Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit

J Bacteriol. 2005 Oct;187(20):7081-9. doi: 10.1128/JB.187.20.7081-7089.2005.


The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: alpha, epsilon, and theta. The alpha subunit contains the polymerase, and the epsilon subunit contains the exonucleolytic proofreading function. The small (8-kDa) theta subunit binds only to epsilon. Its function is not well understood, although it was shown to exert a small stabilizing effect on the epsilon proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, theta yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the theta homolog from bacteriophage P1, named HOT, we constructed a homology model of theta. This model suggested that the unfavorable behavior of theta might arise from exposed hydrophobic residues, particularly toward the end of alpha-helix 3. In gel filtration studies, theta elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of theta in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported theta structure. The new theta structure is expected to provide additional insight into its physiological role and its effect on the epsilon proofreading subunit.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alcohols / metabolism
  • Amino Acid Sequence
  • Chromatography, Gel
  • DNA Polymerase III / chemistry*
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protons
  • Viral Proteins / genetics


  • Alcohols
  • Escherichia coli Proteins
  • Protons
  • Viral Proteins
  • HOT protein, bacteriophage P1
  • holE protein, E coli
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase