Abstract
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.
Highlights
From the *Department of Microbiology and §TheProgram in Cell and Molecular Biology, Duke University Medical Center, Durham, North Carolina 27710
E. coli protein, known as ter, has been shown to be quences within the protein-bindingsites(Hill et al, 1988; recruited in the site-specifictermination of autonomous DNA Lewis et al, 1990),we are confident that thestructure of the replication in the bacterial plasmid R6K
Isopropyl-1-thio-8-D-galactopyranosidweas added to a final concentration of 10 mM to induce the overproduction of the protein
Summary
DNA are required, within terC containing two inverted repeats of 47 and 48 bp centeredon an 8-basepair consensus sequence,eltiocit (known as IRI and IRII,respectively), separated by 59 bp Appears that two dimers of terminator protein bind to each inverted repeat, and that each dimer recognizesa rather conserved 8-bp consensus sequence (Hill et al.,1988; DNA replication can be separated biochemically and phys- Lewis et al, 1990). E. coli protein, known as ter, has been shown to be quences within the protein-bindingsites(Hill et al, 1988; recruited in the site-specifictermination of autonomous DNA Lewis et al, 1990),we are confident that thestructure of the replication in the bacterial plasmid R6K Digested with three restriction enzyme mixtures,BamHI, DdeI/ HindIII, and HinfI/EcoRI, andthe DNA fragments were 3'-end
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