9808412 Wolberger Genes in eukaryotes are frequently regulated by multiprotein complexes that bind DNA in a sequence-specific manner and control mRNA transcription. This type of transcriptional regulation, termed combinatorial control, is thought to make possible the more complex regulatory networks found in higher eukaryotes. The structural studies described in these studies are aimed at gaining insight into the molecular basis of combinatorial control mediated by homeodomain proteins. The 2.35 crystal structure will be determined of the yeast (2 homeodomain bound DNA, thereby extending the resolution and accuracy of the existing model of that complex. The crystallization and x-ray structure determination will also be carried out on an a1/(2-DNA complex containing a triple-alanine mutant with unusual DNA-binding properties. The crystal structure of the Tup1 protein, a general transcriptional repressor that is recruited by (2, will be determined from crystals of a 50 kD fragment of the protein. Analytical ultracentrifugation experiments will also be carried out to determine whether this fragment is capable of binding to the N-terminal tails of histones H3 and H4, an interaction that has been proposed to be part of the mechanism of repression by the Tup1 protein. Attempts will also be made to co-crystallize the Tup1 protein with an a1/(2 N-terminal domain heterodimer, which recruits Tup1 in yeast. To extend these studies of combinatorial control to other proteins, the structure of the human HoxB1/Pbx 1 homeodomain heterodimer bound to DNA will be determined from crystals diffracting to 2.35 resolution. The goal of this research is to provide a deeper understanding of the molecular details of gene regulation by the yeast MAT (2 protein, which is widely regarded as a paradigm for the study of combinatorial control of transcription. The higher resolution structure of (2 alone bound to DNA will help elucidate remaining questions about DNA-binding specificity, while the struct ure of the a1/(2-DNA complex containing a mutant (2 protein with all three major groove-contacting side chains to alanine will make possible an understanding of the unexpected ability of the mutant heterodimer to bind DNA with near-wild type affininty. The structure and solution studies of the Tup1 protein will be the first of a general yeast repression factor and will aid in an understanding of how (2 represses transcription in vivo through recruitment of Tup1. The insights gained from the (2 system will be broadened to other systems through the structural study of the HoxB1/Pbx1-DNA complex, which is a human homologue with many features in common with the yeast a1 and (2 proteins.
