EXCEED THE SPACE PROVIDED. We propose a study of the transcription factor Gata-1 and its specific interactionswith DNA. Gata-1 binds tightly and specifically to DNA at sequences which contain the motif GATA, for which the factor is named. In its biological role in hematopoiesis, Gata-1 is required for bone marrow stem cells to mature into functional blood cells in circulation. Gata-1 functions in the regulation of cell growth, allowing the maturing blood cells to continue cell division, as well as at the level of stimulating transcription of numerous blood-cell specific functional proteins, such as the hemoglobins of oxygen-carrying red blood cells. Thus a more thorough understanding of the Gata-DNAcomplex strengthens our knowledge of blood cell development, both in its normal function as well as in disease states such as leukemia, and also of the general mechanisms by which transcription factor proteins such as Gata-1 recognize specific DNA sequences. The NMR structures of the DMA-binding domain of chicken Gata-1 on DNA suggest that hydrogen bonding as well as hydrophobic contacts drive the specificity of binding. We plan to test the importance of several of thesestructurally observed contacts between Gata-1 amino acids and DNA base pairs via two lines of experimentation: First, we will make us of electrophoretic mobility shift assays to measure Gata/DNA binding in solution. This approach will allow us to make quantitative determinations of equilibrium constants for the complexes formed between Gata-1 protein and DNA substratesof varying sequence, and under varying binding conditions. Secondly, we applied the yeast one-hybrid system, whereby expression of the LacZ gene, whose enzymatic activity has a convenient colorimetric assay, is indicative of Gata-DNA binding. This system will be used as a genetic screen to identify functional mutations to the Gata finger. PERFORMANCE SITE ========================================Section End===========================================
| Vonderfecht, Tyson R; Schroyer, Daniel C; Schenck, Brandy L et al. (2008) Substitution of DNA-contacting amino acids with functional variants in the Gata-1 zinc finger: a structurally and phylogenetically guided mutagenesis. Biochem Biophys Res Commun 369:1052-6 |
| Ghering, Amy B; Jenkins, Lisa M Miller; Schenck, Brandy L et al. (2005) Spectroscopic and functional determination of the interaction of Pb2+ with GATA proteins. J Am Chem Soc 127:3751-9 |
| Mott, Brian H; Bassman, Jennifer; Pikaart, Michael J (2004) A molecular dissection of the interaction between the transcription factor Gata-1 zinc finger and DNA. Biochem Biophys Res Commun 316:910-7 |
