The broad, long term objective of this proposal is to understand the fundamental principles underlying the recognition between the transcriptional factors, a1 and alpha2, for each other and for their target DNA sites. These two regulatory proteins are the prime determinants of cell type in Saccharomyces cerevisiae. In diploid cell types, both a1 and alpha2 are present and form a heterodimer to specifically repress a set of haploid-specific genes. Both proteins contain a homeoJomain DNA-binding motif which uses a helix-turn-helix unit and a flexible amino-terminal arm for DNA recognition. Despite detailed structural studies of this family of proteins, we still do not understand the details underlying the specificity of the homeodomain- DNA association and the effects of heterodimerization on the homeodomain structure and the DNA recognition event.
The specific aims of the research proposed are: (1) to determine detailed solution structures of the functional DNA-binding and heterodimerizing domains of the a1 and alpha2 proteins, both free in solution and as part of the heterodimer complex. Using nuclear magnetic resonance (NMR) spectroscopy, the inherent structural differences between the homeodomains of the two proteins and especially the changes in the a1 homeodomain upon heterodimerization can be studied. Comparison of these structures with the recently published ternary complex will reveal the structural changes that the proteins must undergo to form a specific complex. (2) The dynamics of backbone and sidechain atoms in the a1 and alpha2 homeodomains - in their free forms, bound as the heterodimer and bound to their target DNA sites - will be characterized using heteronuclear NMR methods. Relaxation parameter measurements will highlight the differences in flexibility between the two homeodomain proteins as the specific transcriptional complex is formed incrementally. (3) The energetics associated with complex formation will be characterized using calorimetric methods. These studies will provide a means to gain thermodynamic information on the importance of structural and dynamic changes on the homeodomain complexes formed. Lastly, (4) mutant proteins and different constructs of the proteins offer an opportunity to extend these studies. Structural, dynamic and thermodynamic studies of mutants and differing constructs of the a1 and alpha2 homeodomains will allow insight into the importance of individual interactions and the effects of mutations and surrounding structure on the recognition processes. Currently hundreds of homeodomain proteins have been identified in eukaryotes from yeast to human and play critical roles in development differentiation and cell growth processes. In particular, the a1 protein is related closely to the pbx1 homeodomain of the chimeric fusion protein associated with pre-B cell acute lymphoblastic leukemias. Thus these studies contribute to an increased understanding of oncogenesis in addition to other normal growth and development processes.
