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The role of elF3 a Rps3 in stop codon readthrough
Poncová, Kristýna ; Valášek, Leoš (advisor) ; Vopálenský, Václav (referee) ; Krásný, Libor (referee)
Translation represents a highly regulated, interconnected process of protein synthesis in the cell. It could be divided into 4 phases: initiation, elongation, termination, and ribosomal recycling. Our laboratory is involved in in-depth studies of a complex eukaryotic initiation factor 3 protein (eIF3). We are interested not only in revealing its molecular roles in the translational cycle in general but also in specific mechanisms that allow translational regulation according to specific cellular needs. In the budding yeast, the eIF3 is composed of five essential subunits (a/Tif32, b/Prt1, c/Nip1, g/Tif35 and i/Tif34). In mammals, the protein is even more complex, comprising of 12 subunits (a-i, k-m). eIF3 is a key player not only in translation initiation but also in ribosomal recycling and, surprisingly, in translation termination and stop codon readthrough as well. The latter process harbors important clinical potential, as approximately 1/3 of genetically inherited diseases is caused by the presence of a premature termination codon in the protein-coding region. Therefore, understanding the molecular mechanism underlying this phenomenon provides important tools for the targeted and less toxic drug development approaches needed for patient therapy. In this Ph.D. Thesis, I uncovered the role of...
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Role of small ribosomal proteins forming the decoding site in translation.
Hovorková, Zuzana ; Valášek, Leoš (advisor) ; Hálová, Martina (referee)
Translation is one of the key mechanisms occurring in the cell during every second of its existence. It is a very complex process ensured by three main actors: tRNAs, mRNAs and ribosomes. Despite of being thoroughly studied over decades, the understanding of some of its functional aspects is still rather poor. This bachelor thesis focuses on four small ribosomal proteins listed below that are reaching to the decoding centre of the small ribosomal subunit. It raises awareness of the structure and function of uS12, uS19, eS25 and eS30, their evolution, role within the ribosome, and the influence they have on various stages of translation. In particular, this thesis specifically reviews the importance of these four proteins for the stop codon readthrough. This phenomenon occurs when a near-cognate aminoacyl-tRNA or a natural suppressor tRNA wins with eRF1 over the corresponding stop codon and thus protein synthesis is continued resulting in the existence of a longer protein. It summarizes our current knowledge of its origin, molecular details of its mechanism, its existence in different species, benefits and disadvantages it brings to the life of a cell or even an organism, and finally it sums up all available knowledge for potential future use of readthrough in therapeutics. Key words: translation,...
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The function of ABCF proteins in bacteria
Mičke, Bianka ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
Translation belongs to the most basic processes which happens in the living cells. It is the last step of proteosynthesis when genetic information encoded by the mRNA is transformed into the protein on a ribosome. Organisms have developed a wide range of mechanisms that can regulate it's needs. I focused on one of them - ABCF proteins. This protein group is a member of the ABC transporters superfamily but they haven't a transmembrane domain and their purpose is protect the ribosomes from antibiotics that bind 50S ribosomal subunit or interact with the ribosomes and influence ribosomal functions. Today, we can divide ABCF proteins into the two functional groups: antibiotic resistence proteins (ARE) and proteins with the regulatory functions. The translational regulatory function has been confirmed There is 45 ABCF protein subfamilies spread through the bacteries and eukaryotes but many essential informations like the structure and exact function of them are still missing. My bachelor thesis is analysis and summary of facts that are known about the bacterial ABCF proteins. Key words: ABCF proteins, antibiotic resistence, ARE, translational regulation, ribosome, translation, translational factors
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Regulation of translation in mammalian oocytes and early embryos
Jindrová, Anna ; Šušor, Andrej (advisor) ; Flemr, Matyáš (referee) ; Fulková, Helena (referee)
Fully grown oocytes undergo their further development in the absence of transcription. Completion of meiosis and early embryo development rely on the maternal mRNAs synthetized and stored during earlier development. Thus, the regulation of gene expression in oocytes during that period is controlled almost exclusively at the level of mRNA stabilization and translation. In the same vein, any mRNA metabolism could play a critical function at this stage of development. RNA localization followed by a local translation is a mechanism responsible for the control of spatial and temporal gene expression in the cell. We focused on visualization of mRNA and in situ translation in the mammalian oogenesis and embryogenesis. We characterized localization of global RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. Additionally we visualized specific ribosomal proteins that contribute to translation in the oocyte and embryo. We have shown that the key player of cap-dependent translation mTOR becomes highly active post nuclear envelope breakdown (NEBD) and in turn its substrate, translational repressor 4E-BP1 becomes inactive. Precise localization of inactivated 4E-BP1 at the newly forming spindle of the oocyte indicates the ongoing translation in this area. Furthermore, from...
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Characterization of the ABC-F protein Sco0636 in Streptomyces coelicolor
Pinďáková, Nikola ; Balíková Novotná, Gabriela (advisor) ; Mikušová, Gabriela (referee)
The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...
