How a circularized tmRNA moves through the ribosome

Abstract

During trans-translation, transfer-messenger RNA (tmRNA) and small protein B (SmpB) together rescue ribosomes stalled on a truncated mRNA and tag the nascent polypeptide for degradation. We used cryo-electron microscopy to determine the structures of three key states of the tmRNA-SmpB-ribosome complex during trans translation at resolutions of 3.7 to 4.4 angstroms. The results show how tmRNA and SmpB act specifically on stalled ribosomes and how the circularized complex moves through the ribosome, enabling translation to switch from the old defective message to the reading frame on tmRNA.

Fig. 1. tmRNA-SmpB accommodates into the A-site.

(A) Overview of the ribosomal complex with tmRNA-SmpB occupying the A site of a non-stop ribosome. (B) Aromatic residues of SmpB (teal) interact with decoding center nucleotides (gold). (C) Global superposition of ribosomes, showing the tail of SmpB from E. coli and T. thermophilus bound in the A site (blue vs. purple, respectively) and T. thermophilus SmpB bound in a pre-accommodated state [gray, PDB code 4V8Q (14)]. (D) A single stranded RNA loop from PK2 of tmRNA binds protein uS3 and arginine residues of protein uS3 interact with the phosphate backbone of H5 of tmRNA. (E) Global superimposition of TLD of tmRNA bound to SmpB (left) in the A site (colored) or pre-accommodated (gray) compared to canonical tRNA (right) in the A site (purple) or pre-accommodated (gray).

Fig. 2. tmRNA-SmpB is translocated into the P site of the ribosome.

(A) Overview of the ribosomal complex with tmRNA-SmpB bound in the P site and the MLD occupying the A site. (B) Comparison of SmpB occupying the A site (gray) versus P site (blue). The tail of SmpB flips into the E site, anchoring tmRNA-SmpB in the P site. The conserved glycine residue at the junction between the body and the tail of SmpB is highlighted (red). (C) Helix 5 of tmRNA changes position from the A site complex (gray) to the P site complex (red), allowing the MLD to pass through the space previously occupied by the tail of SmpB. (D) The MLD contacts the junction of the body and tail of SmpB to set the tag reading frame correctly in the A site. The MLD has passed through the A-site latch (inset) when tmRNA-SmpB occupies the P site.

Fig. 3. tmRNA-SmpB is translocated from the P site past the E site.

(A) Overview of the ribosomal complex with tmRNA-SmpB bound on the solvent side of the E site after passing through the ribosome. Canonical tRNA (teal) bound to the resume codon occupies the P site. (B) Density for PK1, the MLD going through the mRNA channel, and H5 shows the MLD fully loaded into the mRNA channel. During the second translocation, the MLD passes through a second latch, this time in the E site (inset). (C) PK2 is anchored to protein uS3, acting as a flexible hinge point during the movement of tmRNA through the ribosome.

Fig. 4. Mechanism of trans-translation.

(1) A 70S ribosome forms a non-stop translation complex when it stalls at the 3′ end of a messenger RNA. (2) Elongation factor Tu (EF-Tu) delivers Ala-tmRNA-SmpB to the ribosome where the C-terminal tail of SmpB forms an alpha helix in the downstream mRNA channel. When trapped in this state the complex is referred to as ‘pre-accommodated’. (3) EF-Tu leaves and tmRNA-SmpB accommodate into the A site. The tail of SmpB remains in the same alpha-helical conformation as in the pre-accommodated state. Analogous to canonical tRNA, TLD-SmpB points the alanine on its 3′CCA into the peptidyl transferase reaction center (PTC) where it joins with the nascent peptide. PK2 interacts with protein uS3, binding tmRNA to the outside of the ribosome and coordinating the position of tmRNA as it moves through the ribosome. (4) Elongation factor G (EF-G) translocates tmRNA-SmpB from the A site into the P site and expels the original mRNA and tRNA. (5) During translocation, the MLD passes through the A-site latch and into the space in the mRNA channel previously occupied by the tail of SmpB. The tail of SmpB flips to the opposite side of the mRNA channel, binding in the E site. (6) Ala-tRNA^Ala decodes the first codon of the reading frame of tmRNA (the “resume” codon) and (7) a peptidyl transferase reaction transfers the peptide from tmRNA to tRNA^Ala. (8) EF-G translocates the peptidyl-tRNA^Ala into the P site and consequently shifts tmRNA-SmpB (9) past the E site to the solvent side of the ribosome. During this second translocation event, the MLD is again loaded into the mRNA channel through a latch, this time at the E site. The MLD is fully loaded into the mRNA channel and translation continues until terminating at a stop codon at the end of the reading frame. (10) The ribosome is released and the peptide is targeted for degradation by proteases that recognize the polypeptide tag.