Crystallization and preliminary structural analysis of the replication terminator protein of Bacillus subtilis.
The replication terminator protein (RTP) is a dimeric molecule that binds specific sequences within the replication terminus of the Bacillus subtilis chromosome and prevents the passage of replication forks. The gene for RTP has been expressed in Escherichia coli, and the protein has been purified in amounts sufficient for structural studies by nuclear magnetic resonance (NMR) and x-ray crystallography. One-dimensional NMR experiments show that the protein has a well-folded compact tertiary structure, as well as a high alpha-helical content. Circular dichroism (CD) studies confirm this finding and show that approximately 32% of the protein is alpha-helical. The terminator protein has been crystallized as monoclinic plates that diffract to better than 2.5 A and are suitable for high resolution structural analysis. Precession photographs show the space group to be C2 with unit cell dimensions a = 77 A, b = 53 A, c = 70 A, and beta = 90 degrees, and two molecules occupy the asymmetric unit. With a view to producing crystals of an RTP.DNA complex, gel-shift assays were performed to establish the shortest sequence of DNA that is required for tight binding to RTP. These clearly show that two turns of DNA are required, centered on an 8-base pair consensus sequence, to elicit relatively stable binding.
Mehta
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Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710.
Bussiere
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Hoffman
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Bastia
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White
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D001345:Autoradiography; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002942:Circular Dichroism; D003460:Crystallization; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C063112:rtP protein, Bacillus subtilis