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.2022 Oct 14;50(18):10201-10211.
doi: 10.1093/nar/gkac576.

Uncovering translation roadblocks during the development of a synthetic tRNA

Affiliations

Uncovering translation roadblocks during the development of a synthetic tRNA

Arjun Prabhakar et al. Nucleic Acids Res..

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.

Plain language summary

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.

© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Graphical Abstract
Graphical Abstract
A single nucleotide mutation in tRNAUTu1 removes the translation roadblock causing ribosome disassembly and permits continued translation.
Figure 1.
Figure 1.
Genetic recoding at stop codons competes with release factors. In the presence of a UAG stop codon, one of two situations can occur: termination or suppression. Termination of translation occurs when release factor 1 (RF1) binds, releasing the polypeptide chain and invoking separation of the ribosomal subunits from the mRNA. Alternatively, in the presence of a suppressor tRNA, the UAG codon can be suppressed, inserting an amino acid and allowing continuation of translation.
Figure 2.
Figure 2.
EF-Tu driven selenocysteine incorporation. (A) Cloverleaf structures ofE. coli tRNASer, tRNAsupD (the amber suppressor of tRNASer) and tRNAUTu1. Residues are colored as follows: acceptor arm (green), D-arm (purple), anticodon arm (red), variable arm (blue), and T-arm (gold). (B) Translation schematic of EF-Tu driven selenocysteine incorporation with tRNAUTu1. tRNAUTu1 is first serylated by seryl-tRNA synthetase (SerRS) to generate Ser-tRNAUTu1. Selenocysteine synthase (SelA) converts the serine to selenocysteine to generate Sec-tRNAUTu1. Aminoacylated tRNAUTu1 (including Ser-tRNAUTu1 which is misincorporated) are substrates for EF-Tu, inserting at the UAG codon for continued translation.
Figure 3.
Figure 3.
Single-molecule characterization of low translation processivity induced by tRNAUTu1. (A) In all single-molecule experiments, 30S preinitiation complexes (PIC) containing Cy3B-30S, fMet-tRNAfMet, and IF2 is immobilized on the surface of the ZMW wells through biotinylated mRNAs. The reaction is started by delivery of BHQ-2-50S, Cy5-TCPhe, EF-G, and one of the serine-charged suppressor tRNA TCs. (B) Expected sequence of fluorescence signals starting with quenching of Cy3B (green) that signals 50S subunit joining (initiation) and six cycles of changes in Cy3B intensity that signal intersubunit rotations during elongation. First and last four elongation cycles are correlated with Cy5 intensity changes that signal Cy5-tRNAPhe binding. (C) Representative single-molecule trace shows successful ribosome translation of all six codons after UAG stop codon suppression by tRNAsupD. (D) Codon survival plot of the fraction of translating ribosomes as a function of number of codons translated. In the presence of 1μM tRNAUTu1 (red,n = 223) ribosome survival (percent of codon 2 ribosomes that survive codon 3) is drastically decreased compared to the same amount of tRNAsupD (blue,n = 186) or tRNASer (black,n = 265).
Figure 4.
Figure 4.
Structural studies of tRNAUTu1 on the ribosome. (A) Structure of the ribosome, highlighting tRNAs in the A, P and E sites. (B) Overlay of A/A (purple, PDB:7UR5) and P/P (orange, PDB:7UR5) state tRNAUTu1 highlights the shift in the variable arm position. (C) Zoom in on the variable arm shows P/P tRNAUTu1 (orange) to rotate 17° upwards from A/A tRNAUTu1 (purple). (D) Overlay of A/A (purple) and P/P (orange) state tRNAUTu1 with A/A state tRNASec (blue, PDB:5LZE) in the context of the ribosome P site. A clash is observed with the A/A state tRNAUTu1 and the A-site finger of the ribosome. A zoom in on the variable arm with respect to the A-site finger shows the shift from the A/A to the P/P state for tRNAUTu1 and compares it with the A/A state of tRNASec (blue). (E) Cloverleaf structure of tRNAUTu1 highlights the position of the residues of interest, C8 and A45. A zoom in on the structure of those residues shows the formation of a hydrogen bond. (F) Cloverleaf structure of tRNASec highlights the corresponding residues of interest from tRNAUTu1. A zoom in on the structure of those residues (PDB:5LZE) shows that there is no base pair present. Instead, the corresponding residues point outwards, away from the tRNA core.
Figure 5.
Figure 5.
Single nucleotide mutation enhances translation processivity. (A) Cloverleaf structure of tRNAUTu1A showing the single nucleotide C to A change from tRNAUTu1 by the red arrow. (B) sfGFP fluorescence readthrough assay (green bars) and GPx1 protein yields (purple dots) demonstrate the increased processivity of tRNAUTu1A compared to tRNAUTu1. Fluorescence reads are shown as an average of four biological replicates (± standard deviation) and protein yields are an average of two biological replicates (± standard deviation). (C) Codon survival curve plotting the fraction of translating ribosomes as a function of number of codons translated highlights the post-suppression survival (percent of codon 2 ribosomes that survive codon 3) advantage in the presence of 1 μM tRNAUTu1A (green,n = 300) over 1 μM tRNAUTu1 (red,n = 223). (D) Pathway map of ribosomes that successfully suppressed a UAG stop codon in the presence of 1 μM tRNAUTu1A.
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