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Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Macíčková Cahová, Hana (referee) ; Valášek, Leoš (referee)
Transcription is the control point of gene expression. This process relies on protein complex of multisubunit RNA polymerases, which are extremely conserved among all cellular organisms. Transciption of extrachromosomal hereditary elements such as organelles, viruses and plasmids is dependent on host cellular RNA polymerases or intrinsic RNA polymerase is contained within these elements. Putative non-canonical two-subunit RNA polymerase is also encoded by linear cytoplasmic plasmids of the yeast Kluyveromyces lactis and most likely transcribes genes of these plasmids. Besides the two subunits of RNA polymerase encoded by linear plasmids of Kluyveromyces lactis there are another two estimated components of the transcription apparatus, namely capping enzyme that adds the cap to 5' mRNA ends and putative DExD/H box helicase. Characterization of the unique and underexplored transcription machinery of Kluyveromyces lactis plasmids was the principal objective of this work. The main goal was to: 1) clarify evolutionary origin of the linear plasmid transcription apparatus; 2) describe architecture of the linear plasmid transcription complex in vivo focused on putative RNA polymerase binding partners; 3) reveal mechanisms of transcription initiation and termination of the yeast linear plasmids. The main...
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Role of RNA helicases in antiviral defense
Krbušek, David ; Vopálenský, Václav (advisor) ; Janovec, Václav (referee)
Hepatitis C virus is an important human pathogen against which there is no immunization yet. This virus is detected by the immune system of the eukaryotic host cell by pattern recognition receptors of the RLR receptor family, which is part of the innate immune system. These RLR receptors detect the presence of hepatitis C virus and initiate a signaling cascade triggering an antiviral immune response. In this thesis, the role of cytoplasmic PRRs involved in antiviral defense during hepatitis C virus infection of eukaryotic cells has been described and determined. Key words Helicase, RIG-I, MDA5, pattern-recognition receptors, HCV, virus
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Interaction of nucleic acids with RNA polymerase
Janoušková, Martina ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Knejzlík, Zdeněk (referee)
Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...
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Bacterial RNA polymerase and molecules affecting its function
Jirát Matějčková, Jitka ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Staněk, David (referee)
RNA polymerase (RNAP) transcribes DNA into RNA and is the only transcriptional enzyme in bacteria. This key enzyme responds to external and internal signals from the cell, resolves the intensity of transcription of individual genes and thus regulates gene expression. RNAP is not only affected by its own subunits, but also protein factors, small molecules or small RNAs (sRNAs). The aim of this Thesis was to contribute to the understanding of the regulation of the RNAP and to add missing fragments to this broad topic. The first part of this Thesis is focused on the influence of selected proteins (δ, YdeB, GreA) on the sensitivity of RNAP to the concentration of the initiating nucleoside triphosphate ([iNTP]) during transcription initiation in Bacillus subtilis. We showed that δ affects the sensitivity of RNAP to [iNTP] at [iNTP]-sensitive promoters, but not at [iNTP]-insensitive promoters neither in vitro nor in vivo. The δ subunit is essential for cell survival during competition with other strains, because it enables the cell to react immediately to changing conditions. Further we showed that YdeB protein does not bind to RNAP in B. subtilis, and has not shown any effect on transcription in vitro. We found that both, GreA and YdeB proteins (unlike δ subunit) were unable to affect RNAP by [iNTP] at...
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Molecular interactions of Staphylococcus aureus with the host
Cabrnochová, Marie ; Melter, Oto (advisor) ; Vopálenský, Václav (referee)
The aim of this thesis is to summarize S. aureus interactions with selected mechanisms of innate host immunity especially interactions with neutrophils and processes on the cell level which lead to host colonization. S. aureus surface proteins MSCRAMM interact with host cell surface proteins such as fibrinogen, keratin and thereby mediate adhesion to the host cell, which is an essential point for colonization of the host cell. The central mechanism of innate immunity against any S. aureus infection is the interaction of the pathogen with neutrophils, which produce neutrophil extracellular traps and phagocytes S. aureus cells. A crucial role in the elimination of bacterial cells in the phagosome of neutrophils is lysis by the antimicrobial peptides and degradation of bacterial biomolecules by the oxygen radicals. S. aureus defence mechanisms against action of immune system are considered to be virulence factors, due to its contribution to the establishment of the infection. These mechanisms are based on cell wall modification, inhibition of neutrophil chemotaxis, and production of enzymes that inhibit the effect of antimicrobial peptides, lysozyme, oxygen and nitrogen radicals. Expression of virulence factors of a particular S. aureus strain and host-specific risk factors can lead through successful...
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Inducible promoters and their use in yeast cell manipulation
Přibáňová, Gabriela ; Palková, Zdena (advisor) ; Vopálenský, Václav (referee)
Promoters which can be regulated by different chemical or physical factors are often used in cell manipulations. This thesis focuses predominantly on promoter systems which use light as an inductor. There are two main approaches to controlling a promoter by light. The first one uses so-called "caged molecules", chemical inducers whose inducing activity is "masked" by a photolabile protecting group. The second approach includes optogenetic systems, which can regulate transcription in cells. These systems are encoded in the DNA of the organism, and light is the only external regulatory stimulus. Photoreceptors that need a specific cofactor (chromophore) are the main components of optogenetic systems. There are several groups of photoreceptors classified by the type of chromophore and photoactivation mechanism. This thesis gives an overview of optogenetic systems used for transcription regulation and focuses on different photoreceptors and induction mechanism used. The systems using photocaged molecules are described as well. Furthermore, the thesis deals with light- systems in yeast as a model organism as well as organism used for biotechnological purposes. Finally, some limitations of light inducible promoters are discussed, including the chromophore type, the wavelength of the light, and the...
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Importance of 5'-end structures of eukaryotic mRNA molecules
Vopálenský, Václav ; Pospíšek, Martin (advisor) ; Lukeš, Julius (referee) ; Staněk, David (referee)
73 V. SHRNUTÍ VÝSLEDKŮ IRESITE: THE DATABASE OF EXPERIMENTALLY VERIFIED IRES STRUCTURES (WWW.IRESITE.ORG) IRESite je kurátorovaná relační databáze typu MySQL-4.1 využívající InnoDB tabulky. Do databáze je možné vložit položky dvojího typu: o natural položky obsahují veškerá experimentální data týkající se určité IRES struktury (kompletní sekvence mRNA s vymezením pozic IRES sekvence a kódovaného proteinu, veškeré proteiny interagující s IRES, případná sekundární struktura) o engineered položky popisují uměle vytvořené plazmidy. Většinou se jedná o bicistronní plazmidy sloužící k testování funkčnosti sekvence, o níž se předpokládá, že obsahuje IRES element, a případné mutantní deriváty této sekvence. Do IRESite se v tomto případě vkládají údaje o sekvenci mRNA s vymezením IRES oblasti, informace o reportérových proteinech, informace o translačních experimentech včetně použitého promotoru a informace o pozitivních a negativních kontrolách. V databázi je nyní uloženo 288 položek; 67 natural (20 virových a 47 buněčných) a 221 engineered. IRESite slouží nejen k ukládání experimentálních dat, ale umožňuje také jejich prohledávání podle klíčových slov, případně následné porovnávání vybraných experimentů. A BIOINFORMATICAL APPROACH TO THE ANALYSIS OF VIRAL AND CELLULAR INTERNAL RIBOSOME ENTRY Publikace...
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