Structural studies of eukaryotic protein synthesis factor complexes eIF2B and eIF2/eIF2B, critical for translational control in eukaryotic cells

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All organisms are composed of cells. Cell growth and cell division are coordinated and controlled by a wide range of signals that ensure they occur at the correct places and times. Hence developing embryos require periods of rapid growth while adults require much slower growth to replace damaged or dying cells. When there is a loss of growth control, diseases such as cancer can develop, while a failure to promote growth when required can cause a failure to repair damaged cells or cause tissue wasting. We have been studying how cells control the conversion of nutrients into the new proteins that are required for life. Proteins perform nearly all cellular functions. Each protein is made from building blocks called amino acids that are linked in chains and folded to make 3-dimensional structures that are important for each to fulfil their individual roles. The instructions required to make each protein correctly are determined by the DNA sequences of the genes in the genome. This is termed 'protein synthesis' and it is the final step in the pathway called 'gene expression' which is critical for ensuring that the correct genes are decoded at the correct place and time. Protein synthesis occurs within 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 adapter molecules called transfer RNAs (tRNAs) that bring the necessary amino acids together. This proposal concerns the initiation phase of protein synthesis in which a dedicated set of factors act that are called protein synthesis initiation factors. They direct the ribosome and a specialised tRNA called initiator 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. This 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. This proposal concerns factors designated eIF2B and eIF2, two factors critical for regulating ribosome binding of Met-tRNAi. eIF2B is known as a factor that 'switches on' its partner eIF2 so that eIF2 can bind to Met-tRNAi and recruit it to ribosomes In this proposal we describe preliminary experiments, which show that the factor eIF2B is twice as big as was previously thought and also provide clues to its overall structure in three dimensions. Because proteins cannot be seen with the naked eye or light microscopes, we propose here to use the technique called cryo electron microscopy to rapidly freeze highly purified protein samples in liquid nitrogen and then magnify them so that we can build a three-dimensional model of eIF2B and study how it binds to its partner protein called eIF2. By providing a detailed understanding of the contribution of eIF2B to the control of protein synthesis it will help understand control of cell growth and provide further insight into vanishing white matter disease, 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 for commercial products or those that grow cells by fermentation because it will allow an improved understanding of protein synthesis mechanism. Detailed structural information will help unravel the precise controls of protein synthesis will assist in the design of optimized commercial protein expression or fermentation systems.