The broad objective of this study is to understand the ability of an organism to assemble a large organelle. Control of assembling large structures poses some formidable problems. A great deal is known about regulatory mechanisms that respond to the level of small molecule ligands. How does an organism assess the degree of completion of a large structure, especially an extracellular structure such as a flagellum? Bacteria can do this. Mutants that lack key components fail to express proteins that would normally be added at later assembly stages. We have evidence that one mechanism involves a regulatory protein, FlgM, that escapes from the cell (and thus can no longer act) through a complete flagellum and is held inside where the structure has not reached a later stage of completion. We propose to investigate the mechanism of FlgM-mediated regulation in the flagellar biosynthetic pathway. The FlgM regulatory protein is a negative regulator of later steps in the flagellar assembly pathway. FlgM prevents late flagellar gene transcription by binding the flagellar-specific transcription factor, &28. FlgM is itself regulated in response to the assembly of an incomplete flagellum known as the basal body intermediate structure. Upon completion of the basal body-hook structure, FlgM is exported through this structure out of the cell. Inhibition of &28-dependent transcription is relieved and genes required for the later assembly stages are expressed allowing completion of the flagellar organelle. We wish to pursue the mechanism through which FlgM inhibits &28-dependent transcription and the mechanism through which FlgM is recognized by the flagellar-specific export apparatus at the proper stage of assembly, and exported out of the cell. The combination of genetic and biochemical approaches to these pursuits should enable the elucidation of just how a cell can accomplish the daunting task of building large organelles and the molecular mechanisms by which they are able to assess the different stages of completion in order to efficiently regulate the many genes involved in the process.
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