stop-carry on translational recoding

Expression of viral proteins frequently includes non-canonical decoding events (‘recoding’) during translation. ‘2A’ oligopeptides drive one such event, termed ‘stop-carry on’ recoding. Nascent 2A peptides interact with the ribosomal exit tunnel to dictate an unusual stop codon-independent termination of translation at the final Pro codon of 2A. Subsequently, translation ‘reinitiates’ on the same codon, two individual proteins being generated from one open reading frame. Many 2A peptides have been identified, and they have a conserved C-terminal motif. Little similarity is present in the N-terminal portions of these peptides, which might suggest that these amino acids are not important in the 2A reaction. However, mutagenesis indicates that identity of the amino acid at nearly all positions of a single 2A peptide is important for activity. Each 2A may then represent a specific solution for positioning the conserved C-terminus within the peptidyl-transferase centre to promote recoding. Nascent 2A peptide:ribosome interactions are suggested to alter ribosomal fine structure to discriminate against prolyl-tRNA and promote termination in the absence of a stop codon. Such structural modifications may account for our observation that replacement of the final Pro codon of 2A with any stop codon both stalls ribosome processivity and inhibits nascent chain release. INTRODUCTION A small number of nascent peptides are known to interact with the ribosomal exit tunnel and/or peptidyl-transferase centre (PTC) to regulate translation of downstream mRNA sequences (1,2). Most of these are encoded by short upstream ORFs (uORFs) in eukaryotes, or leader peptides in bacteria, and typically pause or stall ribosomes in response to small molecule effectors. ‘2A’ peptides, characterized by a C-terminal ‘-D12(V/I)ExNPGP19-’ motif (3,4), are further sequences that pause ribosomes (5–7). However, unlike other characterized ribosomepausing peptides, 2As also drive a translational recoding event, in which two separate proteins (the ‘upstream’ and ‘downstream’ products) are generated from an ORF containing a 2A coding sequence, with a ‘break’ in the polypeptide backbone between the final 2 amino acids, Gly and Pro, of 2A (formally this Pro is the first amino acid of the downstream protein, but is an essential part of active 2A). The 2A reaction has been termed ‘StopGo’ or ‘stop-carry on’ recoding (6,8). 2A peptides are active when transposed into other proteins, i.e. they are autonomous elements, and mediate recoding in all eukaryotic ribosomes tested. The unique activity of 2A peptides has led to their use as tools for co-expression of two (or more) proteins in biomedicine and biotechnology (9). The most studied 2A peptide is found in the Aphthovirus foot-and-mouth disease virus (FMDV). The FMDV RNA genome contains a single, long ORF encoding a polyprotein. 2A directs a key, primary processing event in the resolution of the polyprotein to individual proteins. Modelling of FMDV 2A indicated that it may form an (amphipathic) a-helix over most of its length, with a reverse turn at its C-terminus (5,10). This led to the suggestion that the a-helix, in the ribosomal exit *To whom correspondence should be addressed. Tel: +44 0191 222 7470; Fax: +44 0191 222 7424; Email: [email protected]