We hope to gain in a greater understanding of trp operon regulation at the repressor-operator level. Our immediate goals are to first expand and improve upon the present isolation procedure for obtaining pure trp aporepressor. This will be acomplished by constructing new plasmid bearing strains which produce elevated amounts of aporepressor. The purification procedure will then be enlarged in capacity to yield suitable amounts of repressor for the following proposed studies. We will further characterize the trp aporepressor protein in detail to determine its native size, isoelectric point, the UV-visible spectra and spectrofluorometric properties. Interaction of the trp repressor with the three operators it binds (trp, trpR, aroH) will be examined by the examined by the well established procedures of DNase """"""""foot-printing"""""""" and DMS methylation of the operator DNA fragments in the presence and absence of bound repressor. Using trpR containing DNA fragments in vitro mutagenesis will be carried out to generate regulatory and structurely altered repressor genes. These studies will be designed to obtain altered operators that may result in operon constitutivity in addition to repressor proteins altered for corepressor ligand and operator recognition. The availability of pure aporepressor should permit a detailed study of repressor-operator interaction. Using the existing in vitro transcription repressible assay it should be possible to measure dissociation constants for the trpR and aroH operators in addition to the binding by altered repressors. Traditional nitrocellulose filter binding assays experiments for trp repressor binding studies, previously unsuccessful will be reexamined now that milligram amounts of pure repressor are available. By extending studies on the trp repressor-operator we hope to gain a more detailed understanding of gene control.
