GENETIC ANALYSIS OF REPRESSOR/DNA RECOGNITION

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We will take a molecular genetic approach to understand the structure and function of Escherichia coli Trp aporepressor, a small (107 residue), stable protein that binds tryptophan to form an active repressor (DNA- binding) complex. We are making mutant aporepressors with each of the 2,033 possible single amino acid changes, and analyzing their phenotypes in vivo. From this collection of mutant proteins with single amino acid changes, we should be able to define which residues of aporepressor are critical for folding and dimerization, for binding corepressor (tryptophan), and for binding DNA. In addition, the phenotypes of these single changes will provide us with a database to examine various strategies for the analysis of protein structure and function, including combinatorial cassette mutagenesis and alanine scanning. In addition, we will make multiple Trp aporepressors to examine various aspects of protein structure and function in molecular detail. These include multiple mutants to study the binding of tryptophan and other indole derivatives that act as corepressors using time-resolved fluorescence and NMR spectroscopy, to study DNA binding, and to study unusual structures (e.g., homopolymeric alpha-helices) within the context of this small prokaryotic protein. We will also synthesize covalent Trp repressor, to examine the tryptophan binding pocket by spectroscopic methods in greater detail. The small size and stability of the structurally well-characterized protein, Trp aporepressor, and the multi-faceted nature of its interactions with corepressors and DNA, make the trpR gene a very attractive choice for detailed genetic analysis. Biochemical study of mutant aporepressors will provide us with an unparalleled wealth of information about the structure and function of a single regulatory protein. Understanding the relationship between the structure and function of DNA-binding proteins is central to our understanding of the regulation of gene expression at the molecular level.