GTP-binding to eIF2B as a novel mechanism for G protein activation in protein synthesis initiation

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We study how cells control the conversion of nutrients into the new proteins that are required for life-the growth and division of cells. Proteins are biological machines that perform nearly all cellular functions. Each protein is made from building blocks called amino acids that are linked in chains and folded to make specific 3-dimensional structures that are important for each protein to fulfil their individual roles. The instructions required to make each protein correctly are determined by the DNA sequences of our genes in the genome. Termed 'protein synthesis' this is the final step in the gene expression pathway, which is critical for ensuring that the correct genes are decoded at the correct place and time. Protein synthesis occurs within relatively large and complex molecular machines called ribosomes that decode instructions relayed from the genome within intermediary molecules called messenger RNAs (mRNAs). Human cells each contain over a million ribosomes. mRNA decoding by ribosomes is made possible by the concerted action of 'helpers' called protein synthesis factors and adapters called transfer RNAs (tRNAs). In concert they bring the necessary amino acids together with the instructions to ensure the correct proteins are made at the right time. This proposal concerns the initiation phase of protein synthesis in which a dedicated set of factors act termed protein synthesis initiation factors. They direct the ribosomes and a specialised tRNA that starts proteins with the amino acid methionine (designated Met-tRNAi) to the correct start place on each mRNA. This is critical to make the right proteins in every cell and must be done both accurately and rapidly. Initiation is the most complex phase of protein synthesis and the least well understood at the molecular level. Key to this proposal are factors designated eIF2B, eIF2 and eIF5. These three factors are critical for regulating ribosome binding of Met-tRNAi by controlling the energy charge on the eIF2 factor. The energy status is determined by binding nucleotides called GTP and GDP. When bound to GTP eIF2 is switched on and can bind to Met-tRNAi and when bound to GDP it cannot bind to Met-tRNAi and so is switched off. We have been studying this switching mechanism, as it is central to the control of protein synthesis. eIF5 switches eIF2 off, while eIF2B switches it on. We have recently found that the eIF2B protein also binds GTP. This unexpected finding has implications for how eIF2B works. eIF2B is complex factor and we have identified approximately which part of the protein GTP binds to - called the gamma subunit. We know that the gamma subunit is critical for both eIF2B known functions, first its GDP/GTP exchange function and also a second role we recently found, a function that displaces eIF5 from eIF2 so that eIF2 is free for nucleotide exchange. The experiments we describe in this proposal are aimed at understanding (i) why eIF2B binds GTP, (ii) where exactly it does bind and (iii) how important it is for each of the eIF2B roles in cells. Our idea is that GTP is directly handed from eIF2B to eIF2, which would be a novel way for this type of protein to function. By providing a detailed understanding of the contribution of eIF2B to the control of protein synthesis it will help scientists understand cell growth better and provide further insight into disease mechanisms, for example vanishing white matter disease, a genetic disorder which is caused by mutations in eIF2B. The work may also be of interest to industries eg those that produce specific proteins as drug therapeutics or commercial products or those that grow cells by fermentation, because improved understanding of protein synthesis mechanisms will assist in the design of optimized commercial protein expression or fermentation systems.