CHARACTERIZATION OF MUTANTS AFFECTING THE KRK SEQUENCE IN THE CARBOXYL-TERMINAL DOMAIN OF LAC REPRESSOR

The lac repressor carboxyl-terminal region is required for tetramer as sembly and protein stability. To further investigate this region, espe cially the unusual sequence KRK, four deletion mutants eliminating the carboxyl-terminal 34, 35, 36, and 39 amino acids and five substitutio n mutants at the position of Arg-326, R326K, R326A, R326E, R326L, and R326W, were constructed using site specific mutagenesis. The -34-amino -acid (aa) mutant, missing the most carboxyl-proximal lysine from the KRK sequence, exhibited lower affinity for both operator and inducer a nd lower protein stability than dimeric proteins studied previously. T he -35-aa mutant with RK missing, as well as -36 aa and -39 aa, for wh ich the entire KRK sequence was deleted, yielded inactive polypeptides that could be detected only by monoclonal antibody for lac repressor. In the Arg-326 mutant proteins, operator binding affinity was decreas ed by similar to 6-fold, the shift in inducer binding at elevated pH w as diminished, and protein stability was decreased. Dramatic decreases in protein expression and stability occurred with substitution at pos ition 326 by glutamate, leucine, or tryptophan. These results suggest that Arg 326 plays an important role in the formation of the proper te rtiary structure necessary for inducer and operator affinity and for p rotein stability.