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Analyzing the eukaryotic translation initiation apparatus and new approaches in affinity chromatography

dc.contributor.advisorGörlich, Dirk Prof. Dr.
dc.contributor.authorSeefeldt, Jennifer
dc.titleAnalyzing the eukaryotic translation initiation apparatus and new approaches in affinity chromatographyde
dc.contributor.refereeGörlich, Dirk Prof. Dr.
dc.description.abstractengEukaryotic translation is a multistep process that utilizes 47 individual translation factor subunits. eIF3 is the largest and most complex translation factor, acting during initiation where it promotes assembly of the 43S preinitiation complex and the recruitment of the mRNA to the ribosome. Here we analyzed the native wheat eIF3 complex, showing that this 13 subunit factor forms a stable complex in vivo. We were able to recombinantly express and purify the individual subunits under native conditions. However they failed to spontaneously assemble into the holocomplex, suggesting that the maturation of the eIF3 complex requires factors and mechanisms not available when produced in prokaryotes. Another important aspect of eukaryotic gene expression is the spatial-temporal separation of transcription in the nucleus and translation in the cytoplasm. Cells evolved mechanisms to keep the levels of translation factors in the nuclear interior continually low. Exportins actively transport these proteins to the cytoplasm in a RanGTPase-driven manner. Crm1 is known to interact with almost all translation factors; Exportin 4 specifi cally removes eIF5A from the nucleus. We could show that addition of Crm1 and Exportin 4 to an in vitro translation system decreases translation rates of reporter constructs in a RanGTP dependent manner, supporting the assumption that nuclear transport factors act as compartment speci fic inhibitors of translation. In addition, we present a new tool for a ffinity chromatography based on a MADA activated solid phase and a novel affi nity tag system. The solid phase reacts  specifi cally with thiol groups, allowing to immobilize any ligand that contains an exposed and reduced cysteine. A ffinity resins based on the MADA chemistry compare very favorable to commercially available systems, in particular in terms of reduced background binding under low salt conditions. The streptavidin:biotin system is routinely used as state of the art technique for a ffinity purifi cations. Nonetheless it has severe disadvantages, namely the tetrameric state of streptavidin, poor folding of streptavidin as well as biotin contamination during bacterial expression/purifi cation and bait proteins require covalent modi fication with biotin. We now developed an alternative based on the extremely tight B.amyloliquefaciens Barnase:Barstar complex (KD=10-14M). To allow for separate cytoplasmic expression of these modules, we detoxifi ed the RNase Barnase by a His102Asp mutation and restored high binding strength by a compensatory Cys42Lys mutation on Barnase inhibitor Barstar. The 1.98 A crystal structure indeed shows that the mutated residues form a shielded salt bridge that further stabilizes the complex. Barstar initially showed poor solubility upon overexpression in E.coli. This problem was solved by switching to the Barnase:Barstar pair identifi ed in the hyperthermophilic Bacillus relative Geobacillus
dc.contributor.coRefereeRodnina, Marina Prof. Dr.
dc.contributor.thirdRefereeLipka, Volker Prof. Dr.
dc.subject.engEukaryotic translation initiationde
dc.subject.engeukaryotic translation initiation factor 3 (eIF3)de
dc.subject.engnucleocytoplasmic transportde
dc.subject.engaffinity chromatographyde
dc.subject.engaffinity tag systemsde
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de

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