Abstract
When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.
Highlights
When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA
Each stop codon in the decoding center (DC) is recognized by a stop-codon recognition (SCR) motif in a class-1 RF, and all RFs have a peptidyl transfer center (PTC)-binding GGQ motif, named after its universal Gly–Gly–Gln triplet (GGQ), for coordinated ester bond hydrolysis in the P-site bound peptidyl-tRNA
The RC0 displays no intersubunit rotation, and the tripeptide of its P-site tRNA is seen near the end of the peptide exit tunnel (Supplementary Fig. 1)
Summary
When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. The complete protein is released from the ribosome by a class-1 release factor (RF) recognizing one of the universal stop codons (UAA, UAG, and UGA), signaling the end of the amino acid encoding open reading frame (ORF) of the mRNA. Each stop codon in the decoding center (DC) is recognized by a stop-codon recognition (SCR) motif in a class-1 RF, and all RFs have a peptidyl transfer center (PTC)-binding GGQ motif, named after its universal Gly–Gly–Gln triplet (GGQ), for coordinated ester bond hydrolysis in the P-site bound peptidyl-tRNA. Subsequent high-resolution X-ray crystal[6,7,8,9,10,11,12,13] and cryo-EM14–19 structures of RF-bound 70S ribosomes allowed the modeling of stop-codon recognition by RF1, RF220, eRF121, and GGQ-mediated ester bond hydrolysis[22]
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