Fig. 4: Enzymology characterization of EV71 RdRP and its mutants bearing mutation(s) in the motif G hurdle residues. | Nature Communications

Fig. 4: Enzymology characterization of EV71 RdRP and its mutants bearing mutation(s) in the motif G hurdle residues.

From: Stringent control of the RNA-dependent RNA polymerase translocation revealed by multiple intermediate structures

Fig. 4

a A structure-based sequence alignment of RdRP motif G. Representative RdRPs from positive-strand (+RNA), double-stranded (dsRNA), and negative-strand (-RNA) RNA viruses were chosen for comparison (PICO/PV, EV71: Picornaviridae/poliovirus, enterovirus 71; CALI/NV: Caliciviridae/norovirus; FLAV/JEV, CSFV, HCV: Flaviviridae/Japanese encephalitis, classical swine fever, hepatitis C viruses; LEVI/Qb: Leviviridae/bacteriophage Qβ; PERM/TaV: Permutotetraviridae/thosea asigna virus; CORO/SARS: Coronaviridae/severe acute respiratory syndrome coronavirus; CYST/PHI6: Cystoviridae/bacteriophage ϕ6; REOV/LAM3, ROTA: Reoviridae/reovirus λ3, rotavirus; BIRN/IBDV: Birnaviridae/infectious bursal disease virus; PBIR/hPBV: Picobirnaviridae/human picobirnavirus; OMYX/IBV: Orthomyxoviridae/influenza B virus; RHAB/VSV: Rhabdoviridae/vesicular stomatitis virus; BUNY/LACV: Peribunyaviridae/La Crosse bunyavirus; PNEU/hMPV: Pneumoviridae/human metapneumovirus). Hurdle residues are highlighted by the red box. b The sequences of the T33-Fint/P10 construct used in the stopped-flow assay and the data from the WT construct showing faster fluorescence signal decrease with higher CTP concentration. Fint denotes a fluorescein-labeled nucleotide at an internal position. When GTP and ATP were provided as the only NTP substrates, the 10-mer primer (P10) was extended to a 14-mer product for EC assembly (the incorporated G-A-G-A tetra-nucleotide is boxed). The assembled EC was then used in stopped-flow trials by mixing with CTP for monitoring the single-nucleotide conversion from the 14-mer to a 15-mer. c A comparison of catalytic efficiency for WT and hurdle residue mutants. All constructs were classified into five categories based on their kinetics behaviors (Supplementary Fig. 4). The error bars indicate propagated fitting errors (Supplementary Table 1). d Michaelis–Menten curve fitting of the WT and ST mutant data. KM values with fitting errors were indicated.

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