Project Title:

Structural analysis of the RNA degradosome of Escherichia coli

Project Summary:

In bacteria from highly divergent lineages, RNA turnover involves multi-enzyme complexes, called RNA degradosomes. These assemblies have arisen through both convergent and divergent evolution, indicating a common biological requirement to co-localise enzymes involved in RNA degradation and processing. The RNA degradosome of Escherichia coli is a multi-enzyme complex bound to the inner membrane that plays a fundamental role in the post-transcriptional control of gene expression. The scaffold domain of the RNA degradosome is intrinsically disordered: this feature has been conserved for close to a billion years in different bacterial lineages, indicating that the flexibility must be important for the function of the complex. This property may enable the E. coli RNA degradosome to form dynamically liquid-liquid phase separated droplets, as shown for other bacterial species. However, the flexibility and potential phase-separation behaviour of the RNA degradosome makes it unsuitable for conventional structural studies and requires a different approach for characterisation. Here, cryo-electron tomography (cryo-ET) and other biophysical methods are employed to study the ensemble characteristics of the RNA degradosome of E.coli bound to membranes. The RNA degradosome and its subassemblies have been successfully reconstituted onto membranes to resemble the in vivo conditions found in E.coli. Moreover, the RNA degradosome is known to interact with translating ribosomes and polyribosomes as part of a potential RNA surveillance mechanism. To investigate the structure and the functional meaning of the interaction, cryo-EM Single Particle Analysis (SPA) is being employed to study degradosome/ribosome complexes.