Nucleic Acids Research, 1992, Vol. 20, No. 19 5035-5039
© 1992
MOLECULAR BIOLOGY |
Specificities of three tight-binding Lac repressors
Institut für Genetik der Universität zu Köln Weyertal 121, D-5000 Köln 41, Germany
Received July 21, 1992. Revised September 5, 1992. Accepted September 5, 1992.
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 ß-galactosldase synthesis controled 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 112 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.