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  Ribosome selectivity and nascent chain context in modulating the incorporation of fluorescent non-canonical amino acid into proteins

Thommen, M., Draycheva, A., & Rodnina, M. V. (2022). Ribosome selectivity and nascent chain context in modulating the incorporation of fluorescent non-canonical amino acid into proteins. Scientific Reports, 12: 12848. doi:10.1038/s41598-022-16932-7.

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 Creators:
Thommen, M.1, Author           
Draycheva, A.1, Author           
Rodnina, M. V.1, Author           
Affiliations:
1Department of Physical Biochemistry, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350156              

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 Abstract: Fluorescence reporter groups are important tools to study the structure and dynamics of proteins.
Genetic code reprogramming allows for cotranslational incorporation of non‑canonical amino acids
at any desired position. However, cotranslational incorporation of bulky fluorescence reporter
groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the
cotranslational incorporation of NBD‑, BodipyFL‑ and Atto520‑labeled Cys‑tRNACys into a model
protein using a reconstituted in‑vitro translation system. We show that the modified Cys‑tRNACys can
be rejected during decoding due to the reduced ribosome selectivity for the modified aa‑tRNA and the
competition with native near‑cognate aminoacyl‑tRNAs. Accommodation of the modified Cys‑tRNACys
in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the
positions −1 to −4 upstream of the incorporation site. The incorporation yield depends on the steric
properties of the downstream residue and decreases with the distance from the protein N‑terminus
to the incorporation site. In addition to the full‑length translation product, we find protein fragments
corresponding to the truncated N‑terminal peptide and the C‑terminal fragment starting with a
fluorescence‑labeled Cys arising from a StopGo‑like event due to a defect in peptide bond formation.
The results are important for understanding the reasons for inefficient cotranslational protein labeling
with bulky reporter groups and for designing new approaches to improve the yield of fluorescence‑
labeled protein.

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Language(s): eng - English
 Dates: 2022-07-27
 Publication Status: Published online
 Pages: 14
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1038/s41598-022-16932-7
 Degree: -

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Project name : This work was supported by the fellowship #17C174 from the Novartis Foundation for Medical-Biological Research to MT, the European Research Council (ERC) Advanced Investigator Grant RIBOFOLD to MVR (proposal number n° 787926) and by the Max Planck Society.
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Funding program : -
Funding organization : -
Project name : RIBOFOLD
Grant ID : 787926
Funding program : Horizon 2020 (H2020)
Funding organization : European Commission (EC)

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Title: Scientific Reports
Source Genre: Journal
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Pages: 14 Volume / Issue: 12 Sequence Number: 12848 Start / End Page: - Identifier: ISSN: 2045-2322