Specificities of three tight-binding Lac repressors.
Tight binding mutants of Lac repressor exhibit complex repression phenomena. In this work, in vivo Lac operator binding of three such mutants of E. coli Lac repressor (X86: ser 61-leu, l12: pro 3-tyr and the double mutant l12X86: pro 3-tyr, ser 61-leu) was analyzed. Repression of beta-galactosidase synthesis controlled by ideal lac operator and its 27 symmetric operator variants containing each possible base-pair at each single half-operator position in the presence of the tight-binding Lac repressor mutants was determined. The average increase of repression with all operator variants was about 3 fold with the X86 mutant. It was about 4 fold with the l12 mutant and about 2 fold with the double mutant l12X86 as compared to wildtype Lac repressor. The X86 mutant showed the same increase of affinity to all operator variants, whereas the l12 and l12X86 mutants exhibited lower repression with some variants than with most others. These results suggest that the X86 mutant has gained no additional specificity. In contrast the l12 mutant and the l12X86 mutant exhibit a relaxed specificity for certain base pairs in positions 1 and 3 of lac operator. This suggests that the extreme N-terminus of Lac repressor may interact with the inner base-pairs in the minor groove.
Kolkhof
P
P
Institut für Genetik, Universität zu Köln, Germany.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase