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Composition and adpatation of the E. coli RNA degradosome

University of British Columbia

Although transcription and translation are recognized mechanisms for regulating gene expression, control over RNA stability can also accomplish this task. In E. coli, bulk mRNA degradation is largely carried out by a complex of enzymes called the degradosome. It is composed of the endonuclease RNase E, the exonuclease PNPase, the helicase RhIB, and the glycolytic enzyme enolase. A role in mRNA decay has been assigned to the first three proteins, but the function of enolase is currently unknown. The hypothesis that it senses the metabolic state of the cell and alters the activity of the degradosome accordingly was tested. Assays using model substrates were performed with degradosomes reconstituted from purified components, in the presence and absence of enolase. The presence of this enzyme in the assay had no apparent effect on the activity of either RNase E, PNPase, or RhIB. Simulation of possible signals impinging upon enolase, such as binding of 2-phosphoglycerate or phosphoenolpyruvate, or phosphorylation, exerted only a very modest effect on degradosome activity. While purified RNase E, PNPase, and RhIB reconstitute a complex when incubated together, enolase appears to require the assistance of PNPase and/or another factor to assemble with RNase E. These studies have failed to identify a role for enolase in adapting the activity of the degradosome to metabolic signals. Other mechanisms for altering the function of the degradosome were investigated by studying the complex during and after cold shock. Under those conditions, CsdA, an ATP-dependent RNA helicase enters the complex. Three separate types of experiments support the presence of CsdA in the cold shock degradosome: co-purification, co-immunoprecipitation, and reconstitution from purified components. This enzyme is capable of replacing RhIB in the in vitro degradation of a substrate that requires the coordinated activity of both PNPase and RhIB for degradation. The composition of the degradosome is altered in response to a temperature downshift, and a novel mechanism of regulating the activity of the degradosome, and presumably gene expression, has therefore been discovered.

Thesis/Dissertation

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2009-12-01

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http://hdl.handle.net/2429/16015

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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