Synchrony of harmony and chaos in a dynamic, bacterial riboregulatory machine

Abstract

In all domains of life, proteins involved in the regulation of RNA lifetimes are often found to form multi-enzyme complexes, suggesting a common requirement to colocalise their activities to meet the demands of RNA metabolism 1,2. In diverse bacterial lineages, these multi-enzyme complexes, referred to as “RNA degradosomes”, are composed of ribonucleases, helicases, and other proteins of RNA metabolism. This dissertation focuses on the RNA degradosome of the representative Gram-negative bacterium, Escherichia coli, where the main component of the assembly is RNase E, a homotetrameric endoribonuclease. Each monomer of RNase E can be divided into two domains: the N-terminal domain which carries out the catalytic activity, and the C-terminal domain, which is intrinsically disordered but punctuated by microdomains that correspond to binding sites for the other components of the complex and acts as scaffold domain for the assembly of the RNA degradosome 2,3. The other canonical components of the RNA degradosome of E. coli are the ATP-dependent DEAD box RNA Helicase RhlB, and the second ribonuclease of the complex, the exoribonuclease PNPase. Finally, the last canonical component is the glycolytic enzyme enolase, for which the precise function in the context of the RNA degradosome is not fully understood. The scaffold domain of the RNA degradosome has a conserved propensity for intrinsic disorder character, implicating an important biological function. However, this feature has presented challenges in studying the quaternary organisation of the complex and the cooperation of the different components. This dissertation aims to elucidate some key aspects of the structural organisation of the RNA degradosome of E. coli and its functional implications and focuses on some of the main open questions in the field. The first chapter explores the association of RNase E and PNPase, two of the main ribonucleases involved in RNA metabolism. The interaction between those enzymes has been conserved over evolution 2, but little is known of the details of the interaction, or how it might enable cooperation. In this chapter, evidence is presented of a new, extended binding site for PNPase on RNase E which boosts the binding affinity and has implications for the functional interplay of the two enzymes. Structural and biophysical characterisation of the interaction defines the stoichiometry of the RNA degradosome, where one RNase E tetramer interacts with four PNPase trimers. Activity assays suggest how the interaction can affect cooperation within the RNA degradosome. The second chapter aims to structurally characterise the effect of the binding to the membrane on the scaffold domain of the degradosome and its impact on the ensemble organisation of the nanomachine 4. Finally, the last chapter focuses on the interaction between the RNA degradosome with ribosomes and translating polysomes. The interactions are studied at different stages of transcription in vitro and a hypothesis made that these may represent intermediates that provide an opportunity for RNA surveillance in bacteria.