Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction.
Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.
Gottlieb
P A
PA
Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104.
Wu
S
S
Zhang
X
X
Tecklenburg
M
M
Kuempel
P
P
Hill
T M
TM
D000478:Alkylation; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002876:Chromosomes, Bacterial; D016172:DNA Fingerprinting; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006207:Half-Life; D006878:Hydroxides; D017665:Hydroxyl Radical; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006878:Hydroxides; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D017665:Hydroxyl Radical