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. / Graduation date: 1998
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/34061 |
| Date | 19 June 1997 |
| Creators | Ebel, Wolfgang, 1967- |
| Contributors | Trempy, Janine E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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