Construction of a dimeric repressor: dissection of subunit interfaces in Lac repressor.
Formation of the lactose repressor tetramer is postulated to involve two subunit interfaces, one primarily contributing to monomer-monomer assembly to dimer and the second to dimer-dimer association to tetramer. The latter interface requires a heptad repeat of three leucines at the C-terminus of lac repressor that is presumed to form an abbreviated coiled-coil motif [Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., & Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Krämer, H., & Müller-Hill, B. (1991) New Biol. 3, 57-62; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. To strengthen the dimer-dimer interface, this motif was extended by the addition of one and two leucine heptad repeat units to the C-terminus by site-specific insertion mutagenesis. The tetrameric products displayed operator and inducer affinity essentially indistinguishable from the wild-type repressor. In order to probe the effect of the elongated coiled-coil on assembly of the repressor tetramer, the other of the two postulated subunit interfaces was disrupted by introducing a point mutation (Y282D) that yields a monomeric protein in the wild-type background. Both elongated mutant repressors were able to assemble into dimeric species, apparently due to the strengthened subunit association at the C-terminal region compared to the wild-type repressor. These results further confirm the role of a coiled-coil structure in the formation of tetramer in the lac repressor.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
J
J
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
Surendran
R
R
Lee
J C
JC
Matthews
K S
KS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D004269:DNA, Bacterial; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry*
D001704:Biopolymers; D004269:DNA, Bacterial; D012097:Repressor Proteins