Analyses of RNA in Escherichia coli
Stead, Mark B.
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The maintenance of homeostasis in Escherichia coli is complex. One aspect of control that is often overlooked is post-transcriptional regulation, which is often mediated by ribonucleases that can either destroy or stabilize an RNA species. The work described in this thesis examines the role of ribonucleases both in the control of gene expression and in the processing of ribosomal RNA. During the course of this work, it also became apparent that the RNA extraction methods currently being used for these analyses were not adequate. Accordingly a superior, novel method of extracting RNA was devised. We analyzed the in vivo roles of two endoribonucleases (RNase E and RNase III) using tiling microarrays and demonstrated that both enzymes have considerably larger roles in gene expression than previously envisioned. In fact, we found that RNase E affects ~75% of small RNAs and RNase III affects greater than ten-fold more RNAs than previously thought. In addition, over 300 potentially novel genes were identified, along with the first demonstrated evidence of RNase III cleavage within a protein coding sequence in E. coli. After working on transcriptome-wide gene expression analyses, we realized that no RNA extraction method resulted in the quantitative recovery of all RNA species present in the cell. In fact, we demonstrated that with the most commonly used commercial RNA extraction kits, RNA was selectively lost based on molecule size with some kits retaining only larger species, and some only small species. To circumvent this problem, we developed a very simple, cost-effective RNA extraction method that yields quantitative recovery of nearly 100% of RNA species in the cell, regardless of size, in a little as 15 minutes. This work has also attempted to address long standing knowledge-gaps in the processing of ribosomal RNA, as the enzymes involved in many of the processing steps remain unidentified. While this work remains unfinished, we have constructed the tools required for the analysis of these processing steps and formed the foundation of future work which will resolve the processing pathway involved in ribosomal RNA maturation in E. coli.