A CONSENSUS MOTIF IN THE RFX DNA-BINDING DOMAIN AND BINDING DOMAIN MUTANTS WITH ALTERED SPECIFICITY

The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regu latory functions, in eukaryotic organisms ranging from yeasts to human s. To characterize this novel motif, we have performed a detailed diss ection of the site-specific DNA binding activity of RFX1, a prototypic al member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we ha ve developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure pr ediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These resu lts should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreove r, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interf erence from the ubiquitously expressed endogenous protein. Finally, th e simplicity, efficiency, and versatility of the selection procedure w e have developed make it of general value for the determination of con sensus motifs, and for the isolation of mutants exhibiting altered fun ctional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.