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Helix 69 is key for uniformity during substrate selection on the ribosome.

ABSTRACT: Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.

SUBMITTER: Ortiz-Meoz RF

PROVIDER: S-EPMC3138323 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Helix 69 is key for uniformity during substrate selection on the ribosome.

Ortiz-Meoz Rodrigo F RF Green Rachel R

The Journal of biological chemistry 20110527 29

Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. ...[more]

PMID: 21622559

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Project description:The widespread distribution of pseudouridine (Ψ), an isomer of the canonical uridine base, in RNA indicates its functional importance to the cell. In eukaryotes, it is estimated that around 2% of ribosomal RNA nucleotides are pseudouridines, most of which are located in functional regions of the ribosome. Defects in RNA pseudouridylation induce a range of detrimental effects from compromised cellular protein biosynthesis to disease phenotypes in humans. However, genome-wide changes to mRNA translation profiles by ribosomes lacking specific conserved pseudouridines have not been extensively studied. Here, using a new genomic method called 5PSeq and in vitro biochemistry, we investigated changes in ribosome dynamics and cellular translation profiles upon loss of Ψ2258 and Ψ2260 in helix 69, the two most conserved pseudouridines in the ribosome in yeast cells. We found that inhibiting the formation of these two pseudouridines challenges ribosomes to maintain the correct open reading frame and causes generally faster ribosome dynamics in translation. Furthermore, mutant ribosomes are more prone to pause while translating a subset of GC-rich codons, especially rare codons such as Arg (CGA) and Arg (CGG). These results demonstrate the presence of Ψ2258 and Ψ2260 contributes to the dynamics of the H69 RNA stem-loop, and helps to maintain functional interactions with the tRNAs as they move within the ribosome. The optimality of this ribosome-tRNA interaction is likely to be more critical for those limited tRNAs that decode rare codons. Consistent with the changes in ribosome dynamics, we observe that IRES-mediated translation is compromised in the mutant ribosome. These results explain the importance of Ψ2258 and Ψ2260 in H69 to maintain cellular fitness. The strong conservation of Ψ2258 and Ψ2260 in the ribosomes from bacteria to humans indicates their functional significance in modulating ribosome functions. It's likely that the identified functions of these covalent modifications are conserved across species.

Dataset Information

Helix 69 is key for uniformity during substrate selection on the ribosome.

Publications

Helix 69 is key for uniformity during substrate selection on the ribosome.

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