Effects of dominant-negative lac repressor mutations on operator specificity and protein stability

Abstract

The specific binding of dominant-negative (I- d) lactose ( lac) repressers to wild-type (wt) as well as mutant ( O c) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target. Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322. Twelve of these lacI -d missense mutations were transferred from F' lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13- lacI phages [Mott et al., Nucl. Acids Res. 12 (1984) 4139–4152]. The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-β- d-thiogalactoside (IPTG), as well as binding to wt operator. The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against represser expressed from a chromosomal lacI q gene. Six of the I- d repressors were partially degraded in vivo. All repressors bound IPTG with approximately the affinity of wt repressor. Repressers having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17–25) were examined in vivo for their affinities for a series of single site O c operators. Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator [Ebright, Proc. Natl. Acad. Sci. USA 83 (1986) 303–307], the His-18 represser did not affect specificity. This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions.