Crystallization and preliminary structural analysis of the replication terminator protein of Bacillus subtilis

• Published in: The Journal of biological chemistry Vol. 267; no. 26; pp. 18885 - 18889
• Main Authors: MEHTA, P. P, DIRKSEN ELI BUSSIERE, HOFFMAN, D. W, BASTIA, D, WHITE, S. W
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.09.1992
• Subjects: Autoradiography; Bacillus subtilis; Bacillus subtilis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Biological and medical sciences; Circular Dichroism; crystal structure; Crystallization; DNA, Bacterial; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Magnetic Resonance Spectroscopy; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Protein Conformation; Replication; replication terminator protein; X-Ray Diffraction; Bacillales; Molecular structure; Bacillus subtilis; DNA binding protein; Bacteria; Replication termination; Binding site; NMR spectrometry; Gel retardation technique; Bacillaceae; X ray diffraction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)37043-7

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.