A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go)

Abstract

When a eukaryotic mRNA sequence specifying an amino acid motif known as 2A is directly followed by a proline codon, two nonoverlapping proteins are synthesized. From earlier work, the second protein is known to start with this proline codon and is not created by proteolysis. Here we identify the C-terminal amino acid of an upstream 2A-encoded product from Perina nuda picorna-like virus that is glycine specified by the last codon of the 2A-encoding sequence. This is an example of recoding where 2A promotes unconventional termination after decoding of the glycine codon and continued translation beginning with the 3' adjacent proline codon.

FIGURE 1.

Comparison of the genome organization of Perina nuda picorna-like virus (PnPV) and foot and mouth disease virus (FMDV). The protein products of the single open reading frame are shown as separate boxes. The shaded regions were identified by intact or digested protein analysis of PnPV virions. “L” denotes the leader peptide. Regions encoding 2A sequences are overlined and indicated by arrows. Putative virally encoded protease cleavage sites in PnPV are indicated by “*”. Only one of the virally encoded protease cleavage sites, *, is indicated in FMDV. CP1–4 denotes the four PnPV capsid proteins and VP1–4 denotes the four FMDV capsid proteins.

FIGURE 2.

Analysis of PnPV virion protein digests by FT-ICR. The single 2986 amino acid ORF of PnPV is depicted in sections. Underlined sequences were identified in tryptic or chymotryptic digests of virions. The two 2A sequences are italicized and the most crucial residues for “StopGo” are shown in white with black shading. Putative viral protease recognition sequences are shaded in gray. “pS” indicates phosphorylation on S ₁₁₈₇. The bold “Y” is the residue expected to link the putative VPg to the 5′ end of PnPV RNA.

FIGURE 3.

Electrospray molecular-mass spectra. (A) The mass spectrum of denatured virions showing the four major coat proteins, CP1–4. (B) Expanded views of the regions around the major peaks for CP1–4. Potassium (K) adducts and oxidation products are indicated. Oxidation of cysteine, proline, methionine, or other residues in the protein may have occurred from storage of the virus sample, exposure to reagents, reaction with degraded β-mercaptoethanol in solution, or may be due to post-translational modifications.