Site-specific release of nascent chains from ribosomes at a sense codon

RNA Biology Group and Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; Department of Environmental & Biomolecular Systems, Oregon Health and Science University, Beaverton, OR 97006; School of Biology, Centre for Biomolecular Sciences, Biomolecular Sciences Building, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK; Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, Oregon 97201

^* To whom correspondence should be addressed. Email: Jeremy.Brown{at}ncl.ac.uk.

	Abstract

"2A" oligopeptides are autonomous elements containing a -D(V/I)ExNPGP- motif at their 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 peptidyl transferase centre. 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 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 growth of cells compromised for RF 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.