Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin. Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626886 |
| Date | January 2013 |
| Creators | Murphy, Patrick |
| Contributors | Pavitt, Graham; Heintzen, Christian |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-critical-interactions-between-translation-factors-eif2-and-eif2b(9138d7c8-34b1-4489-8048-a2ac45ef8533).html |
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