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dc.contributor.author
Weisser, Melanie
dc.contributor.supervisor
Ban, Nenad
dc.contributor.supervisor
Leibundgut, Marc
dc.contributor.supervisor
Maier, Timm
dc.contributor.supervisor
Panse, Vikram
dc.date.accessioned
2017-09-18T05:18:15Z
dc.date.available
2017-09-16T17:15:14Z
dc.date.available
2017-09-18T05:18:15Z
dc.date.issued
2017
dc.identifier.uri
http://hdl.handle.net/20.500.11850/185232
dc.identifier.doi
10.3929/ethz-b-000185232
dc.description.abstract
During protein synthesis, the genetic information is translated into a sequence of amino acid building blocks that are sequentially attached to each other by a large ribonucleoprotein complex, the ribosome. One triplet of nucleotides translates into one amino acid. The eukaryotic ribosome can be divided into a small (40S) and a large (60S) subunit. Protein translation is initiated on the small ribosomal subunit upon forming a complex with initiator transfer RNA (tRNA) carrying the first amino acid and messenger RNA (mRNA) containing the nucleotide sequence template. During canonical initiation, the eukaryotic small ribosomal subunit binds to the mRNA in proximity of the capped 5' end. From there it scans along the mRNA until it reaches the correct initiation site for translation where the protein-coding sequence starts. Binding of the initiator tRNA to the start codon on the mRNA determines the reading frame for translation. Once the start codon has been identified correctly, the large ribosomal subunit joins the complex and translation can proceed. All of the above steps are highly regulated and require accessory translation initiation factors as regulatory proteins or regulatory mRNA sequences. In eukaryotes, canonical translation initiation involves at least 12 translation initiation factors (eIF). Two of them, eIF1 and eIF1A, are critical for the fidelity of start codon recognition and for the recruitment of the initiator tRNA to the complex. eIF1 dissociates upon start codon binding, while eIF1A is also important for the association with the large ribosomal subunit at a later stage. In the first part of this thesis, a 3.7 A resolution crystal structure of the canonical eukaryotic 40S-eIF1A-eIF1 initiation complex is described. The structure revealed how the two initiation factors bind to the small ribosomal subunit, how they are positioned with respect to each other and how they influence the conformation of the 40S subunit. The visualization of structural features of the two initiation factors and the conformational changes observed in the decoding center of the small ribosomal subunit provided new insights into the mechanisms of scanning and start codon recognition during eukaryotic translation initiation. In recent years, it became obvious that special sequence motifs or secondary structure elements within the mRNA sequence upstream of the start codon can influence the translation initiation efficiency at the downstream protein-coding open reading frame, allowing an additional level for regulating eukaryotic translation. Approximately half of the mammalian mRNAs possess one or more short upstream open reading frames (uORF), which do not encode for proteins, but regulate the translation efficiency at the downstream main open reading frame. In such a context, translation is terminated at the uORF, but instead of being released, the ribosome remains associated with the mRNA and "re-initiates" translation at the downstream main ORF. In many cases, re-initiation can be mediated by canonical eukaryotic translation initiation factors. However, sets of mRNAs have been identified that encode proteins involved in cell-cycle regulation and that require a different set of translation initiation factors. The protein eIF2D (eukaryotic translation initiation factor 2D, also called ligatin) and the homologous heterodimeric complex of MCT-1/DENR (malignant T-cell-amplified sequence 1) and DENR (density regulated protein) have been identified as translation re-initiation factors. In the second part of this thesis, using single-particle electron cryo-microscopy (cryo-EM), I describe the architecture of re-initiation complexes involving translation initiation factor eIF2D or the heterodimer of MCT-1/DENR, in complex with initiator tRNA and the 40S ribosomal subunit. Furthermore, to enable comprehensive interpretation of the cryo-EM maps for both complexes, I also determined the crystal structure of the C-terminal half of eIF2D for which no structural information had been available. The structures show that these re-initiation factors use loosely connected domains that mimic the action of several canonical initiation factors to provide critical stabilizing interactions for positioning the initiator tRNA on the 40S ribosomal subunit for reinitiation. Taken together, the structural and biochemical results presented in this thesis lay the basis for a better mechanistic understanding of eukaryotic translation initiation and the re-initiation process.
en_US
dc.format
application/pdf
dc.language.iso
en
en_US
dc.publisher
ETH Zurich
en_US
dc.rights.uri
http://rightsstatements.org/page/InC-NC/1.0/
dc.subject
Translation initiation
en_US
dc.title
Structural Insights into Eukaryotic Translation Initiation and Re-Initiation
en_US
dc.type
Doctoral Thesis
dc.rights.license
In Copyright - Non-Commercial Use Permitted
dc.date.published
2017-09-18
ethz.size
179 p.
en_US
ethz.code.ddc
DDC - DDC::5 - Science::570 - Life sciences
ethz.identifier.diss
23889
en_US
ethz.publication.place
Zurich
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02030 - Dep. Biologie / Dep. of Biology::02521 - Inst. f. Molekularbiologie u. Biophysik / Inst. Molecular Biology and Biophysics::03556 - Ban, Nenad / Ban, Nenad
en_US
ethz.leitzahl.certified
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02030 - Dep. Biologie / Dep. of Biology::02521 - Inst. f. Molekularbiologie u. Biophysik / Inst. Molecular Biology and Biophysics::03556 - Ban, Nenad / Ban, Nenad
en_US
ethz.date.deposited
2017-09-16T17:15:15Z
ethz.source
FORM
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2017-09-18T05:18:30Z
ethz.rosetta.lastUpdated
2020-02-15T07:21:58Z
ethz.rosetta.exportRequired
true
ethz.rosetta.versionExported
true
ethz.COinS
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