Ribosome hibernation and its role in the maintenance of the translation machinery: How hibernation factors of E. coli protect ribosomes in adverse conditions

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

Protein synthesis is a fundamental process common to all organisms. Accordingly, the translation machinery is an essential component of a living cell. At the core of this huge apparatus is the ribosome, a ribonucleoprotein particle that catalyses the decoding of the transcribed genetic code into protein. The ribosome is a hub for a plethora of gene regulatory mechanisms and due to the considerable cost of maintaining such a machinery, it is itself subject to intricate systems of regulation that govern the synthesis, upkeep and degradation of its components. Bacteria employ multiple systems of reversible modulation of ribosomes in order to rapidly adapt to the ever-changing environment of their habitats. One of the most important strategies in response to adverse conditions is the conversion of a considerable fraction of available ribosomes into inactive assemblies, a phenomenon called ribosome hibernation. This is mediated by so-called hibernation factors, small, ribosome-associated proteins that are induced by a variety o fenvironmental stresses. Escherichia coli possesses three hibernation factors, ribosome modulation factor (RMF), hibernation promoting factor (HPF) and ribosome associated inhibitor (RaiA). The concerted action of RMF and HPF induces the association of two 70S ribosomes to a 100S complex, while RaiA maintains 70S particles in an inactive state. Over the course of this PhD project I investigated the role of the three hibernation factors of E. coli on a physiological as well as mechanistic level. I attempted to unravel the implications of ribosome hibernation in the adaptation to nutrient starvation, its involvement in the protection of ribosomes as well as the maintenance of nutrient homeostasis and metabolic activity in the cell. I show that hibernation factors RMF, HPF and RaiA jointly protect ribosomes during glucose starvation, thereby maintaining basal ribosome homeostasis. The reservoir of inactive but intact hibernating ribosomes can re-enter translation when nutrients are replenished and ensuresrapid re-entry to exponential growth, thus conferring a considerable fitness advantage. I show that ribosomal RNAs (rRNAs) within assembled 70S ribosomes are increasingly fragmented in the absence of hibernation factors. Mapping accumulating 16S rRNA fragments reveals potential endonuclease cleavage sites at positions crucial for translation initiation and subunit association,which are likely directly protected by binding of hibernation factors at overlapping or adjacent sites. Furthermore, I present evidence that the conserved endoribonuclease YbeY and exonuclease RNase R are involved in the generation of fragmented 16S rRNA in 70S particles and may participate in the downstream degradation of such ribosomes. In addition, I show that the role of ribosome hibernation in efficient regrowth is conserved across various stress conditions, including growth arrest in complex medium and phosphate or nitrogen starvation. When phosphate is limiting, protection of ribosomes and rRNA by hibernation factors has an additional regulatory effect by preventing excessive degradation and subsequent scavenging of rRNA for inorganic phosphate. The accumulation of free phosphate and adenosine triphosphate (ATP) in hibernation-deficient cells leads to deregulation of stationary phase entryand is manifested in increased biomass production and an almost immediate effect on the regrowth capability of cells. To conclude, I present evidence linking the structural basis of ribosome hibernation and the physiological function of this widely conserved mechanism.
ForlagDepartment of Biology, Faculty of Science, University of Copenhagen
Antal sider176
StatusUdgivet - 2020

ID: 251250494