Ribosomes under attack: smFRET analyses of bacterial ribosomes in context of impaired rRNA stability and antimicrobial treatment (360G-Wellcome-213955_Z_18_Z)

Antimicrobial resistance is a major burden on public health and the economy. Current estimates put associated annual costs at EUR 1.5 billion and annual deaths at ~25,000 in the EU and ~700,000 worldwide. Research into resistance mechanisms is urgently needed to tackle this significant threat to society. A major antimicrobial target is the bacterial ribosome, which translates genetic information into polypeptides in a process involving large-scale movements of ribosomal subunits and tRNAs that is inherently asynchronous and heterogeneous. Single-molecule Förster-resonance-energy-transfer (smFRET) enabling analyses of translation-dynamics with single-codon resolution, sub-nanometer accuracy at physiological concentrations in real-time was instrumental in gaining novel insights into antimicrobial interference with ribosome-function. We will implement smFRET to reveal for the first time how the conserved yet previously unrecognised rRNA repair-system RtcAB that increases tolerance to ribosome-targeting antimicrobials affects ribosome-function. Specifically, smFRET will allow us to ask the following questions: Does RtcAB affect the i) conformation and population-dynamics of key states of the translation-apparatus? ii) translation-efficiency and ribosome subunit-rotation? iii) tRNA-selection? iv) ribosome-translocation? and v) ribosome-recycling? The answers to these questions will help us understand how RtcAB contributes to antimicrobial resistance and provide a platform for future studies of the interplay between rRNA repair and ribosome-targeting antimicrobials.