Graduate Thesis Or Dissertation

 

Short lived bacterial regulatory proteins : what determines their fate? Public Deposited

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  • Rapid degradation of certain short lived "timing" proteins is an effective mechanism for cells to control important regulatory pathways. The mechanisms by which regulatory proteases recognize their substrates are not well understood. Escherichia coli Lon, an energy dependent protease highly conserved in many prokaryotes and eukaryotes provides a model system to study protease/substrate interactions. RcsA, a regulator of capsule synthesis, when present in levels high enough to saturate Lon, cannot protect SulA, a cell division inhibitor, from being degraded. These observations suggest Lon recognizes its different substrates with different affinities. The different affinities of these substrates might relate to the role these substrates play in the cell: stabilization of RcsA leads to a nonlethal phenotype (capsule), while stabilization of SulA leads to lethal filamentation. To further examine protease/substrate interactions, targeted mutagenesis was employed to select for mutations in rcsA which give rise to mutant RcsA protein no longer degraded by Lon protease. Two mutants with an increased half-life in the presence of Lon were identified. Their mutations fall into the C-terminal region of RcsA, supporting the hypothesis that this region is involved in the interaction of RcsA with Lon. Stabilization of RcsA was dependent on its partner RcsB; the interaction of RcsA with RcsB is believed to protect RcsA from Lon dependent degradation. However, it was shown that rcsA expression is enhanced in the presence of RcsB, and RcsA protein cannot be detected in strains mutant for RcsB in the presence or absence of Lon. Furthermore, rcsA expression was shown to be activated by RcsA itself: rcsA::lacZ expression is low in the absence of RcsA. A conserved 25 by motif, designated "RcsA-Box" was identified in the promoter region of the rcsA and capsule (cps) genes. This motif was shown to be a likely candidate for RcsA binding: high level expression of both cps::lacZ and rcsA::lacZ fusions was shown to be dependent on the presence of the "RcsA-Box". These studies expand the understanding of the specific interactions between regulatory proteases and their targets, specifically as they relates to complex regulatory networks.
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