The rescue of stalled translation complexes: recoding of a sense to a nonsense codon

Lead Research Organisation: Newcastle University
Department Name: Inst for Cell and Molecular Biosciences

Abstract

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.

Technical Summary

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 discontinuity in the polypeptide chain, and the whole 'stop-carry on' recoding mechanism 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.
 
Description During the time-frame of this application we studied a particular viral peptide (termed 2A) that drives an unusual event during protein synthesis. This is the generation of separate proteins from one single gene (open reading frame). The findings from the work included an understanding of the variability of this sequence, its presence in some organisms, i.e. outwith viruses and mechanistic insight into how it functions. This may be important going forward in trying to utilise this as a target for antiviral drugs that could combat viruses that use this as a way to generate their proteins.
Exploitation Route could be used by companies interested in developing antiviral therapies.
Sectors Healthcare
 
Description 2A peptides, the topic of this research, are important in many laboratories, and also in some biotechnological, pharmaceutical and gene therapy approaches. The publications arising from the grant have, in contributing to understanding of this important means of controlling gene expression, provided impact in these areas. The impact is not though direct and is therefore hard to quantify other than through the citing of publications in further work.
 
Description 2A 
Organisation School of Biology
Department Centre for Biomolecular Sciences; University of St Andrews
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Research program was integrated, so difficult to assign specific contributions. However, our work focussed on yeast genetics/biochemistry, whereas that in St. Andrews focussed on genetics in E. coli and transfection of mammalian cells with various constructs
Collaborator Contribution Fully integrated research program between my laboratory and that of Prof. Martin Ryan to dissect a novel translational recoding event. This has included successful application for 2 BBSRC grants. Paper, conference presentations etc. have all resulted from this.
Impact 18458056
Start Year 2006
 
Description Toes 
Organisation Oregon Health and Science University
Country United States of America 
Sector Academic/University 
PI Contribution Initially we generated and provided constructs that Dr. Sachs used in specific assays (toe-printing of ribosomes). This was followed by myself visiting Dr. Sachs' laboratory to learn the technology for these assays.
Collaborator Contribution Performed specific assays and hosted myself in a visit to transfer technology to my laboratory
Impact 18458056
Start Year 2006