Ribosome stabilisation in latent tuberculosis
File(s)
Author(s)
Koziej, Lukasz
Type
Thesis
Abstract
One third of the world population is infected with asymptomatic and non-infectious latent tuberculosis, which in many cases progresses into active disease later in life. During latent infection Mycobacterium tuberculosis is trapped in a cellular compartment, known as a granuloma. Granulomas provide a growth-limiting environment to the pathogen, due to oxygen depletion (hypoxia) and nutrient deprivation. M. tuberculosis adapts to these conditions by entering a non-replicating persistent state. To sustain this sometimes decade long state while remaining ready emerge, M. tuberculosis must ensure stability of ribosomes, which are responsible for biosynthesis of proteins. Whilst all known bacteria hibernate their ribosomes as 100S ribosome dimers in response to nutrient starvation, the stabilisation mechanism of mycobacterial ribosomes is untypical. M. tuberculosis and its model organism M. smegmatis express two ribosome hibernation promoting factors (HPFs) known as RafS and RafH. We investigated the molecular roles of RafS and RafH proteins using in vitro and in vivo approaches.
We used bioinformatic tools to predict that the C-terminal domain of RafS protein facilitates stabilisation of 100S ribosome dimers, like many other bacteria. We confirmed the existence of these higher order structures in M. smegmatis during prolonged carbon starvation. We used quantitative proteomic approach to discover that excessive degradation of ribosomes in the nongrowing bacteria is prevented by RafS protein.
We confirmed that during hypoxic stasis, mycobacteria-specific RafH protein is expressed to stabilise 70S ribosomes. To investigate the biochemical and structural properties of RafH protein, we formed a complex between purified RafH protein and 70S ribosomes in vitro. We discovered that the C-terminal domain in RafH is important for stabilisation of 70S ribosomes. We used cryoEM to determine the structure of RafH protein in complex with 70S ribosome. In an intermediate resolution electron density map (6.5 A), we confirmed the predicted binding site of RafH protein.
We used bioinformatic tools to predict that the C-terminal domain of RafS protein facilitates stabilisation of 100S ribosome dimers, like many other bacteria. We confirmed the existence of these higher order structures in M. smegmatis during prolonged carbon starvation. We used quantitative proteomic approach to discover that excessive degradation of ribosomes in the nongrowing bacteria is prevented by RafS protein.
We confirmed that during hypoxic stasis, mycobacteria-specific RafH protein is expressed to stabilise 70S ribosomes. To investigate the biochemical and structural properties of RafH protein, we formed a complex between purified RafH protein and 70S ribosomes in vitro. We discovered that the C-terminal domain in RafH is important for stabilisation of 70S ribosomes. We used cryoEM to determine the structure of RafH protein in complex with 70S ribosome. In an intermediate resolution electron density map (6.5 A), we confirmed the predicted binding site of RafH protein.
Version
Open Access
Date Issued
2019-10
Date Awarded
2020-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Williams, Huw
Curry, Stephen
Sponsor
Biotechnology and Biological Sciences Research Council
Grant Number
G98539
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)