Structural basis for stop codon recognition in eukaryotes

Abstract

Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UGA, UAA, or UAG. Release factors recognise stop codons in the ribosomal A site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases1. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognise all three stop codons2. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here, we present electron cryo-microscopy (cryo-EM) structures at 3.5 – 3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A site. Binding of eRF1 flips nucleotide A1825 of 18S rRNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A site, where it is stabilised by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during tRNA selection to drive mRNA compaction. Stop codons are favoured in this compacted mRNA conformation by a hydrogen-bonding network with essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination3,4.

RETRACTED: Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

“…1 D-F), revealed that the absence of the methyl group was sufficient to enable A1824 to move in, whereas A1825 remained out (Fig. 2 C, F, and G), consistent with an eRF1-bound conformation (9) and termination at a PTC. Interestingly, the absence of the 6′ methyl group not only prevented steric blocking of A1824 but also allowed ring I to move partially below the backbone phosphates of the rRNA and make specific interactions that stabilized the "in" position (Fig.…”

“…Critical to accurate codon recognition by both complexes is the conformation of A1824 and A1825, two conserved rRNA residues at helix 44 of the ribosome that can dynamically flip in and out of the helix. Structural studies have shown that binding of eRF1 flips A1825 out to facilitate stacking of termination codon bases and enable accurate recognition of the termination codon, whereas A1824 remains inside the helix (9). By contrast, A1825 and A1824 adopt a flipped-out conformation upon binding of a cognate tRNA complex, enabling them to interact with and stabilize the codon-anticodon duplex and ensuring fidelity of tRNA selection (10)(11)(12).…”

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

“…1A). The latter appeared because of the presence of a stop codon in the A-site, which is known to adopt a compact conformation leading to mRNA retraction into the A-site (31,32). This stop codon conformation is stabilised by eRF1, thus enhancing the 125 nt peak when it binds to the ribosome (30).…”

Advances in the molecular dynamics flexible fitting method for cryo-EM modeling

“…Generally, X-ray crystallography produces atomic-resolution structures (<~3 Å), while cryo-EM densities are resolved typically only at lower resolution (~5-30 Å). However, through recent developments in cryo-EM techniques, high-resolution (~2-5 Å) densities have been obtained [1, 2, 3, 4, 5]. Computational methods that combine information from crystallography and cryo-EM can bridge the resolution gap and hold the promise of generating physiologically accurate, atomic-resolution structures of biomolecular complexes.…”