A NEW LEXA-BASED GENETIC SYSTEM FOR MONITORING AND ANALYZING PROTEIN HETERODIMERIZATION IN ESCHERICHIA-COLI

Interactions between proteins affect a wide variety of biological proc esses, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerizati on of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an alt ered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization d omains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-bindin g domain. Their hetero-association is specifically measured by the tra nscriptional repression of a reporter gene (lacZ) controlled by a hybr id operator containing a wild-type half-site (CTGT) and a mutated oper ator half-site (CCGT). The hybrid operator/lacZ construct was integrat ed into the chromosome of the reporter strain (SU202) to avoid possibl e artefacts due to variations in plasmid copy number. This method shou ld be particularly useful in those cases where one or both partners ar e also able to form homodimers, since the assay described here is sens itive only to the formation of heterodimers. Furthermore, this assay g ives rise to a screenable red/white phenotype on MacConkey-lactose ind icator plates, allowing for a genetic study of the specificity of the interaction.