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Molecular principles of translation reinitiation in mammals
Hronová, Vladislava ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Translation initiation is a multistep process resulting in the formation of the elongation-competent 80S ribosome at the AUG start codon of the mRNA to be translated into a polypeptide chain. This process is orchestrated by numerous proteins called eukaryotic initiation factors (eIFs), out of which the most multitasking one is the eukaryotic initiation factor 3 (eIF3). The main focus of our laboratory aims at the complex characterization of the multisubunit protein eIF3 and the mechanisms of its contribution to various steps of translation initiation. Besides this, we also study one of the gene-specific translational control mechanisms called reinitiation which was, at least in yeast, also shown to be promoted by eIF3. Here I show that the N-terminal domain (NTD) of the largest subunit of yeast eIF3, a/Tif32, plays an important role not only in anchoring the eIF3 complex to the 40S small ribosomal subunit but it also critically contributes to mRNA recruitment to the 43S preinitiation complexes in vivo. The mRNA stabilization role of the a/Tif32-NTD at the mRNA exit channel of the 40S subunit was further confirmed in our following study by biophysical experiments. There, using in vivo approaches, we also demonstrated that mRNAs with longer 5'UTRs are more dependent on the stabilization role of the...
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A study of the HCV IRES variability: An experimental approach coupled with design of a large-scale mutation database
Khawaja, Anas Ahmad ; Pospíšek, Martin (advisor) ; Hirsch, Ivan (referee) ; Valášek, Leoš (referee)
Translation initiation in the hepatitis C virus (HCV) occurs through a cap- independent mechanism that involves an internal ribosome entry site (IRES) capable of interaction with and utilization of the eukaryotic translational machinery. We focused on the structural configuration of the different HCV-IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. For this purpose we introduced into our laboratory, methods such as denaturing gradient and temperature gradient gel electrophoresis for screening the degree of heterogeneity and total amount of HCV-IRES variability accumulated in HCV infected patients over a period of time. The selected samples showed variable migration pattern of the HCV-IRES (from all the patients) visualized in DGGE and TGGE, were sequenced and evaluated for translation efficiency using flow cytometry. In some cases, we discovered that multiple mutations, even those scattered across different domains of HCV-IRES, led to restoration of the HCV-IRES translational activity, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long- range inter- and/or intra-domain functional interactions. We established a large-scale HCV-IRES...
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Functional analysis of eIF3e subunit of human translation initiation factor 3 in living cells.
Šikrová, Darina ; Valášek, Leoš (advisor) ; Šanderová, Hana (referee)
2 Abstract Eukaryotic initiation factor 3 (eIF3) is a critical player involved in many steps of translation initiation, which ultimately result in the formation of the elongation competent 80S ribosome. With its 13 subunits (eIF3a - eIF3m) it is the largest and the most complex translation initiation factor composed of three mutually interconnected modules (i - iii), however, the role of individual subunits involved in its structural integrity and proper function is not fully explored. The eIF3e subunit was shown to be a part of the human eIF3 structural core and to help in the mRNA recruitment to the 43S pre-initiation complex by forming a molecular bridge between the 40S ribosomal subunit and the mRNA cap-binding complex. In this study, we employed siRNA-directed downregulation of eIF3e in HeLa cells and analysed its impact on the overall eIF3 integrity and function in vivo. The eIF3e knock-down (eIF3eK.D. ) led to the severe reduction of protein amounts of other three subunits (eIF3d, k and l), which together with the subunit eIF3c and e form module ii of the eIF3 complex. Remaining module i (composed of a, b, g and i) and iii (containing f, h and m) stayed partially bound perhaps thanks to a bridging effect of eIF3c, and showed reduced binding efficiency towards the 40S subunit compared to control...
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The role of translation initiation factor 3 (eIF3) in translation termination.
