A preliminary CD and NMR study of the Escherichia coli DNA polymerase III Θ subunit
✍ Scribed by Dawei Li; Devon L. Allen; Scott Harvey; Fred W. Perrino; Roel M. Schaaper; Robert E. London
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 144 KB
- Volume
- 36
- Category
- Article
- ISSN
- 0887-3585
No coin nor oath required. For personal study only.
✦ Synopsis
The subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: ␣, ⑀, and , with ␣ possessing the polymerase activity, ⑀ functioning as a proofreading exonuclease, and , a small subunit of 8.9 kD, of undetermined function. The subunit has been expressed in E. coli, and a CD analysis of indicates the presence of a significant amount of secondary structure: D52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, yields a poorly-resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1 H-15 N HSQC analysis of uniformly-15 Nlabeled supports the conclusion that approximately half of the protein is reasonably wellstructured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1 H-15 N HSQC spectra resulted when [U-15 N]was examined in the presence of ⑀186-the Nterminal domain of the ⑀-subunit. Further work is currently in progress to define the solution structure of and the -⑀186 complex.