Abstract
In Escherichia coli cells, there is a protein that specifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks progress of the DNA replication fork. We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plasmid-free cells. With improvement of the promoter region of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2,000-fold. Using these over-producing cells as the enzyme source, the Ter protein was purified to apparent homogeneity. Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene. Footprinting using the purified Ter protein revealed a specific binding to the ter sequences.
Highlights
To block the progress of the DNA replication fork, two factors are required, one of which is the ter sequence on the DNA molecule.Properties of the ter site presentin the E. coli system are as follows. (i) All ter sequences are essentially the same, and their consensus 22-bp‘ sequence is 5’-(A/T)(G/
73 bp apart (Horiuchi and Hidaka, 1988; Hill et al, 1988b); in the E. coli chromosome, four terC sites are located at the opposite region of the unique replication origin anadre arranged as 275 kilobases apart between the nearest pair of the terC sites (Hidaka et al, 1988). (iv) Whenthe terC site is cloned into the ColEl derivative vector in the orientation in which the unidirectional replication fork starting from vector’s origin is blocked at theterC site, presence of the site reduces the copy number of the hybrid plasmid because the site prevents the plasmid from completing DNA replication (Hidaka et al, 1988)
We obtained convincing evidencethat the taugene encodes Thisprotein may be the same as the one purified inour the ter-binding protein (Ter protein)
Summary
To block the progress of the DNA replication fork, two factors are required, one of which is the ter sequence on the DNA molecule.Properties of the ter site presentin the E. coli system are as follows. (i) All ter sequences are essentially the same, and their consensus 22-bp‘ sequence is 5’-(A/T)(G/. To block the progress of the DNA replication fork, two factors are required, one of which is the ter sequence on the DNA molecule.Properties of the ter site presentin the E. coli system are as follows. (iii) A pair of the two ter sites is arranged in an inverted position. (iv) Whenthe terC site is cloned into the ColEl derivative vector in the orientation in which the unidirectional replication fork starting from vector’s origin is blocked at theterC site, presence of the site reduces the copy number of the hybrid plasmid because the site prevents the plasmid from completing DNA replication (Hidaka et al, 1988). The ter-bindingprotein (Ter protein) is another factor essential for termination reaction. In the tau-defective cells, which showed the termination-less phenotype, no terbinding activity was evident, thereby suggesting that tau might be the structuralgene for the Terprotein
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