A quantitative stopped-flow fluorescence assay for measuring polymerase elongation rates

Analytical Biochemistry

Volumn 391, Issue 1, 2009, Pages 45-55

  Gong, Peng ^a   Campagnola, Grace ^a   Peersen, Olve B ^a  

^a Department of Ecosystem Science and Sustainability   (United States)

Author keywords

Activity assay; Fluorescence polarization; Nucleic acid; Poliovirus; Polymerase

Indexed keywords

BIOMOLECULES; ELONGATION; FLUORESCENCE; NUCLEIC ACIDS; POLARIZATION; POLYMERS; REACTION INTERMEDIATES; REACTION KINETICS; RNA; SUBSTRATES;

ACTIVITY ASSAYS; FLUORESCENCE POLARIZATION; HIGH THROUGHPUT SCREENING; NUCLEOTIDE TRIPHOSPHATES; POLIOVIRUS; POLYMERASE; RADIOLABELED SUBSTRATES; TEMPORAL DISTRIBUTION;

POLYETHYLENE TEREPHTHALATES;

DNA POLYMERASE; NUCLEOTIDE TRIPHOSPHATE; RIBONUCLEOTIDE; RNA POLYMERASE; SHORT HAIRPIN RNA; UNCLASSIFIED DRUG;

AMINO ACID SEQUENCE; ARTICLE; CHEMICAL REACTION KINETICS; CONTROLLED STUDY; ENZYME ANALYSIS; ENZYME KINETICS; FLUORESCENCE ANALYSIS; FLUORESCENCE POLARIZATION; HIGH THROUGHPUT SCREENING; MATHEMATICAL MODEL; NONHUMAN; POLIOMYELITIS VIRUS; PRIORITY JOURNAL; PROTEIN SECONDARY STRUCTURE; RNA TRANSLATION;

POLIOVIRUS;

EID: 67349220414     PISSN: 00032697     EISSN: 10960309     Source Type: Journal    
DOI: 10.1016/j.ab.2009.04.035     Document Type: Article

References (20)

1. Johnson K.A. Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates. Methods Enzymol. 249 (1995) 38-61
2. Patel S.S., and Bandwar R.P. Fluorescence methods for studying the kinetics and thermodynamics of transcription initiation. Methods Enzymol. 370 (2003) 668-686
3. Datta K., Johnson N.P., and von Hippel P.H. Mapping the conformation of the nucleic acid framework of the T7 RNA polymerase elongation complex in solution using low-energy CD and fluorescence spectroscopy. J. Mol. Biol. 360 (2006) 800-813
4. Johnson R.S., Strausbauch M., Cooper R., and Register J.K. Rapid kinetic analysis of transcription elongation by Escherichia coli RNA polymerase. J. Mol. Biol. 381 (2008) 1106-1113
5. Upson R.H., Haugland R.P., Malekzadeh M.N., and Haugland R.P. A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal. Biochem. 243 (1996) 41-45
6. Barnett R.E. Effect of monovalent cations on the ouabain inhibition of the sodium and potassium ion activated adenosine triphosphatase. Biochemistry 9 (1970) 4644-4648
7. pol highlights the functional importance of residue 5 in picornavirus polymerases. J. Virol. 82 (2008) 9458-9464
8. Barton D.J., and Flanegan J.B. Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C. J. Virol. 71 (1997) 8482-8489
9. 2+. Biochemistry 43 (2004) 5126-5137
10. Mestas S.P., Sholders A.J., and Peersen O.B. A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity. Anal. Biochem. 365 (2007) 194-200
11. pol) by ubiquitin-protease-mediated cleavage in Escherichia coli. Protein Expr. Purif. 17 (1999) 128-138
12. Thompson A.A., and Peersen O.B. Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase. EMBO J. 23 (2004) 3462-3471
13. Lakowicz J.R. Principles of Fluorescence Spectroscopy (2006), Springer, Science, New York
14. pol): assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub). J. Biol. Chem. 275 (2000) 5329-5336
15. R. Macey, G. Oster, T. Zahnley, Berkeley Madonna User's Guide, University of California, Berkeley, 2000. Available from: .
16. Nazarenko I., Pires R., Lowe B., Obaidy M., and Rashtchian A. Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nucleic Acids Res. 30 (2002) 2089-2195
17. Walter N.G., and Burke J.M. Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates. RNA 3 (1997) 392-404
18. Unruh J.R., Gokulrangan G., Lushington G.H., Johnson C.K., and Wilson G.S. Orientational dynamics and dye-DNA interactions in a dye-labeled DNA aptamer. Biophys. J. 88 (2005) 3455-3465
19. Traut T.W. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140 (1994) 1-22
20. pol) is sufficient for template switching in vitro. J. Biol. Chem. 274 (1999) 2706-2716