Previous research shows that rRNA bases are a key participant in peptide bond formation, and that base oxidation, a molecular consequence of oxidative stress, disrupts that role. By employing a highly sensitive method of atomic mutagenesis, we were able to re-construct ribosomes from scratch with singular oxidations placed site-specifically in their catalytic heart, and study the effect in highly sensitive in vitro assays. We found that indeed, rRNA oxidation in key sites disrupts protein production and this effect contributes to the cytotoxic effects of oxidation. Oxidative stress is a key factor in ageing, Alzheimer’s, inflammation, and neurodegeneration, and a field that we are able to contribute a ribosome perspective to. While data indicates that oxidation generally disrupts ribosome function, the effect is highly site-dependent, and E. coli surprisingly encodes a dedicated enzyme that adds an 5-hydroxyl-group to cytidine C2501 in close proximity the active site. We investigating this dynamic rRNA modification both in vivo through genetic manipulation, modification quantification, and from a mechanistic perspective through in vitro experiments. We found this modification to be highly dynamic and growth phase-dependent, with high oxidation levels helping E. coli adapting to oxidative stress. These findings provided first functional insights into the role of this unique rRNA modification and showcase a novel mechanism for how bacteria adapt to oxidative stress. 

The Willi Group will continue studying not only the effects of oxidative stress on the ribosome, but also the protective mechanisms that enable cells to continue protein synthesis and growth. The main research questions include: 

• What oxidation-protective mechanisms are employed in bacteria? How could we circumvent them for improving anti-bacterial treatments? How can these mechanisms inform antioxidation treatments for humans?

• Could oxidative stress suppress tumor growth, since cancer cells rely on upregulated protein synthesis for rapid cell division?

• Could antioxidants, radical scavengers, or iron-chelators be used to protect rRNA from oxidative damage? How can these compounds work as neuroprotectants?

• Can these compounds be targeted or bound to the ribosome, such as applying a molecular sunscreen? Can sacrificial RNA be added to the cell to harmlessly absorb oxidation that would otherwise damage the ribosome?

• Can what ribosome recycling and repair pathways are involved and can they be modulated to promote turnover of damaged components? 

Our unique perspective, viewing the ribosome at the center of all cellular processes, interfaces with other groups at uLethbridge exploring the dysregulation of protein synthesis during oxidative stress, cancer development, healthy ageing, neurodegenerative disorders, and ribosomopathies.

Further reading:

• Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center, Willi et al, NAR 2018

• Oxidative Stress in Bacteria and the Central Dogma of Molecular Biology, Fasnacht & Polacek, Frontiers 2021

• Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress, Fasnacht ... Willi & Polacek, NAR 2022

• Polacek lab: Atomic mutagenesis of ribosomes

• Limbach lab: Mass Spec of RNA oxidation and modification