Analysis of the enzymes oligoribonuclease (Orn) and RNA pyrophosphohydrolase (RppH) in Escherichia coli K-12 RNA metabolism
Wiese, Nicholas Stacy
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Degradation of mRNA and maturation of stable RNAs play vital roles in controlling gene expression at the post-transcriptional level. In Escherichia coli the enzymes RNA pyrophosphohydrolase (RppH) and oligoribonuclease (Orn) play unique roles in overall RNA metabolism by acting on disparate ends of RNA molecules. The work described in this dissertation further elucidates the functional and physiological role of these two enzymes. We have analyzed the role of RppH in tRNA processing and show that the absence of RppH does not affect the processing of tRNAs by RNase E, contrary to long-held beliefs. We also show that RNase P processing is affected by RppH for a subset of tRNAs, and that this effect is mediated by the presence of the Rph-1 truncated protein. We examined the role of RppH regulation on the entire transcriptome employing high-density tiling microarray analysis. We discovered that RppH is involved in regulating the entire flagellar gene regulatory network, and hypermotility results in strains lacking RppH. As before however, we also observed that like RppH-dependent RNase P processing, hypermotility due to absence of RppH was dependent on the presence of the Rph-1 protein. Lastly, we characterize how the absence of Orn affects cellular functions in E. coli. We constructed a full deletion of orn in a clean genetic background and controlled expression through tightly-regulated expression plasmids. Through numerous assays, we found that the absence of Orn alone does not cause a cessation in growth, but rather that cellular viability is dependent on the absence of either RNase T or PNPase in conjunction with Orn. These results are the first to implicate either RNase T or PNPase as a backup mechanism for oligoribonuclease activity. We also determined the true transcription start site of orn, and have identified the putative promoter sequence, contrary to what is currently annotated. These results suggest that orn regulation is more intricate than previously thought.