PMID:12730189
| Citation |
Joubert, AM, Byrd, AS and LiCata, VJ (2003) Global conformations, hydrodynamics, and X-ray scattering properties of Taq and Escherichia coli DNA polymerases in solution. J. Biol. Chem. 278:25341-7 |
|---|---|
| Abstract |
Escherichia coli polymerase 1 (Pol 1) and Thermus aquaticus Taq polymerase are homologous Type I DNA polymerases, each comprised of a polymerase domain, a proofreading domain (inactive in Taq), and a 5' nuclease domain. "Klenow" and "Klentaq" are the large fragments of Pol 1 and Taq and are functional polymerases lacking the 5' nuclease domain. In the available crystal structures of full-length Taq, the 5' nuclease domain is positioned in two different orientations: in one structure, it is extended out into solution, whereas in the other, it is folded up against the polymerase domain in a more compact structure. Analytical ultracentrifugation experiments report s20,w values of 5.05 for Taq, 4.1 for Klentaq, 5.3 for E. coli Pol 1, and 4.6 for Klenow. Measured partial specific volumes are all quite similar, indicating no significant differences in packing density between the mesophilic and thermophilic proteins. Small angle x-ray scattering studies report radii of gyration of 38.3 A for Taq, 30.7 A for Klentaq, and 30.5 A for Klenow. The hydrodynamic and x-ray scattering properties of the polymerases were also calculated directly from the different crystal structures using the programs HYDROPRO (Garcia De La Torre, J., Huertas, M. L., and Carrasco, B. (2000) Biophys J. 78, 719-730) and CRYSOL (Svergun, D. I., Barberato, C., and Koch, M. H. J. (1995) J. Appl. Crystalogr. 28, 768-773), respectively. The combined experimental and computational characterizations indicate that the full-length polymerases in solution are in a conformation where the 5' nuclease domain is extended into solution. Further, the radius of gyration, and hence the global conformation of Taq polymerase, is not altered by the binding of either matched primer template DNA or ddATP. |
| Links |
PubMed Online version:10.1074/jbc.M302118200 |
| Keywords |
Biophysical Phenomena; Biophysics; Crystallography, X-Ray; DNA/metabolism; DNA-Directed DNA Polymerase/chemistry; DNA-Directed DNA Polymerase/metabolism; Escherichia coli/enzymology; Models, Molecular; Protein Conformation; Scattering, Radiation; Taq Polymerase/chemistry; Taq Polymerase/metabolism; Ultracentrifugation; Water/chemistry; Water/metabolism |
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