PhD

dissertationTranscription factors are modulators of gene expression that bind to regulatory DNA elements. Understanding this control requires an understanding of the physical relationship between transcription factors and DNA. In this thesis I examine DNA binding using the murine Ets-1 transcription factor as a model. The DNA binding domain, termed the ETS domain, binds a 9 to 16 base pair sequence identified by a GGA core. The ETS domain displays a winged-helix-turn-helix motif (winged HTH) composed of three alpha helices and a four-stranded beta sheet. The HTH (helices H2 and H3) forms the hydrophobic core. The third helix, helix HI, packs against one side of the HTH, and the beta sheet, strands S1-S4, packs against the other. ETS domain DNA binding is autoinhibited by an inhibitory module that is composed of three alpha helices that pack against helix HI. The general goal of this work is to elucidate the rules that govern DNA binding affinity, specificity, and regulation by autoinhibition. In Chapter 2, a random mutagenesis screen is used to identify residues critical for DNA binding. In Chapters 4 and 5, the contributions of individual structural elements of the ETS domain are investigated. DNA binding affinities are determined for ETS domains with single or double alanine substitutions in helix HI, the turn, or the wing. These mutations probe contacts mediated by the wing that include a nonsequence specific requirement for DNA flanking the left side of the GGA core. Substitution of amino acids in the turn, between helix H2 and helix H3 of the HTH, reduced DNA binding affinity. Finally an unusual phosphate contact mediated by the N terminus of helix HI is characterized using mutants that alter the apparent positioning of this helix. Evidence for the existence of this contact comes not only from the lower binding activity of helix HI mutant, but also from the partial suppression of a phosphate modification in the right flank of the DNA site by specific mutations. The discovery of the phosphate contact mediated by helix HI, the only element of the ETS domain that is in direct contact with the inhibitory module, identifies a mechanical means by which the inhibitory module can affect DNA binding. Understanding the regulation of DNA binding by autoinhibition is an important step in understanding the determinants of specificity in the ets gene family. In vivo this regulation may be occurring though direct modification of the protein by signaling cascades and interactions between Ets-1 and other partner proteins that release autoinhibition