| Fe-S proteins are ubiquitous in nature and participate in several pathways vital to cellular metabolism. While much is known concerning the function of Fe-S clusters containing proteins, little is known about how Fe-S clusters are assembled into their cognate proteins. The work in this thesis has focused on identifying proteins that facilitate Fe-S cluster assembly in Escherichia coli and how the genes encoding these proteins are regulated.; I have shown that, IscS, is the major in vivo sulfur donor for Fe-S cluster assembly. All Fe-S cluster proteins assayed had substantial defects in activity in an iscS deletion strain, while non Fe-S cluster proteins were unaffected. These results established that IscS is specific for the maturation of Fe-S cluster proteins.; The iscS gene is organized in a gene cluster with three other genes (iscRSUA) and adjacent to the isc genes is a second group of genes (hscBA and fdx) implicated in Fe-S cluster assembly. In addition to an iscS mutant, we have demonstrated that strains mutated for hscA are also deficient in Fe-S cluster assembly, establishing a role for the hscA gene product in this process,; The genomic organization of these two gene clusters is conserved in many bacteria, suggesting that they function in a similar process. Therefore, we have examined the transcriptional organization of this region to gain insight into the physiological role of these gene products. We became interested in the gene upstream of iscS, iscR, because it is homologous to the transcriptional regulator MarA. Upon deletion of iscR, iscS, or hscA the abundance the iscRSUA transcript is substantially increased. The greatest increase in promoter activity results from the loss of iscR, suggesting that IscR represses transcription of the isc operon. Our lab has purified the IscR protein and shown that is contains a Fe-S cluster. Our data suggests a feedback regulatory mechanism whereby the transcriptional activity from the region is influenced by the status of Fe-S cluster assembly, which is conferred through the Fe-S cluster of IscR. Therefore, this work provides a beginning to understanding how Fe-S clusters are assembled and how this process is controlled. |
