Arc repressor is tetrameric when bound to operator DNA.
The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.
Brown
B M
BM
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Bowie
J U
JU
Sauer
R T
RT
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D012483:Salmonella Phages / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA