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. 2024 Mar 4:15:1369760.
doi: 10.3389/fmicb.2024.1369760. eCollection 2024.

Endogenous trans-translation structure visualizes the decoding of the first tmRNA alanine codon

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Endogenous trans-translation structure visualizes the decoding of the first tmRNA alanine codon

David Teran et al. Front Microbiol. .

Abstract

Ribosomes stall on truncated or otherwise damaged mRNAs. Bacteria rely on ribosome rescue mechanisms to replenish the pool of ribosomes available for translation. Trans-translation, the main ribosome-rescue pathway, uses a circular hybrid transfer-messenger RNA (tmRNA) to restart translation and label the resulting peptide for degradation. Previous studies have visualized how tmRNA and its helper protein SmpB interact with the stalled ribosome to establish a new open reading frame. As tmRNA presents the first alanine codon via a non-canonical mRNA path in the ribosome, the incoming alanyl-tRNA must rearrange the tmRNA molecule to read the codon. Here, we describe cryo-EM analyses of an endogenous Escherichia coli ribosome-tmRNA complex with tRNAAla accommodated in the A site. The flexible adenosine-rich tmRNA linker, which connects the mRNA-like domain with the codon, is stabilized by the minor groove of the canonically positioned anticodon stem of tRNAAla. This ribosome complex can also accommodate a tRNA near the E (exit) site, bringing insights into the translocation and dissociation of the tRNA that decoded the defective mRNA prior to tmRNA binding. Together, these structures uncover a key step of ribosome rescue, in which the ribosome starts translating the tmRNA reading frame.

Keywords: A-minor interactions; SmpB; alanyl-tRNA; cryo-EM; endogenous tmRNA; tmRNA decoding.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cryo-EM structure of Escherichia coli 70S•tmRNA complex with tRNAAla in the A site. (A) 3.7-Å cryo-EM density segmented to show the ribosomal subunits (cyan and yellow), tmRNA (red), SmpB (purple) and A-site tRNA (green). (B) Front view of the 70S structure with tmRNA, SmpB and A-tRNA; (C) Relative positions of tmRNA, SmpB and A-tRNA, with tmRNA domains labeled. (D) Close-up view of tmRNA, SmpB and A-tRNA facing the CCA ends of the tRNA and TLD (in the A and P sites, respectively) and SmpB C-terminus in the E site, rendered as molecular surfaces.
Figure 2
Figure 2
Comparison of the 70S•tmRNA•tRNAAla complex with the canonical Escherichia coli 70S elongation complex bound with mRNA and three tRNAs (PDB 6WDE). (A) Superposition of the 70S•tmRNA•tRNAAla structure (colored) with the 70S•mRNA•tRNA3 structure (gray) shows overall similar ribosome conformations except for the 7° tilt of the 30S head domain. Structures were superposed by aligning 23S rRNA (here and in other figures). (B) Close-up view of the similarly positioned tRNA in the A site of the tmRNA-bound (colored) and canonical elongation-state (gray) ribosomes. (C) Close-up view showing different positions of the A-site finger (ASF; H38 of 23S rRNA) in the tmRNA-bound (colored) and canonical elongation-state (gray) ribosomes.
Figure 3
Figure 3
Interactions between A-tRNA and tmRNA. (A) Positions of A-tRNA and tmRNA domains. (B) The tmRNA linker (red) shifts to accommodate the A-site tRNA, relative to its position in the tmRNA-bound complex with a vacant A site (gray backbone and molecular surface) in PDB 7ACJ. (C,D) Cryo-EM density in the decoding center, showing codon-anticodon interactions (C) and interactions with 30S and 50S nucleotides (D). (E) Interactions of the tmRNA linker with the anticodon stem of A-tRNA resemble A-minor interactions (E) and are similar to those between 16S rRNA and tRNAfMet in the P site (F) of initiation and elongation ribosome structures (PDB 6WDE is shown). (G) Close-up view of the A-minor-like interactions between tmRNA nucleotides and A-site tRNA.
Figure 4
Figure 4
Cryo-EM structure of E. coli 70S•tmRNA complex with tRNAs in the A (green) and E (orange) sites. (A) Front view of the 70S structure with tmRNA, SmpB, A-tRNA and E-tRNA rendered as molecular surfaces. (B) Position of the tRNA in the E site of the 70S•tmRNA•tRNAAla complex (colored) differs from that in the canonical 70S•mRNA•tRNA3 structure (gray ribosome and E-tRNA, magenta mRNA and blue P-tRNA). (C,D) Positions of the anticodon stem loops of E-site tRNAs relative to 16S and SmpB (in the 70S•tmRNA•tRNAAla complex) or mRNA (in the 70S•mRNA•tRNA3 complex).

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