A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.
Interactions between proteins affect a wide variety of biological processes, 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 heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.
Dmitrova
M
M
Institut de Biologie Moleculaire et Cellulaire, UPR 9002 du CNRS, Strasbourg, France.
Younès-Cauet
G
G
Oertel-Buchheit
P
P
Porte
D
D
Schnarr
M
M
Granger-Schnarr
M
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003124:Colorimetry; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D016762:Genes, fos; D016758:Genes, jun; D005821:Genetic Techniques; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010641:Phenotype; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein