Mechanism of premature translation termination on a sense codon

Abstract

Accurate translation termination by release factors (RFs) is critical for the integrity of cellular proteomes. Premature termination on sense codons, for example, results in truncated proteins, whose accumulation could be detrimental to the cell. Nevertheless, some sense codons are prone to triggering premature termination, but the structural basis for this is unclear. To investigate premature termination, we determined a cryo-EM structure of the Escherichia coli 70S ribosome bound with RF1 in response to a UAU (Tyr) sense codon. The structure reveals that RF1 recognizes a UAU codon similarly to a UAG stop codon, suggesting that sense codons induce premature termination because they structurally mimic a stop codon. Hydrophobic interaction between the nucleobase of U3 (the third position of the UAU codon) and conserved Ile-196 in RF1 is important for misreading the UAU codon. Analyses of RNA binding in ribonucleoprotein complexes or by amino acids reveal that Ile-U packing is a frequent protein-RNA-binding motif with key functional implications. We discuss parallels with eukaryotic translation termination by the release factor eRF1.

Keywords: 70S ribosome; RNA–protein interaction; hot-spot sense codon; hydrophobic interactions; near-stop codon; ribosome function; ribosome structure; translation regulation; translation release factor; π-stacking.

Figure 1.

A, Michaelis-Menten curves for in vitro formyl-methionine release from fMet-tRNA by RF1 on the stop codon UAA and sense codons UAU, UGG, and AAA (adapted from Refs. 5, 11). B, cryo-EM structure of the 70S·RF1 complex formed on the UAU sense codon. FSC curve is shown for the 70S·RF1 cryo-EM map (lower left). C, local resolution of RF1 in the cryo-EM map, determined using Blocres (76). RF1 is oriented similarly to the view shown in B. The map was sharpened by applying a B-factor of −100 Ų and is shown at 2σ, colored using a resolution scale ranging from 2.8 to 5.3 Å (left). D, cryo-EM map (mesh) in the peptidyl transferase center (PTC). E, cryo-EM map (mesh) in the decoding center (DC). In structural models, the large 50S ribosomal subunit is shown in cyan, small 30S subunit in yellow, RF1 in green, mRNA in dark blue, P-site tRNA in orange, and E-site tRNA in pink. Domains of RF1 are labeled in B and C.

Figure 2.

Recognition of the third nucleotide of the UAU and UAG codons by RF1 and eRF1. A, interactions of E. coli RF1 with U3 of the UAU sense codon in the 70S ribosome (this work). B, interactions of T. thermophilus RF1 with G3 of the UAG stop codon in the 70S ribosome (PDB code 4V7P (9)). Thr-198 forms one of two possible hydrogen bonds with G3 (shown with the dashed and dotted lines). C, interactions of Oryctolagus cuniculus eRF1 with G3 of the UAG stop codon in the 80S ribosome (PDB code 3JAH (19)).

Figure 3.

Distribution of amino acid side chains packing on the four types of RNA nucleotide bases (the more hydrophobic bases U and A are shown in gray and C and G are shown in purple) in three high-resolution ribosome structures from E. coli (PDB code 4YBB (48)), T. thermophilus (PDB code 4Y4P (49)), and S. cerevisiae (PDB code 4V88 (50)).