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3D scene reconstruction using Clifford algebras
Hrubý, Jan ; Návrat, Aleš (referee) ; Hrdina, Jaroslav (advisor)
Tato diplomová práce má za cíl seznámit čtenáře se stále ještě relativně novou a neznámou oblastí matematiky, s geometrickou algebrou. Nejdříve jsou uvedeny základní definice a poté jsou studovány vlastnosti obecné geometrické algebry. Další velká část textu se věnuje Konformní geometrické algebře, která je v současnosti jedna z nejvíce zkoumaných a aplikovaných geometrických algeber. Jsou popsány její algebraické a geometrické vlastnosti, konkrétně schopnost reprezentovat určité geometrické objekty jako vektory. Taktéž umožňuje počítat jejich průniky a konformní transformace. Další část textu je zaměřena na aplikace Konformní geometrické algebry, nejdříve k popisu kinematiky robotické ruky a poté v binokulárním viděni.
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Inhibitors of the eukaryotic translation initiation factor eIF4E1
Pospíšilová, Klára ; Pospíšek, Martin (advisor) ; Roithová, Adriana (referee)
Protein synthesis is one of the most important processes that take place in a cell. Thus there are various mechanisms in a cell that regulate it. If that regulation fails it may lead to serious pathologies. An example of this is an abnormal increase in the production of eukaryotic translation initiation factor 4E1 which occurs in some types of cancer including head and neck squamous cell carcinoma, colorectal cancer, cervical cancer or lung cancer. Enhanced availability of factor 4E1 enables transformed cells to undertake a more intense translation. The expression of individual proteins is not increased to the same extent though. The enhancement in the level of factor 4E1 has a more significant effect on oncogenic proteins. Malignant transformation caused by an incorrect regulation of factor 4E1 can be precluded by an application of compounds that impair the activity of factor 4E1. A summarization of 4E1 inhibitors is the subject of this work.
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Visualization of RNA and its translation in mammalian oocyte
Bašus, Kryštof ; Tětková, Anna (advisor) ; Macůrková, Marie (referee)
Translation is one of the fundamental biological processes. It is assumed that the locali- zation of mRNA and its translation in a certain time and at specific locations plays a role in many cellular processes. The mammalian oocyte becomes transcriptionally inactive when it reaches its full size and utilizes only RNA that has been synthesized and stored in the early stages of development. Thus the regulation of protein synthesis at the translational level is cri- tical for the correct completion of meiotic maturation of oocytes and early development of embryos. To monitor cellular physiology, it is necessary to be able to visualize and monitor spe- cific molecules and processes at single cell level. This is enabled by the development of light microscopy and fluorescence probes that specifically bind to certain organelles, cellular struc- tures, proteins or other molecules. In this thesis I describe selected methods of visualization of RNA, global translation as well as translation of specific transcripts, and proteins. The methods that this thesis describes are RNA FISH, visualization of translation using methionin analogs, FUNCAT, SUnSET, FlAsH, ReAsH, TRICK, SINAPS, FUNCAT-PLA, PURO-PLA. Key words: Translation, RNA, Protein, Visualization, Oocyte
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Eukaryotic translation initiation factor 3 and its role in plant translation regulation
Raabe, Karel ; Michailidis, Christos (advisor) ; Retzer, Katarzyna (referee)
After transcription, mRNA translation is another highly regulated process in gene expression. In plants, translation regulation plays an important role during progamic phase, fertilization and seed development, where synthesized transcripts are stored and selectively translated later in development. Translation regulation is also broadly used in stress responses as a fast and flexible tool to change gene expression; therefore, it plays an essential role in the survival strategy of sessile organisms like plants. Both regulation of the global translational rate as well as selective regulation of specific transcripts modulate the final gene expression response. Most of the regulatory mechanisms are concentrated in the stage of initiation, which is facilitated by several translation initiation factors. Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors, consisting of 12 conserved subunits. Its key function in the initiation is to scaffold the formation of the translation initiation complex and in the scanning mechanism accuracy. In past decades, additional eIF3 functions were discovered acting upon the whole translation cycle, including its importance in global and specific translation regulation. The aim of this work is to review eIF3 functions and to...
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Characterization of eukaryotic translation initiation factor 3 subunits (eIF3) in A. thaliana male gametophyte
Linhart, Filip ; Hafidh, Said (advisor) ; Retzer, Katarzyna (referee)
From RNA-to-protein, translation initiation and protein synthesis is mediated by trans-acting factors that recognize mRNA features common to almost all eukaryotes. Eukaryotic translation initiation factor 3 complex (eIF3) is a highly conserved protein complex that recognizes 5'-CAP elements of the mRNA to initiate translation. eIF3 consists of nine subunits, three of them having two isoforms: eIF3A, eIF2B1, eIF3B2, eIF3C1, eIF3C2, eIF3D, eIF3E, eIF3F, eIF3G1, eIF3G2, eIF3H and eIF3K. This work deals with functional characterization, expression and subcellular localization of eIF3B1, eIF3B2 and eIF3E in Arabidopsis thaliana male gametophyte and interaction of eIF3E with the Constitutive photomorphogenesis 9 (COP9) complex as a regulatory complex of eIF3E post-translational control. Here we show that depletion of eif3b1 or eif3b2 is not gametophytic lethal and that the two protein might function redundantly, whereas, knockout of eIF3E causes male gametophyte lethality. Interestingly, eif3b1 show post-fertilization defects during embryogenesis, suggesting that its redundancy with eIF3B2 is restricted to the gametophyte. Gene expression studies revealed high expression of eIF3 subunits in actively dividing zones of leaf primordia, root meristem and root elongation zones as well as in the vegetative...
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