How oxidation of the ribosome’s active site affects translation

The ribosome is the central ribozyme responsible for protein biosynthesis. The exact working principle of the peptidyl transferase center (PTC), its active site, is still a matter of ongoing debate and research. However, significant contribution to catalysis stems from the ribose-phosphate backbone of rRNA, specifically the 2'OH of A2451 [1]. Recent findings in Alzheimer's disease indicate oxidized ribosomes could be a contributing factor to neurodegenerative diseases [2]. However, the consequences of direct oxidative stress to the ribosome and its effects on translation are unknown. Investigating oxidation in the PTC could provide insight into the mechanism of peptide bond formation, and also elucidate disease-relevant effects of the oxidized translation machinery. Using the technique of atomic mutagenesis, non-natural nucleosides can be introduced at any position of the 23S rRNA during in vitro reconstitution of the ribosome. This method surpasses conventional mutagenesis and effectively enables us to alter single atoms in the context of the ribosome [3]. We replaced the universally conserved PTC nucleobases A2451, A2601, U2585, U2506 and C2063 with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC, respectively. The chemically engineered ribosomes were then studied in various functional assays to investigate the consequences of nucleobase oxidations on translation Incorporation of different oxidized bases into the PTC affected ribosome’s functions in diverse ways. Oxidizing an individual nucleobase either resulted in a radical deceleration of peptide bond formation, alteration of tRNA binding to A- and P-site or even an increase of the translation rate due to facilitated translocation. These results expand our current understanding of the roles of PTC residues in ribosomal function. This study is the first investigation of site-specific oxidation of central PTC residues, and provides diverse, interesting findings for both the ribosome field and research on neurodenenerative disease