Structural basis for ribosome function and inhibition in bacteria. - SUMMARY Protein synthesis is an essential process in all living organisms catalyzed by the ribosome – one of the largest and the most complex macromolecular machines created by nature. Our group uses structural, biochemical, and microbiological techniques to gain insights into the molecular mechanisms of ribosome functioning in bacteria, the modes of action of ribosome-targeting antibiotics, and mechanisms of drug resistance. Interaction of the growing polypeptide chain with the nascent peptide exit tunnel (NPET) in the large ribosomal subunit during protein synthesis plays a fundamental role in the regulation of gene expression as well as in co-translational protein folding. Despite the extreme chemical diversity of its substrates and products, ribosome efficiently makes all cellular proteins. However, some of the peptide sequences were evolutionarily selected to be problematic for the ribosome resulting in translation arrest during their synthesis. The cell often uses this phenomenon of peptide sequence-specific translation arrest, which can occur in a ligand-dependent manner turning the ribosome into a small-molecule sensor, to control its metabolism. Currently, we lack a mechanistic understanding of how interactions of the arrest peptides with NPET and small molecules or antibiotics result in the arrest of translation. This knowledge gap could be addressed by structural studies capturing both arrested and non-arrested Ribosome Nascent chain Complexes (RNCs) containing relevant peptides in the NPET. While many cryo-EM structures of ribosomes carrying various stalling peptides became available in the past years, they all represent arrested RNCs. Because the experimental approach used in these studies for the preparation of RNCs relies on the ability of the ribosome to stall in the presence of a small molecule and a wild-type arrest peptide, using the same methods for the preparation of respective non-arrested RNCs is impossible. Alternatively, the desired arrested or non-arrested RNCs could be assembled from the individually purified components. However, this strategy has remained challenging due to the lack of a reliable method for the large-scale preparation of peptidyl- tRNAs. Recently, we have developed a chemoenzymatic approach based on native chemical ligation reaction for facile semi-synthesis of peptidyl-tRNAs. Most importantly, by determining the first high-resolution structures of non-arrested RNCs, we found that synthetic peptidyl-tRNAs can be efficiently complexed to the ribosome in vitro and yet represent a functionally significant state of the ribosome’s catalytic site. Therefore, our group is best suited to bridge the existing knowledge gap in our understanding of molecular mechanisms underlying antibiotic- or sequence-dependent ribosome stalling by determining high-resolution structures of model RNCs under arresting or non-arresting conditions. Altogether, the proposed project will uncover enigmatic mechanisms of sequence-dependent nascent chain-mediated ribosome stalling, provide the structural basis for the context- specific action of several chemically unrelated classes of ribosomal antibiotics, and reveal mechanisms of action of ABCF ribosome protection proteins upon stalled ribosomes.
