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.