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Publication Date

2009-11-17

Availability

UM campus only

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2009-10-09

First Committee Member

Richard S. Myers

Second Committee Member

Kenneth E. Rudd

Third Committee Member

Chaitanya Jain

Fourth Committee Member

Murray P. Deutscher

Fifth Committee Member

Zhongwei Li

Abstract

Upon encountering stress conditions, cells must rapidly alter their gene expression and re-model their RNA complement to deal with the changing environment. As a consequence, both new RNA transcription as well as RNA degradation must take place. Accordingly, the RNA degradative machinery may adjust to the changes in RNA metabolism. Thus, a study of the response of the three major degradative exoribonucleases in Escherichia coli, polynucleotide phosphorylase, RNase II, and RNase R, to stress is of significant importance. RNase R, a processive 3' to 5' exoribonuclease, is unique among the known E. coli exoribonucleases in its ability to digest through RNAs containing extensive secondary structure without the aid of a helicase. In vivo, RNase R plays important roles in quality control of stable RNA, decay of mRNA with extensive repetitive extragenic palindromic (REP) sequences, cell-cycle regulated degradation of tmRNA in Caulobacter crescentus, as well as processing of rRNA under low temperature in P. syringae. In this dissertation, RNase R was shown to be unusual among the E. coli exoribonucleases in its dramatic response to a variety of stress conditions. Elevation of RNase R activity by as much as 10-fold was observed in response to entry into stationary phase, starvation and cold shock, and an ~3-fold increase was seen during growth in minimal medium compared to rich medium. The elevation in RNase R activity was associated with an increase in RNase R protein. Phenotypes of rnr mutants were also investigated, and RNase R was found to contribute to cell growth and viability. Further investigation of the regulation of RNase R during stress, primarily in stationary phase, revealed a novel regulation mechanism. Despite the large increase in RNase R protein and activity in stationary phase, rnr message actually decreased to only ~14% of its level in exponential phase. Further study revealed that RNase R is highly unstable in exponential phase and becomes stabilized during stationary phase, cold shock, and in minimal medium. Investigation of proteolysis on the unusual instability of RNase R indicated that both Lon and ClpXP play a role. In the absence of Lon, RNase R stability is increased ~10-fold. Based on these results, I propose that the increase in RNase R during stress is due to its enhanced stability under those conditions.

Keywords

Proteolysis; Cold Shock; Stationary Phase; Exoribonuclease

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