tau binds and organizes Escherichia coli replication through distinct domains. Partial proteolysis of terminally tagged tau to determine candidate domains and to assign domain V as the alpha binding domain.
The tau subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the alpha subunit. In addition to playing critical roles in the structural organization of the holoenzyme, tau mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged tau proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the tau subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous delta' subunit, and the third region is near the C-terminal end of the tau-delta' alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). We propose a five-domain structure for the tau protein. Domains I and II are based on the crystallographic structure of delta' by Guenther and colleagues. Domains III-V are based on our protease cleavage results. Using this information, we expressed biotin-tagged tau proteins lacking specific protease-resistant domains and analyzed their binding to the alpha subunit by surface plasmon resonance. Results from these studies indicated that the alpha binding site of tau lies within its C-terminal 147 residues (domain V).
Gao
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Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262.
McHenry
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D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004258:DNA Polymerase III / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D016875:tau Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D016875:tau Proteins; D004258:DNA Polymerase III