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Translation reinitiation mechanism on mRNA of trascriptional activator GCN4.
Vlčková, Vladislava ; Valášek, Leoš (advisor) ; Strachotová, Dita (referee)
Translation reinitiation is a gene-specific translational control mechanism exploiting the ability of some short upstream open reading frames (uORFs) to retain post-termination 40S ribosomal subunit on the mRNA. Reinitiation efficiency depends on cis-acting sequences surrounding the uORF, translation elongation rates on the uORF, selected initiation factors, and the intercistronic distance of the short uORF from the main ORF. Although the precise mechanism of reinitiation is still not known, great progress in elucidating some of its details has been recently made with help of the GCN4 translational control model system. Among them, involvement of eIF3 was shown to play a critical role for efficiency of this process. In particular, it was proposed that eIF3 specifically interacts with sequences located upstream of a reinitiation-permissive uORF upon termination, and that this step is instrumental in stabilizing the 40S ribosomal subunit on the mRNA to allow subsequent resumption of scanning for reinitiation downstream. In this thesis, the current knowledge of the translation reinitiation mechanism is summarized. As a typical example, the yeast transcriptional activator GCN4 has been chosen, the mRNA of which is subjected to a tight translational control via the very reinitiation mechanism.
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Characterization of the molecular mechanism of translation reinitiation in yeast.
Pondělíčková, Vanda ; Valášek, Leoš (advisor) ; Hašek, Jiří (referee) ; Vopálenský, Václav (referee)
Translation initiation is a multi-step process culminating in formation of the elongation- competent 80S ribosome. It requires accurate assembly of small and large ribosomal subunits, mRNA, initiation Met-tRNAi Met and at least 12 eukaryotic initiation factors (eIFs). This phase of protein synthesis is also one of the key points of regulation of gene expression. One of the main aims of our laboratory is a complex characterization of the multiprotein eIF3 complex that has been implicated in most of the steps of translation initiation. For example, we revealed and described its novel role in translation reinitiation (REI), a gene-specific translational control mechanism that among others governs expression of an important yeast transcriptional activator GCN4. Here I present a detailed characterization of the multi-functional N-terminal domain of Tif32 (subunit eIF3a). We demonstrated that the Tif32-NTD functionally interacts with the 5' sequences of short upstream ORF (uORF1) in the GCN4 mRNA leader and thus allows efficient reinitiation downstream of this critical reinitiation-permissive uORF. Four REI- promoting elements (RPEs) were identified in the 5' sequences of uORF1, two of which were shown to work in the Tif32-NTD-dependent manner. The structure of the 5' sequences was determined...
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Translation reinitiation mechanism on mRNA of trascriptional activator GCN4.
Vlčková, Vladislava ; Valášek, Leoš (advisor) ; Strachotová, Dita (referee)
Translation reinitiation is a gene-specific translational control mechanism exploiting the ability of some short upstream open reading frames (uORFs) to retain post-termination 40S ribosomal subunit on the mRNA. Reinitiation efficiency depends on cis-acting sequences surrounding the uORF, translation elongation rates on the uORF, selected initiation factors, and the intercistronic distance of the short uORF from the main ORF. Although the precise mechanism of reinitiation is still not known, great progress in elucidating some of its details has been recently made with help of the GCN4 translational control model system. Among them, involvement of eIF3 was shown to play a critical role for efficiency of this process. In particular, it was proposed that eIF3 specifically interacts with sequences located upstream of a reinitiation-permissive uORF upon termination, and that this step is instrumental in stabilizing the 40S ribosomal subunit on the mRNA to allow subsequent resumption of scanning for reinitiation downstream. In this thesis, the current knowledge of the translation reinitiation mechanism is summarized. As a typical example, the yeast transcriptional activator GCN4 has been chosen, the mRNA of which is subjected to a tight translational control via the very reinitiation mechanism.
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Delece RPS0A-vazebné domény TIF32/eIF3A brání vazbě MFC na 40S ribozóm a blokuje derepresi GCN4 exprese
Szamecz, Bela ; Rutkai, Edit ; Nielsen, K. H. ; Valášek, Leoš
Yeast Initiation factor 3 (eIF3) occurs together with the eIF2.GTP.Met-tRNAiMet ternary complex (TC) and eIFs 1 and 5 in a pre-formed unit designated the multifactor complex (MFC) that was implicated in playing a critical role in ecient recruitment of TC and mRNA to the 40S ribosomes and stimulation of the post-assembly processes such as scanning and AUG recognition. In ecort to identify binding sites of the MFC on the 40S ribosome, we previously demonstrated that deletion of the rst 199 amino acids of the N-terminal domain (NTD) of the TIF32 subunit of eIF3/eIF3a, in the presence of a wild-type gene, completely eliminated association of the mutant MFC with the 40S ribosome without aecting its overall integrity. In addition, we showed that the TIF32-NTD contains a binding site for the C-terminal domain (CTD) of the small ribosomal protein 0A (RPS0A) that is located on the solvent side of the 40S subunit where the main body of eIF3 was proposed to reside
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