Beznosková, Petra ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Protein synthesis is a tightly regulated process of gene expression. Each gene has its start and its stop, which is determined by one of the three stop codons. Many recent articles describe ribosomes that purposely bypass stops on specific mRNAs to extend the nascent polypeptide to alter its properties. It is called programmed stop codon readthrough. Since over 15% of human genetic diseases are caused by so called premature termination codons (PTC) that halt translation and produce truncated proteins, this mechanism has a great potential implication in medical research. Numerous labs search for non-toxic drugs specifically increasing readthrough at PTCs; however, the success of this effort requires identification and understanding of all factors that are involved in this process. Here, we present one such factor eukaryotic initiation factor 3 (eIF3) and describe its ability to induce readthrough on stop codons in termination non-favorable context during programmed readthrough and also the consequences of its action on translation regulation. We additionally analyzed which near-cognate (nc) tRNAs are incorporated at UGA stop codons depending on the nucleotide that immediately follows them (so called +4 base). This way we established new rules for stop codon decoding and identified so called...
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RNA polymerase: The "meeting point" of regulatory networks
Wiedermannová, Jana ; Krásný, Libor (advisor) ; Pospíšek, Martin (referee) ; Valášek, Leoš (referee)
Bacterial RNA polymerase (RNAP) is a multisubunit complex essential for transcription of DNA into RNA. As a key enzyme responsible for regulation of gene expression it interprets regulatory signals from the cell and based on these cues RNAP adjusts transcription level of particular genes. This process is affected both by the regular subunits of RNAP as well as other transcription factors (TFs) directly or indirectly interacting with RNAP. The general focus of this Thesis was to extend the knowledge about the complex transcriptional regulatory networks and about the connections between individual pathways. The main specific topic and the main publication of the thesis are focused on the HelD protein, a novel binding partner of RNAP in Bacillus subtilis. We showed that HelD binds between the secondary channel of RNAP and alpha subunits of the core form of the enzyme. We proved that HelD stimulates transcription in an ATP dependent manner by enhancing transcriptional cycling and elongation. We revealed a new connection in the transcription regulatory machinery when we demonstrated that the stimulatory effect of HelD can be amplified by delta, a small subunit of RNAP specific for gram positive (G+) bacteria. Two other publications of the thesis are dealing with the delta subunit. We solved the 3D...
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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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Functional and biochemical characterization of elF3 and elF3j in Saccharomyces cerevisiae and human cell lines
Wagner, Susan ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
3 Abstract 3.1 english Translation is divided into initiation, elongation, termination and ribosome recycling. One of the largest eukaryotic initiation factors (eIF), eIF3, plays a role in nearly all steps of initiation and was recently also implicated in ribosomal recycling. Here we uncovered novel roles for eIF3 in translation termination in yeast, where the five core eIF3 subunits and their loosely associated eIF3j/HCR1 accessory factor control stop codon read-through in the opposite manner. In addition, we further characterized the function of eIF3j in initiation. Structural analysis revealed that the conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). This binding mode was shown to be conserved in yeast ensuring efficient 40S-binding by eIF3 and stringency of AUG recognition, where j/HCR1 seems to co-operate with eIF1A. We found that the N-terminal half of j/HCR1 in yeast is sufficient for fulfilling all functions of the full-length protein necessary for wild-type growth. Despite the logical dispensability of the j/HCR1 C- terminal half, it was shown that it bears a specific KERR motif that is evolutionary conserved and contained also within the HCR1-like domain of a/TIF32, through which it...
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Mapping the contact points between eukaryotic translation initiation factor eIF3 and the 40S ribosomal subunit.
Kouba, Tomáš ; Valášek, Leoš (advisor) ; Pospíšek, Martin (referee) ; Staněk, David (referee)
Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs) together with the small ribosomal subunit to locate the mRNA's translational start and to properly decode the genetic message that it carries. The largest of these factors, eIF3, forms the scaffold for other initiation factors to promote their spatially coordinated placement on the ribosomal surface. It is our long-standing pursuit to map the 40S-binding site of the yeast multisubunit eIF3 and here we present three new mutual interactions between these two macromolecules (i) The C-terminal region of the eIF3c/NIP1 subunit is comprised of the conserved bipartite PCI domain and we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 in vivo. The extreme C-terminus directly interacts with small subunit ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of ASC1 impairs eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this protein fold in protein-RNA interactions. We conclude that the c/NIP1...
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