INSERTION MUTAGENESIS OF THE LAC REPRESSOR AND ITS IMPLICATIONS FOR STRUCTURE-FUNCTION ANALYSIS

We recently developed a simple technique for the generation of relativ ely large (31-codon) insertion mutations in cloned genes. To test whet her the analysis of such mutations could provide insight into structur e-function relationships in proteins, we examined a set of insertion m utants of the Escherichia coil Inc repressor (LacI). Representatives o f several LacI mutant classes were recovered, including mutants which exhibit fully active, inducer-insensitive, or weak dominant-negative p henotypes. The various properties of the recovered mutants agree with previous biophysical, biochemical, and genetic data for the protein. I n particular, the results support the prior designation of mutationall y tolerant spacer regions of LacI as well as proposed differences in d imerization interactions among regions of the protein core domain. The se findings suggest that the analysis of 31-codon insertion mutations may provide a simple approach for characterizing structure-function re lationships in proteins for which high-resolution structures are not a vailable.