RNA oxidation harms the ribosome and differentially affects the catalytic center

The ribosome is the central ribozyme responsible for protein biosynthesis. In its active site, the peptidyl transferase center (PTC), catalysis relies heavily on the contribution of rRNA, especially the 2’OH of A2451. rRNA bases are oxidized in Alzheimer's, and recent findings indicate this could be a contributing factor to various neurodegenerative diseases. However, the consequences of direct oxidative stress to the ribosome and its effects on translation are unknown. We set out to investigate oxidized nucleobases in the rRNA in general and in the PTC in particular, using extensive chemical engineering experiments. Via the technique of atomic mutagenesis, single modifications 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, and to insert even non-natural nucleosides. We replaced the five 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. We will present our findings on how incorporation of different oxidized bases into the PTC affected ribosome’s functions in diverse ways. For example, individual oxidized nucleobases resulted in either a radical deceleration of peptide bond formation, alteration of tRNA binding to A-site or even an increase of the translation rate. 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 neurodegenerative disease