Helix 69 in 23S rRNA modulates decoding by wild type and suppressor tRNAs

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Helix 69 (h69) of 23S rRNA is crucial for ribosome structure and function, forming inter-subunit bridges and interacting with tRNAs and translation factors.
• Previous studies on h69 mutations yielded conflicting results regarding their impact on tRNA selection and translation fidelity.
• The precise role of h69 in the tRNA selection process and its influence on various translation errors remained unclear.

Purpose of the Study:

• To investigate the in vivo effects of various single- and multi-base mutations in Escherichia coli 23S rRNA Helix 69 on translation errors.
• To elucidate the specific contributions of h69 to tRNA selection, stop codon readthrough, frameshifting, and missense decoding.

Main Methods:

• Systematic introduction of single- and multi-base mutations into Helix 69 of Escherichia coli 23S rRNA.
• In vivo analysis of translation error rates, including stop codon readthrough, frameshifting, and missense decoding, in mutant strains.
• Characterization of the effects of specific h69 mutations on the reading of UGA codons by cognate, near-cognate, and suppressor tRNAs.

Main Results:

• A majority of h69 mutations influenced stop codon readthrough and frameshifting.
• The DeltaA1916 mutation uniquely affected missense decoding.
• Different h69 mutants exhibited varied effects on stop codon readthrough, with some decreasing UGA readthrough by near-cognate tRNAs.
• h69 mutations that reduced UGA readthrough also impacted the reading of UGA by a mutant tRNA with a D arm substitution.

Conclusions:

• Helix 69 interactions with release factors are significant for efficient translation termination.
• h69 interactions with the D arm of A-site tRNA are critical for discriminating between cognate and near-cognate tRNAs.
• The study clarifies the in vivo roles of h69 in bacterial translation fidelity and error regulation.