Ribosome integrity and translational fidelity require accurate modification and processing of rRNA in the yeast Saccharomyces cerevisiae

Abstract

Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. Although kinetic and biochemical studies along with high resolution crystal structures have provided much information about the ribosome, many of the underlying mechanisms of ribosome function are still poorly understood. This work seeks to understand how ribosome structure and function are affected by changes in rRNA as caused by two very different mechanisms. mof6-1, originally isolated as a recessive mutation which promoted increased efficiencies of programmed -1 ribosomal frameshifting, was found to be an allele of RPD3 which encodes a histone deacetylase that is involved in transcriptional activation and silencing. This mutant demonstrated a delay in ribosomal RNA (rRNA) processing leading to changes in reading frame maintenance and ribosomal A-site specific defects. To understand the role of cis-acting changes to rRNA, yeast strains deficient in rRNA modifications in the peptidyl transferase center of the ribosome were monitored for changes in ribosome structure and translational fidelity. Analyses revealed mutant phenotypes including sensitivity to translational inhibitors; changes in reading frame maintenance, nonsense suppression and aa-tRNA selection; and increased rates of A-site tRNA binding to the mutant ribosome. One mutant in particular, spb1DA/snr52Δ, promoted increased rates of programmed -1 ribosomal frameshifting, increased rates of near cognate tRNA selection and A-site tRNA binding. Structural analysis of spb1DA/snr52Δ revealed changes consistent with a more accessible ribosomal A-site. These results suggest that rRNA nucleotide modifications produce small but distinct changes in ribosome structure and function contributing to overall translational fidelity.

Taken together, these data suggest that rRNA, a main component of the ribosome, contributes directly to translational fidelity. Defects in rRNA caused by changes in both its processing and modification can cause changes in reading frame maintenance, nonsense suppression, aa-tRNA selection and binding as well as ribosome structure.