Site-Specific Release of Nascent Chains from Ribosomes at a Sense Codon

Abstract

“2A” oligopeptides are autonomous elements containing a D(V/I)EXNPGP motif at the C terminus. Protein synthesis from an open reading frame containing an internal 2A coding sequence yields two separate polypeptides, corresponding to sequences up to and including 2A and those downstream. We show that the 2A reaction occurs in the ribosomal peptidyltransferase center. Ribosomes pause at the end of the 2A coding sequence, over the glycine and proline codons, and the nascent chain up to and including this glycine is released. Translation-terminating release factors eRF1 and eRF3 play key roles in the reaction. On the depletion of eRF1, a greater proportion of ribosomes extend through the 2A coding sequence, yielding the full-length protein. In contrast, impaired eRF3 GTPase activity leads to many ribosomes failing to translate beyond 2A. Further, high-level expression of a 2A peptide-containing protein inhibits the growth of cells compromised for release factor activity and leads to errors in stop codon recognition. We propose that the nascent 2A peptide interacts with ribosomes to drive a highly unusual and specific “termination” reaction, despite the presence of a proline codon in the A site. After this, the majority of ribosomes continue translation, generating the separate downstream product.

ACKNOWLEDGMENTS

We are indebted to David Bedwell, Charles Cole, Allan Jacobson, Sabine Rospert, Joe Salas-Marco, Ian Stansfield, Mick Tuite, and Nianshu Zhang for strains and/or reagents; David Lydall, Alan Brown, Jeff Errington, laboratory members, and particularly Nicholas Watkins for discussion and/or comment on the manuscript; and Joe Gray and Francesca Harrison (Pinnacle Proteomics Unit, Newcastle University) for MS analysis.

This work was supported by an MRC Senior Non-Clinical Research Fellowship (J.D.B.); BBSRC grants C20418, BB/E009093 (J.D.B.), and C20035, BB/E/01070911 (M.D.R.); and NIH grant GM47498 (M.S.S.).