title: Site-directed mutagenesis supports a three-dimensional model of the runt domain.
abstract: The "runt domain" (RD) is a 128 amino acid region of the Drosophila pair-rule gene runt. This highly conserved region delineates the DNA-binding domain of a new family of transcription factors; the RD proteins. The family includes genes from Drosophila, chicken and mammals that are involved in a wide range of developmental processes, from sex determination and neurogenesis in Drosophila to hematopoiesis and osteoblast differentiation in mouse and human. The RD confers DNA binding ability and mediates the interaction of mammalian RD proteins with the beta-subunit (CBFbeta), which enhances the DNA binding. The primary sequence of RD shows no similarity to other known DNA-binding motifs and its three-dimensional (3D) structure is not known. We employed molecular modeling-based mutagenesis to generate a 3D model of RD. Fold recognition programs identified the palm subdomain of rat DNA polymerase beta as the most likely fold for RD. In the predicted model, the RD region which interacts with DNA contains two arginine residues, R130 and R135, which appear to be in close contact with the major groove of the DNA and to interact with the three essential guanine bases of the core DNA motif PyGPyGGT. We mutated these two R residues and demonstrated that mutations markedly reduced the binding of RD to DNA with no effect on RD interaction with CBFbeta. The data provide important clues about the possible 3D structure of the RD and its interaction with the core DNA motif.
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Levanon
authors.forename: M
authors.initials: M
authors.lastname: Eisenstein
authors.forename: Y
authors.initials: Y
authors.lastname: Groner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C080833:run protein, Drosophila