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Transient kinetic analysis of the elongation mode of Dengue Virus RNA polymerase domain
Author(s) -
Jin Zhinan,
Deval Jerome,
Johnson Kenneth A.,
Swinney David C.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb75
Subject(s) - elongation , polymerase , rna , rna polymerase , chemistry , nucleotide , biophysics , primer (cosmetics) , microbiology and biotechnology , enzyme , biology , biochemistry , gene , materials science , organic chemistry , metallurgy , ultimate tensile strength
Dengue viral RNA polymerase replicates its positive single‐stranded RNA genome in two phases: de novo initiation and elongation. The slow and inefficient initiation during replication masks the elongation mode. The aim of this work was to develop methodology to characterize the mechanism of elongation. Formation of the elongation complex required preincubation of the enzyme with a double stranded RNA (primer/template) for 1 hour at 37 °C in the presence of magnesium. The elongation complex incorporated a correct nucleotide at a rate of ~20 s −1 . The fidelity of the polymerase, ~ 2 x 10 −5 , was determined by measuring the specificity of incorporation of correct and incorrect nucleotides. The reaction pathway comprised of enzyme and RNA binding followed by nucleotide binding and incorporation was studied by transient kinetic methods. The microscopic rate constants for each step of the pathway were obtained by global data fitting and computer simulation.