Characterization of a Novel Eukaryotic DNA-binding Domain

The aim of the proposed studies is to identify amino acids involved in base-specific contacts in the yeast DNA-binding protein RAP1. The RAP1 protein is essential for viability in yeast and functions in both regulation of transcription (as either a repressor or activator) and maintenance of telomere structure. The DNA-binding domain of RAP1 (A central 250 amino acid region) has no similarity to known DNA binding motifs and thus may represent a novel class of DNA binding proteins. The proposed research builds upon a genetic selection in yeast, based on the ability of RAP1 to activate transcription, to isolate mutant proteins with altered DNA binding specificity (ABS mutants). Such mutants are identified as dominant suppressors that allow a non-consensus (mutant) binding site to function as an activation element. Mutant proteins whose function is specific to changes in only one position in the binding site will then be characterized biochemically to determine precisely their sequence specificity of binding. Inferences from initial suppression studies will be extended by alanine substitution mutagenesis of small segments of the DNA-binding domain (-10-15 amino acids) in a systematic search for 'missing contact' mutants. Results from these experiments will be used in saturation mutagenesis experiments designed to obtain further quantitative information concerning specific amino acid- base contacts. %%% Regulation of transcription in eukaryotics is of fundamental importance in both development and in an organism's response to its environment. This study relates to the role of sequence - specific DNA binding regulating proteins in transcriptional regulation.