| Previous studies suggest that the [2Fe-2S] cluster of IscR is necessary for repression of its own promoter that contains a Type 1 IscR binding site (Schwartz 2001, Giel 2006). Recent work indicates that IscR also binds to a second class of binding sites, designated Type 2 sites (Giel 2006). In this thesis, I showed that, surprisingly, the [2Fe-2] cluster of IscR is not necessary for in vivo regulation of promoters containing a Type 2 IscR binding site (PsufA, PyaiU, PhyaA, PnapF, and PhybO). Unlike IscR binding to Type 1 sites, both the wild-type form of IscR containing the Fe-S cluster and a mutant of IscR that lacks the Fe-S cluster bound to the Type 2 site within the hya promoter with similar binding affinities. The effect of mutations within the IscR binding site from the hya promoter was determined by DNA competition assays. These results refined our understanding of the sequence required for IscR binding and allowed us to propose the following binding motif "1Ax3CCx2 Ax7TAxGGx3T25". Finally, IscR appeared to bind the hya site cooperatively as two dimers. Based on this data and the similarities between the biochemical properties of IscR and the transcription factor, QacR, I propose that two dimers of IscR bind on different faces of the DNA helix. In summary, IscR does not require its [2Fe-2S] cluster to regulate promoters containing a Type 2 site, and IscR binding to Type 2 sites is complex in part due to the cooperative binding of two dimers.;Furthermore, I investigated what factors lead to promoters containing a Type 2 site to being regulated to a greater extent under aerobic than under anaerobic growth conditions (Giel 2006). One possible explanation is that other transcription factors compete with IscR for regulation under anaerobic conditions. I found that ArcA and AppY both prevent repression by IscR at Type 2 containing PhyaA· However, the decreased regulation is also due to the 10-fold decrease in wild-type IscR protein under anaerobic conditions because increasing the levels of IscR results in repression of PhyaA·. Thus, IscR levels may be insufficient to bind to some Type 2 sites under anaerobic conditions and/or to compete with binding of other transcription factors. |
