Transcription of genetic information in all organisms must be responsive to extracellular and intracellular signals such as levels of metabolytes, cell-cell interactions, and occupancy of cell surface receptors by ligands. Multifunctional transcription regulatory proteins provide an efficient means of directly linking these signals to the transcription initiation process. Quantitative studies of these transcription factors will reveal the molecular switching mechanisms that enable intimate coupling of an array of physiological signals to genetic regulation. In E.coli, production of the nutrient, biotin, is linked to cellular demand by BirA, a multi-functional transcription factor. BirA is an allosteric DMA binding protein and the enzyme that catalyzes posttranslational addition of biotin to a carboxylase. The intermediate in the biotin transfer reaction, bio-5'-AMP, also activates the protein for site-specific DNA binding by dramatically enhancing the energetics of BirA homodimerization. Thus, elucidation of the consequences of bio-5'-AMP binding for the structural and dynamic properties of the represser monomer will provide insight into the pathway of allosteric activation. These studies will employ stopped-flow fluorescence measurements of effector binding and structural analysis of the complexes using H/D exchange coupled to mass spectrometry. The functional switch of BirA from transcription represser to enzyme is hypothesized to occur via switching of protein partners. Moreover, as the same surface of the represser is proposed to be utilized for the two protein:protein interactions, molecular mimicry is anticipated to contribute to the switching process. Thermodynamic and kinetic measurements of the alternative protein:protein interactions will reveal the molecular basis of the use of a single surface for the alternative interactions that are central to this transcriptional switch. Surface Plasmon Resonance, steady-state and stopped-flow fluorescence and quench-flow methods will be used for these measurements. Structural analysis of the relevant complexes will be carried out using x-ray crystallography.
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