Mechanism of ribosome rescue by alternative ribosome-rescue factor B

Abstract

Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria.

Ribosome Rescue Pathways in Bacteria

“…As for trans -translation, the release activity decreased with increasing length of the mRNA, with a significant drop for mRNAs with more than nine nucleotides downstream of the P-site ( Ivanova et al, 2004 ; Feaga et al, 2016 ). By contrast, ArfB and the tmRNA⋅SmpB complex were shown to associate with the ribosome independently of the mRNA length ( Kurita et al, 2014b ; Chan et al, 2020 ). This indicates that the mRNA and the C-terminal tails of ArfB and SmpB can compete with the mRNA for binding to the mRNA channel.…”

“…Alternative ribosome rescue factor B consists of 140 amino acids, and has a NTD (residues 1–100) and a C-terminal tail (residues 115–140), connected via a ∼12 amino acid long flexible linker ( Gagnon et al, 2012 ; Chan et al, 2020 ). The NTD is homologous to domain III of bacterial class I RFs, including the GGQ motif, whereas further class I RF domains, like the codon recognition superdomain II/IV, are absent ( Singarapu et al, 2008 ; Chadani et al, 2011b ; Gagnon et al, 2012 ; Kogure et al, 2014 ; Chan et al, 2020 ). As for class I RFs mutation of the GGQ (G 25 G 26 Q 27 ) motif abolished peptidyl-tRNA hydrolysis activity ( Korostelev et al, 2008 ; Chadani et al, 2011b ; Handa et al, 2011 ; Santos et al, 2013 ).…”

“…As for class I RFs mutation of the GGQ (G 25 G 26 Q 27 ) motif abolished peptidyl-tRNA hydrolysis activity ( Korostelev et al, 2008 ; Chadani et al, 2011b ; Handa et al, 2011 ; Santos et al, 2013 ). The C-terminal tail has some similarities to the C-terminal tail of SmpB, as it contains positively charged amino acids, stays unstructured in solution and engages an α-helical conformation upon binding to the ribosome ( Chadani et al, 2011b ; Gagnon et al, 2012 ; Kogure et al, 2014 ; Chan et al, 2020 ). Sucrose density gradient centrifugation showed that ArfB is associated with 70S ribosomes, as well as polysomes ( Chadani et al, 2011b ; Handa et al, 2011 ), and that removing 10 C-terminal residues abolished binding to the ribosome ( Handa et al, 2011 ).…”

“…The second intermediate shows that the NTD is still bound to H69, but is rotated so that the GGQ loop is directed toward the large subunit A-site ( Figure 4C ), priming ArfB for insertion into the PTC to perform hydrolysis. Eventually, the NTD is placed in the A-site of the large subunit and accommodates into the PTC ( Figure 4D ), which is induced upon binding of the NTD ( Gagnon et al, 2012 ; Carbone et al, 2020 ; Chan et al, 2020 ). The GGQ motif adopts an analogous conformation to the GGQ motif of bacterial class I RFs and mediates hydrolysis of the ester bond between the peptide and the P-tRNA.…”

“…The GGQ motif adopts an analogous conformation to the GGQ motif of bacterial class I RFs and mediates hydrolysis of the ester bond between the peptide and the P-tRNA. During all stages the position of the tail overlaps with the path of a full-length mRNA, as well as with C-terminal tail of SmpB and the C-terminus of ArfA, indicating that all of these systems monitor the channel ( Gagnon et al, 2012 ; Neubauer et al, 2012 ; James et al, 2016 ; Demo et al, 2017 ; Huter et al, 2017c ; Zeng et al, 2017 ; Chan et al, 2020 ). As for trans -translation, the release activity decreased with increasing length of the mRNA, with a significant drop for mRNAs with more than nine nucleotides downstream of the P-site ( Ivanova et al, 2004 ; Feaga et al, 2016 ).…”