Translational Fidelity Mutations in 18S rRNA Affect the Catalytic Activity of Ribosomes and the Oxidative Balance of Yeast Cells

Abstract

The function of mutations rdn1A, rdn1T, and rdn2 in 18S rRNA of Saccharomyces cerevisiae is investigated. The mutations correspond to substitutions C1054A, C1054U in helix 34, and G517A in helix 18 of 16S rRNA in Escherichia coli, respectively, in which the first and third mutations caused nonsense suppression, while C1054U caused no suppression. In yeast, rdn1A caused phenotypic suppression at nonsense codons, whereas rdn1T and rdn2 caused antisuppression. We provide in vitro evidence that, in addition, rdn1A decreases translational accuracy at sense codons as well, by a factor of 8, accompanied by extreme sensitivity to paromomycin, compatible with its error-prone character. Mutations rdn1T andrdn2 exhibit hyperaccuracy and paromomycin resistance. Thus, mutations in conserved rRNA regions may affect the same functions in the various species but in opposite directions. Mutation rdn1A, but not rdn1T or rdn2, affected also the catalytic activity of the ribosome, a 60S subunit activity. The rate of peptide bond formation was reduced to half its normal value, indicating a communication between the two subunits. Moreover, error-prone mutation rdn1A was less susceptible to oxidative modifications than wild type, indicated by decreased lipid peroxidation and nonprotein/protein disulfides, as well as by increased protein thiols. In contrast, hyperaccurate mutations rdn1T and rdn2 displayed increased oxidative stress. Our results suggest that the cells may consume more energy to achieve hyperaccuracy leading to increased oxidative modifications.

Is Ribosome Synthesis Controlled by Pol I Transcription?

“…The reasons why post-diauxic culture cells show a greater resistance to hydrogen peroxide when containing a larger amount of assembled ribosome 1 are unclear, but may indicate that control of the protein biosynthesis machinery is important for the oxidative equilibrium of the cell, in agreement with recent data showing that changes in translational fidelity affect this balance. 48 In conclusion, the recent characterization of the CARA strain strongly supports the emerging concept that Pol I activity is a key element for the coordinated synthesis of ribosome components. To dissect the molecular mechanisms involved, and more specifically to understand how the level of Pol I transcription impacts on Pol II transcription will certainly represent a major breakthrough.…”

The look-ahead effect of phenotypic mutations

“…Few mutations may be sufficient to alter the fidelity of a ribosome, for example, a single mutation in the S5 ribosomal protein in E. coli increases frameshifting and nonsense mutations [ 38 ]. In yeast, mutations in the 18S RNA have been found that both increase and decrease translational fidelity [ 39 ].…”

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

“…This helix has also been implicated in decoding in both the A and P sites during elongation and initiation, respectively, and in the translocation of mRNA and tRNAs through the ribosome (O'Connor et al 1997;Matassova et al 2001;Konstantinidis et al 2006). Given the importance of the flexibility of this region in allowing conformational changes during each of these processes, it seems possible that yeIF4B binding could induce a rearrangement in h34 that promotes mRNA recruitment.…”