Dissecting the contributions of proteolysis, iron-sulfur cluster assembly, and transcriptional repression on the regulation of the Escherichia coli transcription factor FNR.

摘要

In Escherichia coli, the transcription factor FNR senses O2 and responds by altering the expression of genes required for adaptation to O2-limiting conditions. FNR is active under anaerobic conditions, when it contains an O2-labile [4Fe-4S] cluster, which is required for FNR activity. Under aerobic conditions, the cluster is destroyed, thereby inactivating FNR. The work in this thesis describes additional regulatory processes that contribute to the ability of FNR to optimally sense and respond to O2.; To investigate the role of proteolysis, the degradation rate of FNR was measured. These studies showed that inactive, clusterless FNR is specifically but slowly proteolyzed by the ClpXP protease, whereas dimeric, [4Fe-4S]-FNR escapes degradation. Varying FNR levels over a small range had a direct effect on the efficiency of FNR inactivation in aerobic cells, suggesting that proteolysis maintains appropriate FNR levels for its efficient inactivation by O 2. Finally, proteolysis was used to demonstrate that O2-inactivated FNR (apo-FNR) in aerobic cells is reactivated to [4Fe-4S]-FNR.; To understand the pathways required for FNR Fe-S cluster assembly, the contributions of the Isc and Suf Fe-S cluster biogenesis pathways were evaluated. By monitoring the activity of an FNR mutant that contains an O2-resistant Fe-S cluster in strains lacking either Isc or Suf, I found that Isc is the major pathway by which newly synthesized FNR acquires Fe-S clusters under aerobic and anaerobic conditions. However, under anaerobic conditions, a second Fe-S cluster biogenesis pathway can also provide Fe-S clusters for FNR. In addition, the re-conversion of apo-FNR in aerobic cells to [4Fe-4S]-FNR is mediated by Isc.; Finally, the regulation of fnr expression was examined. Using in vitro and in vivo approaches, it was demonstrated that FNR represses its own synthesis by binding to a single site within Pfnr. The finding that IHF plays a role in activating Pfnr suggests an additional level of regulation. In summary, this thesis provides evidence which suggests that proteolysis, Fe-S cluster assembly, and transcriptional regulation are additional processes that contribute to the regulation of FNR as an O2-sensor.