Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome

Abstract

Quality-control mechanisms intervene appropriately when defective translation events occur to preserve the intergrity of protein synthesis. Rescuing the ribosome translating on mRNAs absent of a stop codon is one of the co-translational quality control pathways in the cell. In many bacteria, ArfA recognizes stalled ribosome and recruits release factor RF2, which catalyzes the termination of protein synthesis 1–3. While an induced-fit mechanism of ArfA/RF2-mediated nonstop mRNA surveillance has been reported 4, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report a cryoEM structure of ArfA and RF2 in complex with the 70S ribosome that is bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to promote RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Further, binding of the ArfA and RF2 induces conformational changes in the ribosomal decoding center in a way that is similar to other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 attain a catalytically competent conformation for peptide release. Insights gained from this investigation provide a framework to understand recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.

ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

“…The E. coli ArfA-mediated hydrolysis of GFP-tRNA was only observed when the ribosomes and RFs were both derived from E. coli (lanes 15 and 16). Thus, E. coli ArfA is incompatible with the B. subtilis ribosomes and RF2, analogous to that observed previously between E. coli ArfA and T. thermophilus RF2 36,37 . The high specificity of molecular interactions involving the RF-dependent rescue factors is in contrast to the broader interactions in the tmRNA- and ArfB-based rescue pathways (see Discussion).…”

“…Indeed, F. tularensis ArfT can work with F. tularensis RF1 or RF2, but not E. coli RFs 24 . Also, E. coli ArfA fails to recruit Thermus thermophilus RF2 36,37 . We tested the compatibility of B. subtilis ResQ and E. coli ArfA with heterologous RFs in vitro.…”

Mechanism of ribosome rescue by ArfA and RF2

“…After submission of this manuscript, several other structures of 70S•ArfA•RF2 complexes with RF2 in the extended conformation were reported (James et al, 2016; Huter et al, 2017; Ma et al, 2017; Zeng et al, 2017). Structure I, however, was not found in these studies.…”

“…Following the submission of our manuscript, several groups reported 70S•ArfA•RF2 cryo-EM structures (James et al, 2016; Huter et al, 2017; Ma et al, 2017; Zeng et al, 2017), but each dataset contained a single ArfA•RF2 conformation (similar to our Structure I and Structure II), unlike our dataset, which contained both ArfA•RF2 states. We hypothesize that this difference may be due to several factors.…”

The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

“…Further cryo-EM work showed that ribosome-bound RF1 and RF2 have extended structures 2,3 , facilitating coordinated codon recognition in DC and ester bond hydrolysis in PTC. Subsequent high-resolution X-ray crystal 7,16,17,18,19,20,21,22 and cryo-EM 5,6,23,24,25,26 structures of RF-bound 70S ribosomes allowed the modeling of stop-codon recognition by RF1, RF2 27 , eRF1 28 and GGQ-induction of ester bond hydrolysis 28 .…”