Structural and functional characterisation of the protein-protein and protein-RNA interactions in the RNA degradosome

The C-terminal domain of RNase E is intrinsically unstructured, but small segments of 13 to 80 residues are predicted to have propensity for defined conformation and evidence presented here indicates that they function in nucleic acid binding and protein-protein interactions in the degradosome. Binding of two of these ordered regions to their predicted partners, enolase and PNPase, has been demonstrated using non-dissociating nano-flow mass spectrometry (MS). Binding of helicase to an arginine rich domain of RNase E (residues 628-843) has also been shown by MS and other approaches. The binding stoichiometry for the various degradosome components with minimal binding regions on RNase E is also provided by non-dissociating MS, and these data provide insight into the organisation of the degradosome. The crystal structure of E. coli enolase in complex with a synthetic peptide corresponding to the proposed recognition in the degradosome (RNase E residues 833-850) has been solved to 1.6Å resolution. The crystal structure reveals a 1:1 complex of the peptide with the enolase dimer. It is predicted that enolase and RNase E interact specifically and stably in many Gram-negative pathogens, with implications for a common mode of regulated turnover of targeted transcripts. An analysis of the metabolome data from wild type E. coli cells and the degradosome mutant strains grown normally and under phosphosugar stress signify the importance of coordinated RNA degradation by the organisation of a degradosome machinery in regulating cellular responses to physiological stress stimuli.EThOS - Electronic Theses Online ServiceGBUnited Kingdo