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delta subunit of bacterial RNA pol and its role in regulation of gene expression in B. subtilis
Dvořáček, Lukáš ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee)
Delta subunit of bacterial RNA pol and its role in regulation of gene expression in B. subtilis. In this work I focus on regulation of eubacterial gene expression. First, I describe recent knowledge about a key stage of gene expression - transcription, focusing on regulation of trancription iniciation via small effector molecules (guanosine tetraphosphate, initiating nucleoside triphosphate) that are important for the regulation of ribosomal RNA. Second, in the experimental part of my work, I focus on the role of the _ protein, a subunit of RNA polymarase in gram positive bacteria, in transcription iniciation and its effects on regulation of RNA polymerase by the concentration of initiating nucleoside triphosphates.
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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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Role of Smarca5 (Snf2h) during transcription of transfected DNA template.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Smetana, Karel (referee)
Cellular and tissue characteristics are results of dynamic regulation of gene expression. DNA wrapped into proteins, referred to as chromatin, requires involvement of mechanisms guiding accessibility of specific sequences. In higher organisms, chromatin remodeling proteins are indispensable in regulating chromatin structure including ISWI ATPase SMARCA5. SMARCA5 is involved in almost any transaction on DNA including transcription, however precise in vivo role of SMARCA5 in these processes remains unknown. To advance understanding of specific role of SMARCA5 in the development of chromatin structure during transcription we devised cellular model in which SMARAC5 level is manipulated while chromatin structure development and transcriptional response are monitored. Our data indicate that the transfected DNA template that is transcribed is enriched with histone H3 and its specific methylation of Histone H3 lysine (K) 4, a mark of active chromatin structure. Overexpression of SMARCA5 results within the reporter gene coding sequence in ~2,5-3 fold increase of both H3 occupancy an its modification H3K4Me3. Increased DNA template commitment into chromatinization is associated with repression of reporter gene expression. These results are supported by studies indicating dynamic development of nucleosomal...
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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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Molecular mechanisms of interactions- interactions of constitutive androstane receptor with selected stilbene compounds
Linhartová, Lenka ; Pávek, Petr (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lenka Linhartová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Molecular mechanisms of intractions - interactions of constitutive androstane receptor with selected stilbene compounds Constitutive androstane receptor (CAR), member of nuclear receptors family, is a major regulator of gene expression of phase I and II enzymes metabolizing endobiotics and xenobiotics. Changes in its activity can lead to pharmacokinetic drug interactions, ineffective treatment or higher toxicity of drugs simultaneously administered with CAR ligands. Recently another effects of this receptor, especially in homeostasis of bile acids, lipids and glucose have been discovered and CAR is now considered as a potential drug target for the treatment of metabolic diseases. Stilbenes represent a small group of plant polyphenols with typical 1,2-diphenylethylene nucleus. The most famous member is resveratrol, which has attracted great attention thanks to its antioxidant, anti-inflammatory, antiproliferative and cardioprotective effects. Others stilbene compounds such as pterostilben, piceatannol or pinosylvin have shown similar health beneficial effects as well. The aim of this diploma thesis was...
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Structural studies of transcription factors implicated in regulation of metabolism of pancreatic beta cells
Duchoslav, Vojtěch ; Maloy Řezáčová, Pavlína (advisor) ; Vaněk, Ondřej (referee)
Nkx6.1 is a homeodomain protein (37.8 kDa) and an important transcription factor, which regulates transcription of key genes in pancreatic ß-cells. Insufficient expression of this protein leads to reduced glucose uptake from blood as a consequence of suppressed transcription of the glucose transporter Glut2 and impaired glucose metabolism. Furthermore, the proliferation of pancreatic ß-cells is suppressed due to insufficient transcription of Cyclin D2, a protein regulating the mitosis. Moreover, the biosynthesis of insulin is impaired duet he diminished transcription of the genes coding for Ero1lb a Slc30a8, which as a consequence leads to reduced production of the mature insulin. Nkx6.1 could play a role in the pathogenesis of the type 2 diabetes , where ß-cells show diminished ability to compensate high demand for insulin. This malfunction is the cause of an insufficient ability to secrete insulin and death of pancreatic cells. Perhaps driven by misregulation of transcription of the genes that are involved in the mentioned processes. Nkx6.1 recognizes a strictly conserved 8-base pair DNA sequence (TTAATTAC). Its binding to DNA is regulated by an acidic domain at the C-terminus. Within the bachelor thesis, the resonances were assigned to the backbone atoms of the Nkx6.1 protein using nuclear...
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Localization matters: function of paxillin and phopholipids in the cell nucleus
Marášek, Pavel ; Hozák, Pavel (advisor) ; Půta, František (referee) ; Žárský, Viktor (referee)
(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...
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Human endogenous retrovirus ERVWE1: transcriptional activation and modifications of promoter DNA methylation
Dobšová, Martina ; Trejbalová, Kateřina (advisor) ; Španielová, Hana (referee)
Endogenous retrovirus ERVWE1 is an integral part of the human genome. In the course of evolution, a protein encoded by the env gene of this retrovirus - Syncytin-1 - has gained unique function in human development. It mediates cell-to-cell fusion of placental cytotrophoblasts. Receptor that binds to Syncytin-1 is expressed in different cell types. Syncytin-1-mediated fusion is essential in placenta, but it can cause disruption of tissue integrity in other cell types. ERVWE1 expression is regulated by promoter DNA methylation, transcription factor GCM1 and efficient mRNA splicing. This thesis concerns the ERVWE1 expression and its regulation in non-placental tissues. It was found out that the moderate GCM1 overexpression was not sufficient to induce Syncytin-1 expression. Neither treatment with DNA demethylation agent 5-azacytidine nor with Syncytin-1 activator forskolin was able to manage Syncytin-1 expression. This thesis extends previous findings concerning high syncytin-1 expression in seminomas. In same tissues, there was found elevated TET1 expression on mRNA level in comparison with controls. The presence of the TET1 demethylation enzyme can influence ERVWE1 promoter DNA methylation. Previously unreported splicing variant of TET1 has been found during the construction of human TET1 expression...
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