Control of trp operon expression in E. coli is a paradigm of genetic regulation of microbial metabolism. Negative control through the action of trp repressor in response to tryptophan availability is the predominant regulatory mechanism imposed. trp repressor is a DNA-binding transcription factor that contains the helix-turn-helix structural motif. DNA-binding transcription factors regulate many normal and disease related biological processes. In fact, certain critical events in development result from the action of homeodomain containing proteins. The homeodomain has been shown to contain the helix-turn-helix structural motif. Additionally, cellular regulatory proteins such as Myc, Fos, Jun and Myb, and the cognate viral regulators can be oncogenic if aberrantly expressed or mutated. The goal of our research is a full understanding of the molecular mechanisms utilized by DNA-binding regulatory proteins to perform their biological functions. Currently, we are focusing our efforts on a well-characterized model protein, the trp repressor of E. coli. The proposed research seeks to address specific outstanding questions concerning trp repressor function. Previously, we determined that one type of structural domain, the NH2-terminal arm, is involved in association of the repressor with operator to form complexes. This domain has not been resolved in structural studies. We will perform a comprehensive mutational analysis of the arm domain in order to determine the functional size of the arms and the important characteristics of the arm amino acid residues. Further, the NH2-terminal arms may mediate a sliding mechanism of operator search. We will test the wild type trp repressor and arm mutant repressors for this important mechanism. We will also use a newly developed PCR-based technique to test the model of """"""""indirect readout"""""""" proposed to mediate specificity of trp repressor/operator binding. We will identify and isolate all DNAs in a pool of random DNA that trp repressor can bind with high affinity, characterize the binding site within each DNA and the affinity of repressor for each. This work will enhance our understanding of the fundamental principles that mediate interactions between regulatory transcription factors and chromosomal binding sites.
| Czernik, P J; Shin, D S; Hurlburt, B K (1994) Functional selection and characterization of DNA binding sites for trp repressor of Escherichia coli. J Biol Chem 269:27869-75 |
