title: eIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics.
abstract: Translation of messenger RNA (mRNA) into proteins is key to eukaryotic gene expression and begins when initiation factor-2 (eIF2) delivers methionyl initiator tRNA (Met-tRNAi (Met)) to ribosomes. This first step is controlled by eIF2B mediating guanine nucleotide exchange on eIF2. We isolated eIF2 from yeast and used mass spectrometry to study the intact complex, and found that eIF2β is the most labile of the three subunits (eIF2α/β/γ). We then compared conformational dynamics of the ternary complex eIF2:GTP:Met-tRNAi (Met) with apo eIF2 using comparative chemical cross-linking. Results revealed high conformational dynamics for eIF2α in apo eIF2 while in the ternary complex all three subunits are constrained. Novel post-translational modifications identified here in both eIF2 and eIF2B were combined with established sites, and located within protein sequences and homology models. We found clustering at subunit interfaces and highly phosphorylated unstructured regions, at the N-terminus of eIF2β, and also between the eIF2Bε core and catalytic domains. We propose that modifications of these unstructured regions have a key role in regulating interactions between eIF2 and eIF2B, as well as other eIFs.
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, UK.
authors.forename: Victoria
authors.initials: V
authors.lastname: Beilsten-Edmands
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, UK.
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Gordiyenko
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, UK.
authors.forename: Jocky Ck
authors.initials: JC
authors.lastname: Kung
authors.affiliation: Department of Chemistry, University of Oxford, Oxford, UK; Department of Biochemistry, University of Oxford, Oxford, UK.
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, UK.
authors.forename: Carla
authors.initials: C
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, UK.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
keywords: acetylation; eukaryotic translation initiation; mass spectrometry; phosphorylation