Structural Study of eIF2B by Electron Microscopy

  • Yu Zhou

    Student thesis: Phd

    Abstract

    In eukaryotic translation initiation, eIF2B, a 295 kDa multisubunit (from alpha to ε) complex,is the guanine nucleotide exchange factor (GEF) of eIF2, a GTP binding protein, and hasmultiple roles in regulating the level of active eIF2-GTP-Met-tRNAi ternary complexes inthe cytoplasm. Mutations in eIF2B subunits affect global protein synthesis and, in human,are responsible to cause a genetically inherited lethal childhood brain disease calledLeukoencephalopathy with Vanishing White Matter (VWM). Although the genetic aspectseIF2B have been widely studied over decades, detailed structural knowledge only becameavailable in recent years but is still limited. This study aims to gain structural insights intoyeast eIF2B by a range of electron microscopy techniques to improve our understandingtowards its GEF activity with eIF2 and regulatory response.By performing size-exclusion chromatography and multi-angle static light scattering (SECMALS), it was found that eIF2B is a stable dimer of pentamers (~600 kDa). Negativestaining (25.8 A) and cryo-EM (12.1 A) eIF2B decamer models that showed 2-foldrotational symmetry were generated by single particle reconstruction. Homology modelingof yeast eIF2B subunits revealed an eIF2B(alphaβdelta)2 hexameric core and two separate arm-likeeIF2Bγε catalytic domains with potential flexibility. To constrain subunit position in thearm structure, Ni-NTA-Nanogold labeling against the multihistidine tag of eIF2Bγ wasperformed. In addition, genetic approaches were applied to eliminate synthesis of eIF2Balpha(34 kDa) and eIF2B(βγdeltaε)2 octamer complexes (532 kDa) were purified by SEC-MALSand analysed by negative staining single particle reconstruction. It was speculated thatdeletion of eIF2Balpha might have triggered significant conformational rearrangement that ledto high uniformity in the 2D class averages. A hypothetical model was thus proposed forthe octamer where the two arm-like domains clamp together to form a compact structure.
    Date of Award1 Aug 2016
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorGraham Pavitt (Supervisor) & Alan Roseman (Supervisor)

    Keywords

    • eIF2B
    • cryo-EM

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