REGULATION OF eIF2B BY PHOSPHORYLATION A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2013 REHANA KOUSAR Table of contents Table of figures .............................................................................................................. 5 Table of tables ................................................................................................................ 7 Abstract .......................................................................................................................... 8 Declaration ..................................................................................................................... 9 Copyright ..................................................................................................................... 10 Acknowledgements ...................................................................................................... 12 List of abbreviations .................................................................................................... 13 Communications .......................................................................................................... 15 1 Introduction .......................................................................................................... 17 1.1 Prokaryotic translation initiation ...................................................................... 18 1.2 Eukaryotic translation ...................................................................................... 21 1.2.1 Translation initiation ................................................................................ 21 1.2.2 IRES mediated translation initiation ........................................................ 27 1.2.3 CITE assisted translation initiation .......................................................... 28 1.2.4 Translation elongation ............................................................................. 29 1.2.5 Translation termination ............................................................................ 31 1.3 Targets of translational control ........................................................................ 32 1.3.1 Cis-acting elements and translation regulation ........................................ 33 1.3.2 Trans-acting factors and translation regulation ....................................... 37 1.4 Control of initiation factors activity by phosphorylation ................................. 38 1.5 eIF2: function, structure and control ................................................................ 39 1.5.1 Localization of eIF2 ................................................................................. 39 1.5.2 Role of eIF2 ............................................................................................. 40 1.5.3 Structure of eIF2 ...................................................................................... 40 1.5.4 eIF2 kinases ............................................................................................. 42 1.5.5 eIF2α(p) and expression of GCN4 ........................................................... 48 1.6 Target of Rapamycin pathway ......................................................................... 49 1.6.1 Structure of TOR ...................................................................................... 51 1.6.2 TOR complexes ....................................................................................... 51 1.6.3 Rapamycin sensitive TORC1 ................................................................... 52 1.6.4 Rapamycin insensitive TORC2 ................................................................ 57 1.7 eIF2B-GEF for eIF2 ......................................................................................... 59 1.7.1 Localization of eIF2B .............................................................................. 60 1.7.2 Subunit structure of eIF2B ....................................................................... 60 1.7.3 Mechanisms of eIF2B catalysed reaction ................................................ 65 1.7.4 Regulation of eIF2B activity .................................................................... 67 1.7.5 Mammalian eIF2B kinases ...................................................................... 68 1.7.6 eIF2B and Human Disease ....................................................................... 71 1.8 Introduction to current study ............................................................................ 72 1.8.1 The aims and objective(s) of the research ................................................ 73 1.8.2 Structure of thesis .................................................................................... 73 2 MATERIALS AND METHODS ......................................................................... 75 2.1 Yeast strains ..................................................................................................... 75 2.1.1 S. cerevisiae strains .................................................................................. 75 2.1.2 List of plasmids ........................................................................................ 77 2.2 Growth media ................................................................................................... 77 2.2.1 Yeast growth media ................................................................................. 77 Regulation of eIF2B by Phosphorylation 2 Table of contents 2.3 List of E. coli strains ........................................................................................ 80 2.3.1 Luria-Bertani medium for E. coli ............................................................. 80 2.4 S. cerevisiae strains growth and maintenance .................................................. 81 2.4.1 Strain maintenance ................................................................................... 81 2.4.2 Reviving the strains from -80˚C .............................................................. 81 2.5 DNA recombinant methods ............................................................................. 82 2.5.1 Plasmid DNA isolation from E. coli ........................................................ 82 2.5.2 Site directed mutagenesis ......................................................................... 82 2.5.3 Transformation of E. coli ......................................................................... 83 2.5.4 Restriction digestion ................................................................................ 85 2.5.5 Agarose gel electrophoresis ..................................................................... 85 2.5.6 DNA sequencing ...................................................................................... 86 2.5.7 Lithium acetate aided yeast transformation ............................................. 87 2.5.8 Plasmid shuffling ..................................................................................... 88 2.6 Growth assays .................................................................................................. 90 2.6.1 Yeast quantitative growth assays ............................................................. 90 2.7 Polysome profiling ........................................................................................... 93 2.7.1 Whole cells extract preparation ............................................................... 94 2.7.2 Sucrose density gradients preparation ..................................................... 95 2.7.3 Procedure of sucrose gradient sedimentation .......................................... 95 2.8 Biochemical methods ....................................................................................... 96 2.8.1 Cell lysis and protein extraction (small scale) ......................................... 96 2.8.2 Quantification of protein in whole cells extract-Bradford assay ............. 97 2.8.3 Purification of FLAG-tagged eIF2B ........................................................ 97 2.8.4 Dephosphorylation of purified protein ................................................... 100 2.9 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ..... ........................................................................................................................ 100 2.9.1 Sample Preparation for SDS-PAGE ...................................................... 100 2.9.2 SDS-PAGE ............................................................................................ 100 2.9.3 Gel staining ............................................................................................ 101 2.10 Western blotting ............................................................................................. 101 2.10.1 Western transfer ................................................................................. 102 2.10.2 Immunoblotting and protein detection ............................................... 102 2.11 Antibodies ...................................................................................................... 104 2.12 Bioinformatics ................................................................................................ 106 3 Optimising conditions to evaluate the roles of multiple phosphorylation sites in eIF2B .................................................................................................................. 108 3.1 Background of current project ......................................................................