Wild-type operator binding and altered cooperativity for inducer binding of lac repressor dimer mutant R3.
Substitution of the C-terminal leucine heptad repeat region of the normally tetrameric lactose repressor by the leucine heptad repeat dimerization domain of GCN4 protein resulted in cell extracts containing protein, designated R3, which behaved as a dimer based on gel retardation analysis of DNA binding (Alberti, S., Oehler, S., von Wilcken-Bergmann, B., and Müller-Hill, B. (1993) EMBO J. 12, 3227-3236). We have purified this R3 protein and characterized its properties in comparison with the wild-type repressor. R3 protein elutes from a molecular sieve with a Stokes radius characteristic of a dimer and a deduced molecular mass of 66 kDa. Unlike other dimeric repressors, produced by deletion or mutation in the leucine heptad repeat region, which display reduced apparent operator affinity, R3 binds to operator DNA sequences with wild-type equilibrium and kinetic properties. Although inducer affinity at neutral pH is similar for R3 and wild-type protein, at elevated pH the R3 protein undergoes a slightly smaller decrease in affinity and exhibits minimal cooperativity in sugar binding compared with the wild-type protein. Interestingly, in the presence of operator DNA, a state in which inducer binding to wild-type repressor is also of reduced affinity and slightly cooperative, R3 binding affinity is decreased to a greater extent, and the protein displays higher cooperativity than wild-type repressor. Consistent with inducer binding data in the presence of operator, the release of operator from R3 protein requires a higher sugar concentration than wild-type protein. These results are interpreted in the context of alterations involving the subunit interface which affect the allosteric behavior of the repressor protein.
Chen
J
J
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
Alberti
S
S
Matthews
K S
KS
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside