Abstract
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.
Highlights
The region that is involved in the formation of tetramerThe expression of proteins required for the metabolism of species from dimer (or in “long axis” dimer formation)is at the lactose in Escherichia coliis regulatedby the binding of lactose C terminus where the leucine heptad repeats essentialfor tetrepressor protein to the operatorDNA sequence that lies adja- ramer formation are found
Substitution of the C-terminal leucineheptad repeat ley et al, 1975; OGorman et al, 1980a, 1980b; Culard and region of the normally tetrameric lactose repressor by Maurizot, 1981, 1982; Whitson and Matthews, 1986)
The expression of proteins required for the metabolism of species from dimeris at the lactose in Escherichia coliis regulatedby the binding of lactose C terminus where the leucine heptad repeats essentialfor tetrepressor protein to the operatorDNA sequence that lies adja- ramer formation are found
Summary
The expression of proteins required for the metabolism of species from dimer (or in “long axis” dimer formation)is at the lactose in Escherichia coliis regulatedby the binding of lactose C terminus where the leucine heptad repeats essentialfor tetrepressor protein to the operatorDNA sequence that lies adja- ramer formation are found Et al (1993) have suggested that theC-terminal leucineheptad repeats in thelactose repressor form a four-helical antiparallel bundle (Fig. 112)C. onsistent with this hypothesis, replacement of the heptad repeat region in the lactose repressor by the GCN4 leucine heptad repeat sequences,presumably able to form only dyadic structures (O’Shea et al, 19911, resulted in a dimeric species as assayed by the inability of cell extracts containing thisprotein to generatelooped complexes with operator DNAby gel mobility shift analyses (Alberti et al, 1993). The dissociation rate constants of the repressor-operator complexes were determined by filter binding methods (Riggs et al, 1970). Panel B, aminoacid sequences of the C-terminal leucine heptad repeats Repressor and 32P-labeled 40-mer operator (1:l ratio) were equilibrated in thewild-type, -11 aa, andR3 lac repressors. The protein blots were reactedmownoitchlonal monium sulfatea t 37% saturation,followed by passage through a phos- antibody B-2 as described by Sams et al (1985)
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