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The role of the N-terminal domain of the a/TIF32 subunit of eIF3 in mRNA recruitment to the 43S pre-initiation complexes.
Vlčková, Vladislava ; Valášek, Leoš (advisor) ; Mašek, Tomáš (referee)
Translation initiation is a complex process which results in the assembly of the elongation competent 80S ribosome from the 40S and 60S ribosomal subunits, the initiator tRNA and mRNA, and is orchestrated by numerous eukaryotic initiation factors (eIFs). Although it represents one of the most regulated processes of gene expression, the exact mechanism of one of the key steps of translation initiation - mRNA recruitment to the 43S pre-initiation complex (PIC) - is still only poorly understood. Recent studies indicated that besides eIF4F and poly(A)-binding protein, also eIF3 might play an important, if not crucial, role in this step. In our laboratory, we recently identified a 10 Ala substitution (Box37) in the a/TIF32 subunit of Saccharomyces cerevisiae eIF3, which interfered with translation initiation rates. Detailed analysis showed that this mutation significantly reduces the amounts of model mRNA in the gradient fractions containing 48S PICs as the only detectable effect in vivo. Moreover, a recently solved crystal structure of the N-terminal part of a/TIF32 pointed to two Box37 residues, Arg363 and Lys364, both proposed to contribute to one of the positive, potentially RNA-binding areas on the a/TIF32 surface. The fact that also their substitutions with alanines severely impaired the mRNA recruitment...
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Dissection of eIF3 functional domains promoting the 48S pre-initiation complex assembly
Beznosková, Petra ; Valášek, Leoš (advisor) ; Novotný, Marian (referee)
In eukaryotes, translation initiation is guided by up to twelve protein initiation factors (eIFs) and begins with the formation of the 43S pre-initiation complex (PIC) composed of the small ribosomal subunit (40S), eIF2.GTP/Met-tRNAi Met ternary complex, and eIFs 1, 1A, 3 and 5. The 43S PIC subsequently interacts with the 5'end of an mRNA (an mRNA recruitment step) and thus formed 48S PIC travels in 5' to 3' direction along the mRNA leader sequence to locate the AUG start codon (this presumably linear movement is generally known as scanning). Start site selection results in the dissociation of the initiation factors and joining of the large (60S) ribosomal subunit to form the 80S initiation complex poised for elongation. Eukaryotic initiation factor 3 (eIF3) plays a critical role in most of these events; however, the molecular details of most of its contributions are still unknown to us. Previous in vivo studies generated numerous mutations in all eIF3 subunits with specific defects either in the PICs assembly or in the following steps such as scanning, AUG recognition, etc. To understand the exact role of eIF3 in this intriguing process at the molecular level, we have embarked on a study that aims to dissect the individual functions of each eIF3 subunit in translation initiation using the purified...
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The role of eIF3 in mRNA recruitment to the 43S pre-initiation complex
Beznosková, Petra ; Mašek, Tomáš (referee) ; Valášek, Leoš (advisor)
Translation initiation in eukaryotes plays an important role in gene expression and relies on interactions between many eukaryotic initiation factors, small and large ribosomal subunits, mRNA and initiator tRNA. The largest of these initiation factors, the eukaryotic initiation factor 3 (eIF3), participates in the most of translation initiation reactions. In yeast, eIF3 occurs together with eIF1, eIF5 and ternary complex (TC, eIF2-GTP-Met-tRNAi Met complex) in the multifactor complex (MFC) and promotes formation of the 43S preinitiation complexes. eIF3 also stimulates mRNA recruitment to the 43S preinitiation complexes, scanning the 5' untranslated region of mRNA for AUG recognition and has an important role in the gene-specific translational control mechanism called reinitiation involving short upstream open reading frames (uORFs).
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