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Searching for interaction partners associated with the transcriptional apparatus of yeast linear plasmids
Ľalíková, Kristýna ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
The pGKL plasmids are a type of yeast linear double-stranded DNA plasmids found in the cytoplasm of yeast Kluyveromyces lactis. The plasmid gene transcription process involves the plasmid transcription apparatus, which shows similarity to the transcription apparatus of poxviruses. Poxviruses cause a range of serious diseases. Thus pGKL plasmids offer a safe way to study some of the mechanisms of these viruses without the risk of infection. The plasmid transcription apparatus comprises non-canonical RNA polymerase, helicase, and capping enzyme as its main components. Although RNA polymerase has been thoroughly characterized and described, little focus has been on the interactions between proteins within the transcription apparatus. Therefore, this study aims to shed light on this fundamental aspect of the gene expression of pGKL plasmids. The main goal of the thesis was to prepare a system that would facilitate the search for interaction partners of the pGKL transcription apparatus and verify the interactions between chosen proteins in yeast cells. The foundation of this system relies on the use of optimized genes and the yeast two-hybrid system method. The development of the experimental method has begun successfully, and it's expected to conclude soon, although further optimizations are still...
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Where transcription meets translation
Hegrová, Karolína ; Krásný, Libor (advisor) ; Mašek, Tomáš (referee)
Transcription and translation are key steps in gene expression. The RNA polymerase (RNAP) plays a major role in the transcription process, while the ribosome is involved in translation. In bacteria, these two processes are not separated. RNAP and the ribosome interact, and its called transcription- translation coupling. In this thesis, I discuss the mechanism of transcription and translation, with the main focus on transcription-translation interactions. I divide these interactions into indirect, which are caused by regulátory molecules, and direct, where the ribosome directly binds with RNAP. When physical binding occurs, either a tight junction between these molecules occurs or a bridge is formed by transcription factors. Then I describe regulatory function of this connection and explain the exceptions where transcription and translation don't link. In the last part of the thesis, I focus on elongation factor Tu (EF-Tu), its important role in metabolism, its interactions with MreB protein, and how this factor is used by some bacteriophages. Finally, I mention its possible role in transcription-translation interactions. Key words: transcription, translation, transcription-translation coupling, RNA polymerase, ribosome, transcription factors, EF-Tu
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Non-template activities of DNA/RNA polymerases and their significance
Pokorná, Kristýna ; Vopálenský, Václav (advisor) ; Pospíšil, Jiří (referee)
Non-templated addition of adenosine on the 3' end of the product of several DNA polymerases commonly used in molecular biology has been a problematic yet unexplained phenomenon for a few decades now. The only other instance where adenosine is added to the 3' end of an information molecule in a cell is polyadenylation. At the same time, several RNA viruses appear to perform similar action to either edit or polyadenylate their mRNAs. This work focuses on the details of these three highlighted processes and relationship between them and has found out that all of them appear to stem from a common conserved property of the ancestral polymerase. Key words: DNA polymerase, RNA polymerase, non-templated addition, Taq polymerase, polyadenylation
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Structural characterization of influenza A polymerase PA subunit domains in complex with novel inhibitors
Radilová, Kateřina ; Kožíšek, Milan (advisor) ; Rumlová, Michaela (referee) ; Obšil, Tomáš (referee)
Influenza RNA-dependent RNA polymerase is a heterotrimeric complex and has an essential role in the life cycle of the virus. It is responsible for viral replication and transcription. One of its subunits, the polymerase acidic protein, interacts with the PB1 subunit via a crucial protein- protein interaction at its C-terminal domain. This 310 helix-mediated intersubunit interaction is required for the whole heterotrimer assembly. The N-terminal domain carries the endonuclease active site with two manganese ions. Both domains are considered promising drug targets. Current strategies to fight the influenza virus are limited to seasonal vaccines, and there are only a few anti-influenza drugs targeting mostly other viral proteins. Many used antivirals are susceptible to rapid resistance mutations development or cause severe side effects. This thesis provides structural insights into the two domains of the PA subunit. The first part is devoted to the characterization and optimization of a PB1-derived minimal peptide interacting with the C-terminal domain. Results from this part may be considered as a starting point for the rational design of first-in-class anti-influenza inhibitors of the PA-PB1 protein-protein interaction. In the other half, we have explored the inhibitory potency of flavonoids and...
