title: The Escherichia coli primosomal DnaT protein exists in solution as a monomer-trimer equilibrium system.
abstract: The oligomerization reaction of the Escherichia coli DnaT protein has been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. In solution, DnaT exists as a monomer-trimer equilibrium system. At the estimated concentration in the E. coli cell, DnaT forms a mixture of the monomer and trimer states with a 3:1 molar ratio. In spite of the modest affinity, the trimerization is a highly cooperative process, without the detectable presence of the intervening dimer. The DnaT monomer consists of a large N-terminal core domain and a small C-terminal region. The removal of the C-terminal region dramatically affects the oligomerization process. The isolated N-terminal domain forms a dimer instead of the trimer. These results indicate that the DnaT monomer possesses two structurally different, interacting sites. One site is located on the N-terminal domain, and two monomers, in the trimer, are associated through their binding sites located on that domain. The C-terminal region forms the other interacting site. The third monomer is engaged through the C-terminal regions. Surprisingly, the high affinity of the N-terminal domain dimer indicates that the DnaT monomer undergoes a conformational transition upon oligomerization, involving the C-terminal region. These data and the high specificity of the trimerization reaction, i.e., lack of any oligomers higher than a trimer, indicate that each monomer in the trimer is in contact with the two remaining monomers. A model of the global structure of the DnaT trimer based on the thermodynamic and hydrodynamic data is discussed.
authors: Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston , 301 University Boulevard, Galveston, Texas 77555-1053, United States.
authors: Michal R
authors: MR
authors: Szymanski
authors: Maria J
authors: MJ
authors: Jezewska
authors: Wlodzimierz
authors: W
authors: Bujalowski
mesh_terms: D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D014461:Ultracentrifugation
chemical_list: D004268:DNA-Binding Proteins; C104198:DnaT protein, E coli; D029968:Escherichia coli Proteins