A SIMPLE SCREENING FOR MUTANT-DNA BINDING-PROTEINS - APPLICATION TO MURINE TRANSCRIPTION FACTOR PEBP2-ALPHA SUBUNIT, A FOUNDING MEMBER OF THE RUNT DOMAIN PROTEIN FAMILY

Mouse transcription factor PEBP2. (polyomavirus enhancer-binding prote in (2) is composed of two distinct subunits alpha and beta. The a subu nit has an ability to bind the specific DNA sequences, which is enhanc ed by formation of a heterodimer with the beta subunit. The DNA bindin g and heterodimerization activities of the a subunit are both localize d within a 128-amino-acid (aa) region termed as the Runt domain for it s homology to the Drosophila segmentation gene runt. To characterize t he molecular determinants for these activities, the Runt domain was ra ndomly mutagenized and produced in E. coli as a secreted form. Using E . coli culture supernatant, the DNA binding and heterodimerization of mutant Runt domains were analyzed by gel retardation assay. Nine rando mly picked single-aa substitution mutants showed various functional al terations in DNA binding and heterodimerization either separately or s imultaneously. This observation suggests that the structure of Runt do main is highly ordered and is quite sensitive to modulations in its pr imary structure. The method presented here provides a simple and quick method to characterize a large number of mutant DNA binding proteins.