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The omega subunit of Bacillus subtilis RNA polymerase.
Mikesková, Klára ; Krásný, Libor (advisor) ; Nešvera, Jan (referee)
Transcription is catalysed by the enzyme RNA polymerase (RNAP). RNAP contains a core made up of two α subunits, one of each β, β'and ω. These subunits are conserved in all bacteria. The ω subunit is a small subunit with a molecular weight of 7.6 kDa that binds β'. ω is important for the folding and integrity of RNAP and promoter selection. This was shown by experiments performed with Gram-negative bacteria but the knowledge about in Gram-positive bacteria is minimal. In my Diploma Thesis, I characterized from the model Gram-positive bacterium from the phylum Firmicutes, Bacillus subtilis. First, I prepared various expression strains for isolation of Bacillus subtilis ω. Then, I successfully isolated the ω subunit, which was the main initial aim of this Diploma Thesis. Subsequently, I tested the influence of the ω subunit on in vitro transcription by RNAP associated with the primary σA factor and alternative σF and σE factors that regulate sporulation in Bacillus subtilis. I also evaluated the effect of , a small RNAP subunit found in Firmicutes, both alone and in combination with . The experiments revealed that ω stimulated transcription both from vegetative promoters and sporulation-related promoters. Moreover, this stimulation was synergistically amplified by the δ subunit. This nicely...
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The delta subunit of RNA polymerase from gram positive bacteria
Matějčková, Jitka ; Krásný, Libor (advisor) ; Beranová, Jana (referee)
1 Abstrakt Aby bakteriální buňka přežila neustále se měnící podmínky, musí se na ně adaptovat. Tato adaptace je podmíněna změnou genové exprese. Klíčovým krokem genové exprese je transkripce. Hlavním enzymem bakteriální transkripce je RNA polymerasa (RNAP), což je esenciální vícepodjednotkový enzym. RNAP je nejvíce prostudována u Escherichia coli, modelového organismu gram negativních bakterií. Porovnala jsem E. coli a Bacillus subtilis (zástupce gram pozitivních bakterií) a shrnula jsem rozdíly v RNAP a transkripci. Jejich RNA polymerasy se liší přítomností podjednotky δ u gram pozitivních bakterií. Tato podjednotka zvyšuje promotorovou selektivitu, recykluje jádro RNAP a celkově stimuluje syntézu RNA. Podjednotka δ ovlivňuje sporulaci a virulenci některých bakterií. V této práci jsem shromáždila současné poznatky o jednotlivých částech genové exprese, zejména o regulaci iniciace transkripce a o podjednotce δ RNAP.
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Factors interacting with bacterial RNA polymerase and their effect on the regulation of transcription initiation
Ramaniuk, Volha ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Valášek, Leoš (referee)
(ENGLISH) The bacterial cell needs to regulate its gene expression in response to changing environmental conditions. RNA polymerase (RNAP) is the pivotal enzyme of this process and its activity is controlled by a number of auxiliary factors. Here I focus on RNAP-associating factors involved in regulation of transcription in G+ bacteria: factors, initiating nucleoside triphosphates (iNTPs), HelD, δ and small RNA Ms1. The main emphasis is on σ factors from Bacillus subtilis. σ factors allow RNAP to specifically recognize promoter DNA. In my first project I set up in vitro transcription systems with purified alternative σ factors, σB , σD , σH , σI from B. subtilis. Using these systems, I studied the effect of initiating NTP concentration ([iNTP]) on transcription initiation. I showed that promoters of alternative factors are often regulated by [iNTP]. In the next project I comprehensively characterized one of the least explored alternative factors from B. subtilis, I . I identified ~130 genes affected by I , though only 16 of them were directly affected. Moreover, I discovered that I is involved in iron metabolism. Finally, I showed that I binding requires not only the conserved -35 and -10 hexamers, but also extended -35 and -10 elements located in the spacer region. In collaboration with...
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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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