The long term goal of this project is to understand the biochemical and molecular events that allow cells to sense and adapt to changes in oxygen in their environment. Since oxygen is essential for the viability of many organisms, this problem has broad biological significance. To address this problem, we are studying the Escherichia coli transcription factor FNR that globally regulates gene expression in response to oxygen deprivation. The experiments in this proposal will answer two central questions regarding how FNR regulates transcription of its target genes under anaerobic conditions. The observation that FNR activity is regulated by oxygen availability has provided us the opportunity to dissect a cellular sensing mechanism for oxygen. Our data indicate that this protein contains a [4Fe-4S] cluster which appears to act as an oxygen sensor. The proposed experiments will demonstrate whether oxygen directly controls the activity of FNR by causing the oxidative degradation of this Fe-S cluster. To determine how the Fe-S cluster affects FNR activity, the rate of decrease in dimerization and DNA binding will be compared to the rate of Fe-S cluster loss following exposure of FNR to oxygen. To test the idea that instability of this Fe-S cluster to oxygen is physiologically relevant, the rates of FNR inactivation in vitro and in vivo will be compared for WT FNR and a series of FNR* mutant proteins which have increased activity in the presence of oxygen. If FNR* mutant substitutions are identified that alter the stability of the Fe-S cluster, we would conclude that oxygen dependent inactivation of the Fe-S cluster regulates the activity of FNR in vivo. To determine whether oxygen or the oxygen radical superoxide is more effective in inactivating FNR, the effect of lowering superoxide levels on FNR function in vivo and in vitro will be assayed. Determining whether the FNR [4Fe-4S] cluster is sensitive to superoxide is of fundamental importance because such a result would indicate a link between the signaling systems for oxidative stress and those of oxygen deprivation. Furthermore, the potential use of an Fe-S cluster as an oxygen or superoxide sensor in FNR adds to a growing list of the versatile functions that these metal centers can provide in biology. Another goal of our experiments is to determine how FNR activates transcription at its target promoters and define whether both the sigma7O and alpha-subunit of RNA polymerase are required for FNR-dependent transcription activation. Defining this function of FNR is of great physiological importance since this protein is a global regulator of many anaerobically induced genes. In addition, the use of co-activators like NarL and CAP at some FNR-dependent promoters allows E.coli to integrate additional environmental signals such as changes in nitrate and cAMP in the absence of oxygen. Our experiments should provide the necessary foundation for elucidating the interactions of FNR at these more complex promoters.
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