Specificity & Regulatory Properties of trp Repressor

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This is a proposal to study the molecular basis for the specific recognition of nucleic acids by proteins, and the factors involved their regulatory interactions, both in vivo and in vitro. The work will focus on the structurally well- characterized trp repressor of E. coli, and will be concentrated in five areas. 1. A thorough study of operator DNA structure/function relations will be made including their role in the recognition process. An oligonucleotide representing the operator will be synthesized, along with a control DNA of identical composition but unrelated sequence. The average solution conformation of these DNAs will be defer mined using circular dichroism (CD). The thermodynamics of the helix-coil transition for these DNAs will be determined using CD, and the results will be compared to those obtained in the presence of the repressor. 2..Development of new reliable, quantitative assays for the binding reactions of trp repressor will be undertaken. First, it will be ascertained whether protein binding causes a change in the CD spectrum of the DNA; this assay is likely to be suitable only for nonspecific binding. In an effort to assay specific binding, fluorescent probes will be attached to the DNA at sites where they may be quenched by the bound protein without interfering greatly with its binding. 3. The affinity and specificity of trp repressor for binding to DNA will be determined under the exact conditions that have been used for crystallization of the complex. The results should establish whether these conditions are compatible with operator-specific binding, and would thus resolve a major controversy engendered by the crystal structure. 4. A complete analysis of the biochemical and thermodynamic characteristics of the in vitro binding reactions of the repressor will be made. Salt concentration and temperature dependence for binding to specific and nonspecific DNA and to tryptophan will be measured, and related to what is known about the structure of the protein. We will be used to establish whether an order-disorder transition explains the large CP for DNA binding found in the temperature studies. 5. In vivo and in vitro experiments will be carried out to determine the detailed molecular mechanism of transcriptional control by trp repressor. The question of whether the effect of repressor on RNA polymerase is the same at the three known sites of regulation, and which step in the polymerase mechanisms is affected at each site will be investigated. The affinities of the three operators in vivo will be compared, ant it will be determined if repressor and polymerase communicate to modulate each other's interactions with the DNA.