This is a competing continuation application for a grant that has supported our efforts toward understanding the 5S DNA and 5S ribosomal RNA binding properties of the zinc finger protein TFIIIA. During the previous grant period we also began to analyze another important class of DNA-binding transcription factors, the basic helix-loop-helix family, to understand the molecular basis for dimerization and DNA binding specificity. These studies have been made possible through the use of solid phase peptide synthesis methods and a new powerful approach for chemical mutagenesis of protein domains. This application now represents a collaborative effort between the Gottesfeld laboratory and the laboratory of Dr. Philip Dawson, an expert in the chemical synthesis of proteins. The Dawson laboratory has developed methods for the synthesis of libraries of peptide analogues including """"""""protein signature analysis."""""""" This synthetic scheme allows for the generation of large numbers of """"""""mutant"""""""" polypeptides and the rapid identification of critical residues for biological function. These methods will be applied to the RNA-specific zinc fingers of TFIIIA in order to understand the molecular basis for RNA-binding and to basic helix-loop-helix proteins to understand the structural basis for dimerization and DNA-binding specificity. Quantitative nucleic acid binding experiments, using gel mobility shift assays and fluorescence anisotropy, will be used to assess the energetic significance of the mutants identified in the combinatorial screens. Additionally, the energetics of bHLH dimerization with wild-type and mutant peptides will be assessed using fluorescence and sedimentation methods. These studies will shed light on the molecular mechanisms involved in regulation of two of the most important classes of transcriptional regulatory proteins. It is well established that mutations and gene rearrangements involving zinc finger and bHLH proteins underlie a variety of human malignancies. Thus, understanding the basis for nucleic acid recognition and dimerization specificity may provide the necessary background for development of the novel therapeutic approaches.
