Direct entry by RNase E

The rapid degradation of mRNA is central to the control of gene expression. In Escherichia coli (E. coli), the initiation of mRNA degradation is performed by RNase E, an essential endonuclease that is also involved in the processing of stable RNAs. Previous studies have found that the recognition of a 5’-monophosphate group by RNase E can stimulate RNA cleavage. However, more recent work has shown that this 5’ monophosphate-dependent pathway may not be the major mechanism by which RNase E initiates mRNA decay, and that another pathway exists that has been termed ‘direct’ entry. However, the biochemical nature of this pathway has remained hypothetical and the exact importance of direct entry in RNA decay has yet to be established. The first results chapter presented here provides biochemical confirmation that direct entry cleavage by RNase E involves the simultaneous interaction of two or more unpaired regions of RNA. In addition, evidence is provided that shows that a 5’-monophosphate group does not enhance the catalytic activity of RNase E, a previous suggestion that contradicts the direct entry model. The second results chapter addresses the importance of direct entry in the cleavage of the E. coli transcriptome. It was found that a large proportion of cleavages dependent on RNase E were also sites of direct entry cleavage. The third results chapter provides evidence that post-transcriptional gene regulation by RNase E may extend beyond its ability to make endonucleolytic cleavages, by preventing the association of competing ribosomes to the mRNA and/or sequestering it to the inner membrane whereby the RNA decay machinery is associated. In summary, this thesis has shown that direct entry by RNase E is a simple yet prevalent mechanism involved in initiating mRNA decay in E. coli that is not exclusive with the 5’ monophosphate-dependent pathway.