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Binding with intent to destroy : RssB adaptor function in Clp-XP-mediated proteolysis of sigmaS

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dc.contributor.advisor Tania A. Baker. en_US
dc.contributor.author Ebrahim, Shamsah en_US
dc.contributor.other Massachusetts Institute of Technology. Dept. of Biology. en_US
dc.date.accessioned 2008-03-27T18:29:05Z
dc.date.available 2008-03-27T18:29:05Z
dc.date.copyright 2007 en_US
dc.date.issued 2007 en_US
dc.identifier.uri http://hdl.handle.net/1721.1/40959
dc.description Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007. en_US
dc.description Includes bibliographical references (p. 113-121). en_US
dc.description.abstract Severe stress results in global changes in the bacterial proteome. To respond effectively, new proteins must be synthesized, others destroyed. Coordinated changes in protein synthesis often result from the engagement of an alternate a subunit with the RNA core polymerase. The as subunit of the RNA polymerase is a master stress response regulator in E. coli. Under satisfactory growth conditions, proteolysis keeps the levels of as low; upon stress, various mechanisms converge to raise the levels as. In this work we focus on the facilitated delivery of as to the ClpXP protease. Proteolysis of as by ClpXP requires the accessory factor RssB. RssB is a two-component response regulator. Not surprisingly, its activity is positively regulated by phosphorylation of a conserved aspartate in its receiver domain. Whereas most response regulators are transcription factors, however, RssB is an adaptor protein. RssB binds to as, promoting a conformational change in as that exposes its N-terminal ClpX recognition motif. RssB also contacts ClpXP itself, enabling the formation of a quaternary degradative complex. Following the degradation of as, RssB is released. This work addresses two outstanding questions regarding RssB-mediated proteolysis of [sigma]s: 1) the requirement of RssB phosphorylation for [sigma]s degradation; and 2) the mechanism of RssB interaction with ClpXP. Previous studies have shown that phosphorylation of RssB increases its affinity for [sigma]s. Initially, phosphorylation of RssB was thought to be a pre-requisite for [sigma]s binding; more recently this has come under some debate. Our data demonstrate that phosphorylation is not strictly required for RssB function. en_US
dc.description.abstract (cont.) Using both wild-type RssB and an unphosphorylatable variant, we show that the impact of phosphorylation on RssB activity is condition-dependent. With regards to the interaction between RssB and ClpXP, we demonstrate that critical contacts are made between the N-terminal domain of ClpX and a ClpX-binding sequence located, not at the C-terminus of RssB [sigma]s previously predicted, but within the inter-domain linker of RssB. This sequence motif is similar to those used by the other ClpXP adaptor proteins, all of which make contact with the ClpX N-domain. These results support a model in which adaptor proteins interact with a common binding site on ClpX. Indeed, we show that competition between SspB and RssB can occur in vivo and in vitro. We postulate that competition between adaptors is important in modulating substrate prioritization in times of stress. en_US
dc.description.statementofresponsibility by Shamsah Ebrahim. en_US
dc.format.extent 121 p. en_US
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. en_US
dc.rights.uri http://dspace.mit.edu/handle/1721.1/7582 en_US
dc.subject Biology. en_US
dc.title Binding with intent to destroy : RssB adaptor function in Clp-XP-mediated proteolysis of sigmaS en_US
dc.title.alternative RssB adaptor function in Clp-XP-mediated proteolysis of sigmaS en_US
dc.type Thesis en_US
dc.description.degree Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Dept. of Biology. en_US
dc.identifier.oclc 213081197 en_US


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