanese encephalitis virus
S604A	site-directed mutagenesis, the mutant's elongation activity is marginally reduced compared to the wild-type enzyme	Japanese encephalitis virus
S604A-D541A	site-directed mutagenesis, the mutant shows no detectable primer extension activity	Japanese encephalitis virus
S799A	site-directed mutagenesis, the mutant catalyzes elongation almost identically to the wild-type enzyme	Japanese encephalitis virus
S799Y	site-directed mutagenesis, the mutant catalyzes elongation almost identically to the wild-type enzyme	Japanese encephalitis virus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	dissociation constants for the interaction of jRdRp with RNA in the presence/absence of NTPs and Mn2+	Japanese encephalitis virus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mn2+	required, GTP binding reduces the affinity of jRdRp for RNA in the absence of Mn2+, binding affinity of jRdRp toward RNA in the presence of GTP/ATP is restored when Mn2+ ion is included in the reaction buffer	Japanese encephalitis virus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
additional information	Japanese encephalitis virus	flaviviral RNA-dependent RNA polymerases 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. Flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. 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	?	-	?
additional information	Japanese encephalitis virus P20778	flaviviral RNA-dependent RNA polymerases 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. Flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. 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	?	-	?
nucleoside triphosphate + RNAn	Japanese encephalitis virus	-	diphosphate + RNAn+1	-	?
nucleoside triphosphate + RNAn	Japanese encephalitis virus P20778	-	diphosphate + RNAn+1	-	?

Organism

Organism	UniProt	Comment	Textmining
Japanese encephalitis virus	O90417	polyprotein; a flavivirus	-
Japanese encephalitis virus P20778	O90417	polyprotein; a flavivirus	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally GST-tagged enzyme from Escherichia coli strain C41(DE3) by glutathione affinity chromatography and gel filtration	Japanese encephalitis virus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	flaviviral RNA-dependent RNA polymerases 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. Flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. 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	Japanese encephalitis virus	?	-	?
additional information	ATP and GTP enzyme binding structures, detailed overview. The triphosphate moiety of GTP interacts with the side chains of basic residues from motif F (R460, K463, K471 and R474) and from motif E (R734 and R742)	Japanese encephalitis virus	?	-	?
additional information	flaviviral RNA-dependent RNA polymerases 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. Flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. 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	Japanese encephalitis virus P20778	?	-	?
additional information	ATP and GTP enzyme binding structures, detailed overview. The triphosphate moiety of GTP interacts with the side chains of basic residues from motif F (R460, K463, K471 and R474) and from motif E (R734 and R742)	Japanese encephalitis virus P20778	?	-	?
nucleoside triphosphate + RNAn	-	Japanese encephalitis virus	diphosphate + RNAn+1	-	?
nucleoside triphosphate + RNAn	primer-free initiation assay with 13-nt RNA template, and ATP, CTP, FAM-UTP, and GTP, and additionally with a primer (5'-GUUCACACAGAUAAACUUCU-3') with a 6-FAM-labeled at the 5'-end in the primer extension assay	Japanese encephalitis virus	diphosphate + RNAn+1	-	?
nucleoside triphosphate + RNAn	-	Japanese encephalitis virus P20778	diphosphate + RNAn+1	-	?
nucleoside triphosphate + RNAn	primer-free initiation assay with 13-nt RNA template, and ATP, CTP, FAM-UTP, and GTP, and additionally with a primer (5'-GUUCACACAGAUAAACUUCU-3') with a 6-FAM-labeled at the 5'-end in the primer extension assay	Japanese encephalitis virus P20778	diphosphate + RNAn+1	-	?

Synonyms

Synonyms	Comment	Organism
jRdRp	-	Japanese encephalitis virus
RDRP	-	Japanese encephalitis virus
RNA-dependent RNA polymerase	-	Japanese encephalitis virus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	37	assay at	Japanese encephalitis virus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Japanese encephalitis virus