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bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.29.609187
preprint
1
Article
Single-Molecule Studies of Cognate and Near-Cognate Elongation in an in vitro Eukaryotic Translation System
Fritsch Clark http://orcid.org/0000-0002-7507-8967

Bhattacharya Arpan http://orcid.org/0000-0002-0460-7876

Ng Martin Y. http://orcid.org/0000-0001-5601-8857

Li Hong http://orcid.org/0000-0002-8259-1961

Nelson Philip C. http://orcid.org/0000-0002-1782-3076

Cooperman Barry S.
Goldman Yale E. http://orcid.org/0000-0002-2492-9194

30 8 2024
2024.08.29.609187https://creativecommons.org/licenses/by-nd/4.0/ This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, and only so long as attribution is given to the creator. The license allows for commercial use.
http://biorxiv.org/lookup/doi/10.1101/2024.08.29.609187
nihpp-2024.08.29.609187.pdf
The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with an in vitro eukaryotic translation system to investigate tRNA selection and subsequent steps during peptide elongation. We compared accommodation of a tryptophan-aminoacyl-tRNA into the ribosomal A-site containing either a cognate or near-cognate codon and unexpectedly found that, following an initial slow sampling event, subsequent near-cognate sampling events proceeded more rapidly than the initial event. Further, we found a strong negative correlation between the concentration of near-cognate aminoacyl-tRNA and the efficiency of tRNA accommodation. These novel characteristics of near-cognate interaction with the eukaryotic ribosome suggest that rejection of a near-cognate tRNAs leads to formation of an altered ribosomal conformation that assists in rejecting subsequent incorrect tRNA interactions.
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pmc
