Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

36-POS Board 36 NTP-ASE ACTIVITY OF ZIKA VIRUS NS3 HELICASE AND ITS COUPLING WITH RNA UNWINDING ACTIVITY Evelyn Mikkelsen 1 ; Leopoldo G Gebhard 2 ; Leila A Cababie 1 ; Jeremías J Incicco 1 ; Rolando C Rossi 1 ; Andrea V Gamarnik 3 ; Sergio B Kaufman 1 ; 1 Instituto de Química y Fisicoquímica Biológicas & Depto. de Ciencias Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires-CONICET, Ciudad de Buenos Aires, Argentina 2 Universidad Nacional de Quilmes-CONICET, Depto. de Ciencia y Tecnología, Quilmes, Argentina 3 Fundación Instituto Leloir-IIBBA-CONICET, Ciudad de Buenos Aires, Argentina Zika virus (ZIKV) nonstructural protein 3 (NS3) is a motor protein that unwinds double- stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates (NTPs). The aim of this work is to characterize the NTPase activity of ZIKV NS3 helicase, to determine the specificity order for the substrates: ATP, CTP, GTP and UTP, and to establish the ability of these four substrates to drive the unwinding activity. To initiate the characterization of NS3 NTPase activity we studied the hydrolysis of NTPs under steady-state conditions. Initial rates of reaction were determined measuring the Pi released from NTP hydrolysis. The substrate curves obtained for ATP, GTP, CTP and UTP were, in all cases, well described by hyperbolic functions whose parameter values (k cat and K M ) were obtained by non-linear regression analysis. The order of specificity of NS3 for these nucleotides, which was evaluated according to the value of the ratio k cat /K M , was ATP > GTP > CTP ≅ UTP. Additionally, different nucleotides were tested as substrates for the RNA unwinding activity of NS3 helicase. RNA unwinding reactions were evaluated by fluorescence spectroscopy using fluorescently labeled RNA oligonucleotides. Results indicate that NS3 helicase acts as an energy transducer using any of the four nucleotides tested as substrates. That is, in all four cases the helicase couples its catalytic properties (NTPase activity) with the ability to perform the mechanical work of translocation along single stranded RNA and unwinding of double stranded RNA.

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