The side-chain of the amino acid residue in position 110 of the Lac repressor influences its allosteric equilibrium.
Binding of the Lac repressor to its operator DNA controls the expression of the genes of the lac operon of Escherichia coli. Lac repressor's affinity for the lac operator is diminished by an inducer that affects the structure of the repressor tetramer. Here we report the cloning and sequencing of the mutant Lac repressor i-t gene, whose product, the LacR-t repressor, shows a higher affinity for the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and a lower affinity for the lac operator than the wild-type repressor. We show that the altered phenotype is due to a single amino acid residue replacement; the alanine residue at position 110 in the wild-type is replaced by threonine in i-t. Other amino acid residues in position 110 have been shown to result in an i-s phenotype. For the i-s-substitution of alanine 110 with lysine we demonstrate an increase in the affinity for operator DNA and a decrease in the affinity for IPTG. Thus, A110--> K shows the opposite effect to A110-->T on the repressor protein. We explain the phenotype of the LacR mutants by displacements of the conformational equilibrium for the dimeric repressor unit between RR (high operator affinity, low inducer affinity) and R*R* (low operator affinity, high inducer affinity) towards R*R* in the i-t and towards RR in the i-s mutant in position 110 with respect to the wild-type. The putative structures of the wild-type and mutant Lac repressors confirm this conclusion.
Müller-Hartmann
H
H
Institut für Genetik, Universität zu Köln, Germany.
Müller-Hill
B
B
D000494:Allosteric Regulation; D000596:Amino Acids / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics*; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*
D000596:Amino Acids; D012097:Repressor Proteins