Biochemical analysis of translational recording driven by 2A peptide

Abstract

2A/2A-like peptides are short sequences (20-30 amino acids) encoded predominantly within open reading frames (ORFs) of RNA viruses. They drive a non-canonical translation, in which the nascent chain is released from the ribosome at a sense (proline) codon, followed by continued translation to generate a separate downstream protein, initiated from the same proline codon. The aim of this study is to investigate the role of ribosomal factors in the 2A reaction in Saccharomyces cerevisiae cells. Results obtained showed that reduced activity of eRF1/3 inhibits the 2A reaction. This inhibition did not strongly correlate with the effect that mutations have on termination at stop codons. In particular, several mutations within the NIKS motif, which is essential for stop codon recognition, had minimal effect on the 2A reaction. To confirm these results, we developed a new reporter to investigate the 2A activity, where the green fluorescent protein (GFP) sequence was separated with a 2A sequence, between residues 157 and 158. This reporter was utilised to confirm the effects of eRF1 mutations, previously assessed by immunoprecipitation, and results, observed by flow cytometry, revealed consistency in terms of the role of eRFs in the 2A reaction. In summary, these observations provide evidences supporting recruitment of eRFs to the ribosome to drive the non-canonical termination event that releases the first part of the 2A reaction.