Uncovering translation roadblocks during the development of a synthetic tRNA.
Nucleic Acids Res
; 50(18): 10201-10211, 2022 10 14.
Article
em En
| MEDLINE
| ID: mdl-35882385
ABSTRACT
Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
Continued expansion of the genetic code has required the use of synthetic tRNAs for decoding. Some of these synthetic tRNAs have unique structural features that are not observed in canonical tRNAs. Here, the authors applied single-molecule, biochemical and structural methods to determine whether these distinct features were deleterious for efficient protein translation on the ribosome. With a focus on selenocysteine insertion, the authors explored an allo-tRNA with a 9/3 acceptor domain. They observed a translational roadblock that occurred in A to P site tRNA translocation. This block was mediated by a tertiary interaction across the tRNA core, directing the variable arm position into an unfavorable conformation. A single-nucleotide mutation disrupted this interaction, providing flexibility in the variable arm and promoting efficient protein production.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ribossomos
/
Biossíntese de Proteínas
/
RNA de Transferência
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Estados Unidos