Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

Abstract

Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2 m ), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k cat by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the k cat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.

Mechanism of ribosome rescue by ArfA and RF2

“…Structures I and II are in agreement with previous biochemical and mutagenesis studies, as we have described above (Chadani et al, 2010, 2012; Shimizu, 2012; Kurita et al, 2014; Zeng and Jin, 2016). An ArfA-inactivating mutation has been identified (Chadani et al, 2010).…”

“…Similarly, a combination of ArfA and RF1 did not result in [S 35 ]-fMet release, consistent with the requirement for RF2. By contrast, efficient release was observed in 10 min after addition of ArfA and RF2 in combination, consistent with published data (Zeng and Jin, 2016). The time of the cryo-EM sample incubation prior to freezing (15 min, as described in the previous section) was therefore sufficient for achieving equilibrium and peptide release, suggesting that the cryo-EM structures represent interconverting equilibrium states.…”

“…The mid-region of ArfA (His21 to Glu30; E. coli numbering is used) lies in the A site (Figure 1C and D). ArfA leaves a ~12 Å gap in the codon-binding region, sufficient to accommodate one or two nucleotides of mRNA following the P-site codon but not a longer mRNA, consistent with the reduced efficiency of ArfA-mediated release on mRNAs that extend three or more nucleotides beyond the P site (Shimizu, 2012; Zeng and Jin, 2016).

10.7554/eLife.23687.009Figure 2.Structure and sequence of ArfA.( A ) Close-up view of the intersubunit space and mRNA tunnel occupied by ArfA in Structure II.

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“…SmpB bound with tmRNA in preparation for trans-translation, forms a shorter α-helix, well-resolved near the mRNA entry at the 30S solvent surface (Neubauer et al, 2012). SmpB is similar to ArfA in that both proteins are sensitive to the mRNA occupancy of the A site (Ivanova et al, 2004; Shimizu, 2012; Zeng and Jin, 2016), whereas ArfB can function on stalled ribosomes with either the vacant or occupied A site (Handa et al, 2011). Diverged sequences, structures and binding modes of these proteins likely reflect distinct affinities and sensitivities to stress conditions, in keeping with their roles in mediating distinct ribosome rescue pathways (Figure 2—figure supplement 1).…”

Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner

“…Western blot was done as previously described (Coller and Parker 2005;Sweet et al 2012;Zeng and Jin 2016). Briefly, fractions from polysome profiling were resolved by SDS-PAGE; and proteins in unfixed gels were transferred to nitrocellulose membranes (GenScript) for 30 min at 100 V in a Mini Trans-Blot apparatus (Bio-Rad).…”