The rescue of stalled translational conplexes: recoding of a sense to nonsense codon

Ribosomes synthesise all the proteins in the cell by reading or 'translating' information in messenger RNA (mRNA), which in turn is a copy of information stored in the cell's DNA. Accurate translation by ribosomes relies on them obeying certain rules, the genetic code, in which each triplet of nucleotides is always read in the same way to initiate, continue or terminate synthesis of a completed protein. The fidelity of translation is extremely high, and very few errors are made. Surprisingly however, on occasion ribosomes are prompted to disregard this code, often by particular sequences in the mRNA that is being read or the protein sequence that has just been synthesized by the ribosome and is still inside it. Such 'recoding' events are often found in viral RNAs and allow viruses to generate all the proteins they need from very compact, efficient genomes. Some recoding events also take place on cellular mRNAs and can be very important for correct expression of proteins. In many cases recoding events allow the normal 'stop' signals that signify the end of a protein to be bypassed, leading to extension of the protein. We have uncovered a novel recoding event in which the ribosome is prompted to stop translation and then restart / without the normal signals for either / thereby generating 2 separate proteins from one mRNA. This is dictated by a short peptide sequences termed '2A' from viruses and provides both an important and convenient tool for co-expression of more than one protein without the need for multiple mRNAs, and also the possibility of insight into how the ribosome works. Understanding this event may have another important repercussions as it may allow development of antiviral strategies, aimed at inhibiting this recoding event during viral infection. Our aim is therefore to understand reaction dictated by 2A in detail, identify all the factors that are required and the cellular functions that it impinges on. Thus far we have found that the 2A peptide causes ribosomes to pause, and that the 'release factors' that normally catalyse termination of translation at a stop signal are required for the abnormal termination reaction that takes place at 2A. We will investigate the interactions of release factors with ribosomes paused at 2A and attempt to determine what factors contribute to the pause.

Progress and fidelity of translation depends critically on binding of appropriate factors to the ribosomal A site. Elongation and termination factors, as well as perhaps factors involved in RNA turnover, all bind this critical position at which information in mRNA is decoded. The sets of interactions and steps in recognition that lead to cognate binding of factors to the A site are being elucidated, not least through structural biology, often allied with biochemical studies. Key information on interactions of factors with the ribosome can also be gained from situations where the ribosome is prompted not to obey the normal rules for interpreting information in mRNA. Such 'recoding' events,often avoid termination at a stop codon, by read-through or frame-shifting. We have uncovered a novel recoding mechanism, in which the ribosome pauses, apparently recruits release factors, and undergoes terminated on a sense codon, before continuing past the site via a reinitiation event. Combined this generates a break in the polypeptide chain, and the whole 'stop-carry on' recoding mechanims is directed in cis by a short ~18 amino acid peptide, termed 2A, within the exit tunnel of the ribosome. We now propose to dissect the requirements for RF in the recoding at 2A, with the aim of understanding the interactions and functions required. Further we will analyse the pause in more detail to determine what contributes to it. Like many recoding mechanisms stop-carry on is used by viruses. Important experiments will therefore also examine the recoding event in the context of a viral infective cycle / does the outcome of recoding change through infection, is this/can this be influenced by the levels of RF and other factors in the cell? Finally, we will initiate experiments through which we hope to identify factors that participate in other stages of 2A reaction.