Cloning and characterization of several dominant-negative and tight-binding mutants of lac repressor

Abstract

Using both general recombination and molecular cloning techniques, 13 I −, I −d and I tb missense mutations in the lacI gene were transferred from F' lad q episomes to ColE1 derivative plasmids. Two deletion derivatives of the lacI genes encoding the wild-type (wt) and the tight-binding (I tb) B3 and B5 repressors were also constructed. The mutant repressers were examined for polypeptide size and stability, and for binding to the inducer isopropyl-β- d-thiogalactoside (IPTG). Several of the I −d repressors were shown to be partially degraded in vivo, in confirmation of earlier results based on [ 14C]IPTG binding [Miwa and Sadler, J. Mol. Biol. 117 (1977) 843-868]. The sizes of polypeptides produced by lacI deletion derivatives were consistent with expectations based on the extent of deletion and the location of termination sites within the plasmid sequence. The first 400 bp of several mutant lacI genes were sequenced. Our wt lacI gene differs from another wt lacI sequence (Farabaugh, 1978), containing a single bp change that results in an Ala to Thr substitution at amino acid (aa) 109. We identified bp substitutions and the resultant aa changes for two I tb and two I −d genes ; the positions correlated with prior genetic mapping data. Three of these new changes were in the N-terminal domain (headpiece) of repressor, with one change in the core domain at aa 99.