Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation
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Abstract
Recent findings in Alzheimer's disease indicate oxidized ribosomes could be a contributing factor to neurodegenerative diseases [1]. However, the consequences of direct oxidative stress to the ribosome and its effects on translation are unknown. Additionally, the exact mechanism of peptide bond formation in the ribosomal peptidyl transferase center (PTC) is still a matter of ongoing debate and research. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone of rRNA, specifically 2'OH of A2451 [2]. Site-specific oxidation of central PTC residues could contribute to our understanding of the chemistry of peptide bond formation, and furthermore elucidate the disease-relevant effects of oxidative stress on the translation machinery.
So far, no conventional replacement or even removal of the PTC bases has been able to affect in vitro translation. Using the technique of atomic mutagenesis, modified bases can be introduced at any position in the 23S rRNA via short synthetic oligonucleotides. This method surpasses conventional mutagenesis and effectively enables us to alter single atoms in the context of the ribosome [3]. Reconstituting ribosomes in vitro using this approach, we replaced the universally conserved PTC bases A2451, U2585 and C2063 with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC respectively. To investigate the consequences on translation, the chemically engineered ribosomes were then studied in various functional assays.
Incorporation of different oxidized bases into the 70S ribosome affected its functions in different ways. Oxidizing a single nucleobase either resulted in radical deceleration of peptide bond formation (8-oxorA2451), alteration of tRNA binding to A- and P-site (5-HOrU2585) or even an increase of translation rate due to facilitated translocation (5-HOrC2063). These results are in accordance with our current understanding of the roles of said rRNA residues in ribosomal function. Since this is the first time specific rRNA oxidations were functionally assessed in vitro, these findings provide insight into the possible effects of oxidative stress on translation.
So far, no conventional replacement or even removal of the PTC bases has been able to affect in vitro translation. Using the technique of atomic mutagenesis, modified bases can be introduced at any position in the 23S rRNA via short synthetic oligonucleotides. This method surpasses conventional mutagenesis and effectively enables us to alter single atoms in the context of the ribosome [3]. Reconstituting ribosomes in vitro using this approach, we replaced the universally conserved PTC bases A2451, U2585 and C2063 with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC respectively. To investigate the consequences on translation, the chemically engineered ribosomes were then studied in various functional assays.
Incorporation of different oxidized bases into the 70S ribosome affected its functions in different ways. Oxidizing a single nucleobase either resulted in radical deceleration of peptide bond formation (8-oxorA2451), alteration of tRNA binding to A- and P-site (5-HOrU2585) or even an increase of translation rate due to facilitated translocation (5-HOrC2063). These results are in accordance with our current understanding of the roles of said rRNA residues in ribosomal function. Since this is the first time specific rRNA oxidations were functionally assessed in vitro, these findings provide insight into the possible effects of oxidative stress on translation.
Date Issued
2016-07-09
Publication Type
Conference Item
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en
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