Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli.
It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.
D000595:Amino Acid Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D011233:Precipitin Tests; D012097:Repressor Proteins / Q000378:metabolism*; D013045:Species Specificity; D014157:Transcription Factors / Q000378:metabolism*
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors