A quantitative stopped-flow fluorescence assay for measuring polymerase elongation rates.
The measurement of nucleic acid polymerase elongation rates is often done via a lengthy experimental process involving radiolabeled substrates, quenched elongation experiments, electrophoretic product separation, and band quantitation. In this work, we describe an alternative real-time stopped-flow assay for obtaining kinetic parameters for elongation of extended sequences. The assay builds on our earlier PETE (polymerase elongation template element) assay designed for high-throughput screening purposes [S.P. Mestas, A.J. Sholders, O.B. Peersen, A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity, Anal. Biochem. 365 (2007) 194-200] and relies on measuring how long it takes a polymerase to reach the end of a defined length template. Using poliovirus polymerase and self-priming hairpin RNA substrates with 6- to 26-nt-long templating regions, we demonstrate that the assay can be used to determine V(max) rates for elongation and apparent K(m) values for nucleotide triphosphate (NTP) use. Modeling the reaction kinetics as a series of irreversible steps allows us to numerically fit the entire time-based dataset by properly accounting for the temporal distribution of intermediate species. This enables us to determine average elongation rates over heterogeneous templating regions that mimic viral genome substrates. The assay is easily extendable to other RNA and DNA polymerases, can accommodate secondary structures in the template, and can in principle be used for any enzyme traversing along an extended substrate.
Gong
Peng
P
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
Campagnola
Grace
G
Peersen
Olve B
OB
D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D021141:Nucleic Acid Amplification Techniques; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry; D013696:Temperature
D005456:Fluorescent Dyes; D012313:RNA; D012321:DNA-Directed RNA Polymerases