A common mechanism utilized by organisms to respond to metabolic demands is through changes in transcription of specific genes. This response can occur through complex signal transduction processes or through simpler mechanisms involving binding of metabolites to transcription factors to regulate their DNA binding function. One paradigm for achieving communication of metabolic requirements to gene expression is through the direct use of metabolic enzymes in regulating gene transcription. In this proposal experiments are described to elucidate the mechanism by which the Escherichia coli biotin protein ligase, BirA, responds to the demand for biotin at the molecular level. BirA funnels biotin into metabolism by catalyzing its linkage to the biotin-dependent carboxylase, acetyl CoA carboxylase, and binds sequence specifically to the biotin operator sequence to regulate transcription of the biotin biosynthetic genes. The function of this regulatory circuit reflects several properties including allosteric activation by a small molecule, use of a single protein surface for multiple interactions and kinetic control of protein function. In the proposed studies the molecular mechanism of the BirA functional switch will be elucidated using combined biophysical and biochemical measurements. These include equilibrium measurements of ligand binding by Isothermal Titration Calorimetry and protein association by sedimentation equilibrium, measurements of H-D exchange coupled to mass spectrometry, and kinetic measurements of protein association. The combined studies will reveal the basic molecular and cellular mechanisms of communication between metabolism and transcription.
The ability of any organism to respond to changes in metabolite levels at the transcriptional level is essential for viability. In addition, several diseases, including diabetes, obesity and cancer are now known to involve communication of metabolic status to transcription. Elucidation of the molecular mechanisms by which this communication occurs will lead to development of new methods for intervening, when possible, in the process.
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