Interaction between translation factor eIF2gamma and its regulatory proteins

Interactions between proteins modulate essential functions of cells ensuring that they can grow and perform their tasks at the correct time and place. Each protein within a cell is like a piece within a complex machine. In a machine each part must be in the correct place and act at the correct time in concert with the other parts for the machine to function properly. The same is true for proteins within cells, but unlike most machines and their parts, proteins are continually renewed, move and can be modified by interaction with many other cell components to alter their function and/or location. Many proteins act together within complexes composed of proteins or with other cell components (lipids, DNA or small molecules for example) to coordinate a common function. One group of proteins relevant to this proposal is required make (or synthesize) all new proteins in each cell. These are called 'protein synthesis factors'. Understanding how protein synthesis factors function and interact with each other is therefore fundamental to understanding how proteins are made in all cells in all organisms. By improving our understanding how processes such as protein synthesis work in normal cells it can help scientists understand diseases in which this process is altered, or how infectious agents such as viruses are able to hijack plant, animal or human cells and cause infectious diseases. In work leading up to this proposal we identified a novel interaction between two protein synthesis factors known as eIF2 and eIF2B that is critical for protein synthesis. Mutations in eIF2B that impair this interaction are lethal in a yeast cellular model. We propose to study where on the surface of eIF2 is important for this interaction and to compare this with binding to eIF5, another important ligand for eIF2. We intend to study these interactions using a combination of genetic experiments using yeast cells as a model simple cell system and biochemical experiments with purified proteins in test-tubes to understand the biological significance of these interactions for the mechanism and regulation of protein synthesis.

Protein synthesis is a Key phase in the gene expression pathway that allows rapid global and gene-specific temporal and spatial control of protein production. In addition to simply generating biomass, translational control is important for a wide-range of responses. One factor whose activities are critical for translation and its control is eukaryotic initiation factor (eIF) 2. eIF2 is a general eukaryotic protein synthesis initiation factor that functions to deliver tRNAiMet to 40S ribosome/mRNA complexes. Upon tRNAiMet-AUG start codon recognition GTP bound to eIF2 is hydrolysed to GDP by eIF5 releasing these factors as an eIF2-GDP/eIF5 complex. eIF2B is the guanine-nucleotide exchange factor that must interact with eIF2 to promote GDP/GTP exchange to initiate a new round of translation. Thus eIF2 must interact with a variety of RNA, nucleotide and protein ligands during this cycle. The gamma subunit of eIF2 is well established as being critical for the tRNAiMet and GDP/GTP binding activities. We have now obtained direct evidence that eIF2gamma is also important for interaction with the catalytic centre of eIF2B in addition to eIF5. We propose here a series of yeast genetic and complementary biochemical experiments to determine how eIF2gamma interacts with eIF2B and eIF5. Specifically, two complementary genetic approaches will be used. Firstly, a site-directed mutagenesis strategy targeting surface residue clusters. Secondly, a complementary suppressor screen to identify mutations that specifically suppress the lethal phenotype of a mutant eIF2B allele that interacts only weakly with eIF2gamma. Next we will perform a detailed genetic and biochemical characterization of the mutants isolated with complementary in vivo and in vitro experiments to correlate the protein-protein interactions with the phenotypes observed.