STRUCTURAL STUDIES OF EUKARYOTIC REPRESSORS

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The physical interactions of proteins involved in eukaryotic gene expression will be studied using nuclear magnetic resonance (NMR) techniques. Two proteins, alpha2, and alpha1, from the yeast Saccharomyces cerevisiae, act together to bind DNA operators and repress transcription of haploid specific genes. Both proteins contain homeo domains, DNA-binding motifs found in many eukaryotic transcriptional regulators. These two proteins from a ternary complex with DNA containing the operator sequences characteristic of haploid specific genes. This ternary complex contains one monomer each of alpha1 and alpha2 in addition to the operator DNA. In the absence of alpha2, the alpha1 protein has very poor if any affinity for the operator DNA. Fragment of alpha2 (alpha128-210) and alpha1 (alpha166-126), containing the homeodomains form heterodimeric complex in solution. This hetrodimer retains high operator affinity (Kd=-10-10M), suggesting significant cooperative interactions between the homeodomains. Using NMR methods we have demonstrated that the C-terminus of the alpha2 fragment changes structure from a disordered state in the free protein to a helix in the heterodimer that interacts with the homeodomain of alpha1. The protein fragments will be studied to determine their solution structures, to identify the intermolecular contacts of the heterodimer, and to characterize the ability of the complex to bind its specific DNA recognition sequence. Isotope labeling approaches will be used to simplify the interpretation of the NMR- spectra and to identify protein-protein contacts. These studies will provide some of the first detailed three dimensional information concerning the way in which different proteins interact in a combination fashion to regulate gene expression in higher organisms.