For many organisms, the ability to sense and adapt to changes in oxygen tension in the environment is crucial to their survival. For example, excess oxygen creates reactive oxygen species that can damage most macromolecules, whereas oxygen deprivation can result in energy starvation and consequently inhibit cell growth. Although oxygen plays a pivotal role in many biological systems, the molecular mechanism by which oxygen is sensed by cells is poorly understood. Our long range goal is to identify how cells sense and respond to changes in environmental oxygen. The Escherichia coli transcription factor Fnr provides an ideal system in which to investigate this question since it globally regulates gene expression in response to oxygen deprivation. Under anaerobic conditions, Fnr stimulates transcription of anaerobic respiratory enzymes, and it also represses synthesis of at least two aerobic respiratory complexes. Fnr levels are not oxygen regulated, but Fnr activity is regulated by oxygen deprivation. The central questions in this field are the identities of the physiological signal which results from oxygen deprivation and the effector molecule which converts Fnr from its inactive form in aerobic cells to an active transcription under anaerobic conditions. Therefore, the ultimate goal of our experiments is to biochemically define how Fnr activity is regulated by oxygen deprivation. The eventual realization of this goal will require identifying the effector for Fnr, developing an in vitro system to monitor Fnr-specific DNA interactions, and determining if the conformation of Fnr is altered by effector binding. I have selected Fnr* mutants that activate transcription of an Fnr target operon in the presence of oxygen. These Fnr* mutants provide a unique opportunity to develop an in vitro system to study Fnr-DNA interactions because they bypass the need for the unknown effector which has made previous analysis of the wild type protein difficult. I will confirm that the phenotype of these Fnr* mutants is correlated with their unique ability to bind to target sequences in the presence of oxygen in vivo. I will purify Fnr* proteins to determine if they contain any bound prosthetic groups and identify the effector. In addition, I will develop an in vitro system to determine if effector binding alters wild type Fnr conformation. A genetic analysis will begin tracing steps in the proposed signal transduction pathway from the physiological signal induced by oxygen deprivation to effector binding and Fnr activation. Finally, analysis of Fnr mutants will allow us to determine regions of this protein that allow it to respond to oxygen deprivation. This interdisciplinary approach will allow me to demonstrate how oxygen deprivation regulates Fnr activity.
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