Transcriptional activator proteins that bind to specific DNA sequences and elevate the levels of transcription regulate many cellular processes, including development and differentiation. The long range goal of this proposal is to understand the stereochemical details of how transcriptional activator proteins target their specific DNA sequences. In this competitive renewal application, we will continue to pursue this goal by correlating the high-resolution structures of protein/DNA complexes with functional studies. We will focus on two model systems to address two fundamental questions underlying protein- DNA recognition, (1) how does a family of related DNA binding proteins distinguish among different DNA target sequences, and (2) how is the DNA-binding specificity of a family of related DNA binding proteins modulated by the nearby binding of a DNA-binding cofactor. To address the first question, we will focus on a group of fungal-specific proteins that contain a conserved Zn2 Cys6 binuclear cluster. We have previously determined the structures of the DNA complexes with the Ga14, Ppr1, Put3 and Hap1 members of this family, revealing how these proteins recognize a unique inter-half-site separation between two common DNA half-sites. In aims 1-3 of this proposal, we will extend our studies to Lue3, Pdr3, and Uga3 and continue our studies with Hap1 to discover how this family of proteins bind half-sites of different base-pair identity and polarity. To address the second question, we will focus on a group of mammalian ternary complex factors (TCF's) that contain a conserved ETS DNA-binding domain and that interact with DNA bound serum response factor (SRF) at the c-fos promoter. We have previously determined the structures of the TCF proteins, SAP-1 and Elk-1 bound to DNA, revealing how two proteins that have identical DNA recognition helices can bind different DNA sequences. In aims 4 and 5 of this proposal, we will determine the structure and DNA-binding properties of the ternary TCF/SRF/DNA complex to reveal how SRF modulates the DNA binding properties of the TCF protein. Taken together, the studies proposed here will illustrate how proteins that contain a conserved Zn2Cys6 domain can be configured to bind distinct DNA target sequences, and how the DNA-binding properties of TCF proteins are modulated by the nearby binding of the SRF protein at the c-fos promoter. Both systems provide excellent models for understanding detailed stereochemical issues underlying the sequence-specific DNA binding activity of transcriptional activator proteins in eukaryotes.