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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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The role of pre-mRNA splicing in human hereditary diseases
Malinová, Anna ; Staněk, David (advisor) ; Vanáčová, Štěpánka (referee) ; Krásný, Libor (referee)
U5 small ribonucleoprotein particle (U5 snRNP) is a crucial component of the spliceosome, the complex responsible for pre-mRNA splicing. Despite the importance of U5 snRNP, not much is known about its biogenesis. When we depleted one of the core U5 components, protein PRPF8, the other U5-specific proteins do not associate with U5 snRNA and the incomplete U5 was accumulated in nuclear structures known as Cajal bodies. To further clarify the role of PRPF8 in U5 snRNP assembly, we studied PRPF8 mutations that cause an autosomal dominant retinal disorder, retinitis pigmentosa (RP). We prepared eight different PRPF8 variants carrying RP-associated mutations and expressed them stably in human cell culture. We showed that most mutations interfere with the assembly of snRNPs which consequently leads to reduced efficiency of splicing. The mutant PRPF8 together with EFTUD2 are stalled in the cytoplasm in a form of U5 snRNP assembly intermediate. Strikingly, we identified several chaperons including the HSP90/R2TP complex and ZNHIT2 as new PRPF8's interactors and potential U5 snRNP assembly factors. Our results further imply that these chaperons preferentially bind the unassembled U5 complexes and that HSP90 is required for stability of...
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Stress proteins in the cytoplasmic membrane fraction of Bacillus subtilis
Šemberová, Lenka ; Svobodová, Jaroslava (advisor) ; Krásný, Libor (referee)
Stress proteins in the cytoplasmic membrane fraction of Bacillus subtilis A primary habitat of the Gram-positive bacterium Bacillus subtilis is the upper layer of the soil. Within this ecosystem, B. subtilis experiences a wide variety of environmental challenges and nutrient limitations that induce several mechanisms to help the cells to survive. A process known as a general stress response belongs among them. Expression of about 150 genes is enhanced and their products called general stress proteins (GSP) minimize the cell damage. Although the size and structure, as well as the regulation, of many stress proteins have been fairly well elucidated the information about stress membrane proteins is very limited. We characterized the membrane proteome of Bacillus subtilis 168 trp2- exposed to acidic pH and ethanol. Cells were 1) grown under optimum conditions in complex medium (pH 7.0) at 40řC with aeration; 2) grown in complex medium at pH 5.0 for 20 hours or 3) challenged with 3% v/v ethanol for 30 minutes during exponential growth. The cultures were harvested in the mid-exponential phase by rapid filtration. Isolated membrane fractions were analysed by an optimized two-dimensional gel electrophoresis. Two alternative methods of protein detection were compared: silver staining and radioactive labeling with...
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Transcription factors CSL and their role in the yeast Schizosaccharomyces pombe
Oravcová, Martina ; Převorovský, Martin (advisor) ; Heidingsfeld, Olga (referee) ; Krásný, Libor (referee)
Proteins of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) act as effectors of the Notch signalling pathway in metazoan organisms. They function as repressors or activators of gene transcription in the framework of this pathway and influence many developmental processes. Metazoan CSL proteins can regulate gene expression Notch-independently as well. Notch-independent functions of CSL proteins might be evolutionarily ancestral and in cells and organisms may be important equally as Notch-dependent functions. Presence of CSL proteins was identified in several fungal species, organisms lacking the Notch signalling pathway components and most of known metazoan interacting partners of CSL proteins. CSL paralogs of the fission yeast Schizosaccharomyces pombe, cbf11 and cbf12, are non-essential genes encoding proteins localized in the nucleus of the cell. They exert antagonistic effects on regulation of processes like coordination of nuclear and cellular division and cell cycle progression, ploidy maintenance, cell adhesion and other. In this study, we have proved that both CSL paralogs are able to sequence-specifically bind the CSL-response element DNA in vitro and Cbf11 in vivo as well. Both proteins could activate gene expression in vivo and perform the function of transcription factors....
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Use of transcriptomics to study mechanism of the action of complex mixtures of organic compounds occurring in the ambient air focusing on polycyclic aromatic hydrocarbons
Líbalová, Helena ; Topinka, Jan (advisor) ; Krásný, Libor (referee) ; Postlerová, Pavla (referee)
Polycyclic aromatic hydrocarbons (PAH) represent a large group of organic compounds occuring as pollutants in ambient air. Besides their genotoxic effect, some of them are known to be complete carcinogens and act via nongenotoxic and tumor promoting mechanism. Although effects of many individual compounds are well-documented, human exposure to polycyclic aromatic hydrocarbons in ambient air occurs through complex mixtures and only few studies describe the behavior of PAH in real complex mixtures. The first part of the thesis is dealing with the global gene expression changes in human embryonic lung fibroblasts (HEL) as a consequence of the effect of complex mixtures containing PAH extracted from the respirable airborne particles PM2.5. These particles were collected in 4 localities in the Czech republic (Ostrava - Bartovice, Ostrava - Poruba, Karviná, Třeboň) differing in the level of the air pollution. Gene expression changes induced by three subtoxic concentrations of organic extracts (EOM - extractable organic matter) from each locality after 24 hour incubation were examined by microarray analysis. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to interpret gene expression data. In each locality we identified several deregulated signaling pathways...
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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Effect of knock out of yxkO gene on environmental stress adaptation in genus Bacillus
Tkadlec, Jan ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...
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Factors interacting with bacterial RNA polymerase
Sudzinová, Petra ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
The bacterial cell must be able to rapidly change its gene expression to survive unstable external conditions. Transcription is the key level that affects gene expression. The pivotal enzyme of transcription is RNA polymerase (RNAP). Activity of RNAP is tightly regulated by transcription factors (TFs). These factors affect RNAP in different ways. This work presents an overview of various proteins and others factors, description of their effects on transcription and also mechanisms of their actions. TFs could be divided according to various criteria. In this work, TFs are divided according to how they interact with RNAP: TFs interacting only with RNAP; TFs binding simultaneously DNA and RNAP; TFs interacting with RNA and RNAP. This work presents a comprehensive overview of various TFs that are involved in the bacterial cell's reprogramming of gene expression that is required to withstand the changes in the environment.
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Factors affecting gene expression in Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Vohradský, Jiří (referee)
Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...
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Molecular mechanism of quality control during snRNP biogenesis
Klimešová, Klára ; Staněk, David (advisor) ; Krásný, Libor (referee) ; Vomastek, Tomáš (referee)
The spliceosome is one of the largest and most dynamic molecular machines in the cell. The central part of the complex is formed by five small nuclear ribonucleoproteins (snRNPs) which are generated in a multi-step biogenesis pathway. Moreover, the snRNPs undergo extensive rearrangements during the splicing and require reassembly after every intron removal. Both de novo assembly and post-splicing recycling of snRNPs are guided and facilitated by specific chaperones. Here, I reveal molecular details of function of two snRNP chaperones, SART3 and TSSC4. While TSSC4 is a previously uncharacterized protein, SART3 has been described before as a U6 snRNP-specific factor which assists in association of U6 and U4 particles into di-snRNP, and is important for the U4/U6 snRNP recycling. However, the mechanism of its function has been unclear. Here, I provide an evidence that SART3 interacts with a post-splicing complex and propose that SART3 could promote its disassembly. Our data further suggest that SART3 binds U6 snRNP already within the post-splicing complex and thus participates in the whole recycling phase of U6 snRNP. Then, I show that TSSC4 is a novel U5 snRNP-specific chaperone which promotes an assembly of U5 and U4/U6 snRNPs into a splicing-competent tri-snRNP particle. We identified...
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