Processes that regulate gene transcription are fundamental in controlling the behavior of cells of all types. In eukaryotes regulation occurs through cooperative formation of very large complexes, which make detailed characterization of the processes very difficult. In prokaryotes regulation is simpler, and many components of the molecular machinery that execute the processes have been structurally characterized, including the RNA polymerase and its complex with a70. Our studies of transcriptional activators and a54 expand the understanding of bacterial transcriptional regulation. In bacteria sensing and responding to environmental changes often occurs through two-component systems, kinase - receiver domain pairs, most of which lead to altered levels of transcription. These systems are relatively simple, allowing study of the basic the processes involved in control and initiation of transcription. Our long term goal is to provide a comprehensive molecular level understanding of a two- component system that acts through a54 polymerase. We will continue structural work on the transcriptional activators NtrC, NtrC1 and NtrC4, to understand how the phosphorylation driven conformational change leads to assembly of the active oligomer and subsequent interaction with a54, how ATP hydrolysis changes the conformation of the NtrCs and, thereby that of the a54-polymerase to enable opening of the DMA, the basis for sequence specific DMA binding by these proteins and how cooperativity in DMA binding occurs. We will also determine structures of a54 domains determine how they interact, and to allow us to model NtrC-a54 complexes that are being determined by others at low resolution. We will also analyze a homolog NtrC2, which contains a GAP domain instead of a receiver domain to regulate it, to see if the regulatory mechanism is the same as in NtrC1. We will use NMR spectroscopy to determine structures and probe dynamics of domains, and x-ray crystallography to determine structures of larger complexes as needed. Two component systems occur in prokaryotes and lower eukaryotes and, hence, provide possible targets for future drug development, which the structural understanding obtained through this work would aid. Other parts of the proteins being studied are very similar to human proteins that help reorganize protein complexes. The details of how these work are not yet clear, and what is learned from structural studies of these bacterial versions will provide insights into how these function. Gene regulation is so central to the processes of life that it is important to understand it in great detail, to be able to understand the implications of alterations in the process through effects of disease or the environment. ? ?
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