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Factors interacting with bacterial RNA polymerase and their effect on the regulation of transcription initiation
Ramaniuk, Volha
(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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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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The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid- producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
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Effect of promoter sequence on utilization of NAD+ as a substrate for transcription initiation by RNA polymerase
Pinkas, Daniel ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
For a long time, 5' cap has been thought to be privilege only for eukaryotic organisms in form of 7-methylguanosine cap at the end of mRNA. This was changed only a few years ago. By using methods liquid chromatography and mass spectrometry a new molecule associated with RNA of Escherichia coli has been found. This molecule turned out to be nicotinamide adenine dinucleotide (NAD+ ) attached to 5' end of some small regulatory RNAs (sRNA). Later it has been shown, that RNA polymerase can attach NAD+ at 5' of RNA ab initio, meaning that RNA polymerase can utilize NAD+ as a substrate for transcription initiation. To some extent substrate for transcription initiation is chosen based on promoter sequence. Crucial requirement is presence of adenine at +1 position of DNA coding strand. This thesis focuses on promoter sequence requirements for transcription initiation with NAD+ . As a template for transcription four promoters with different modifications and their chimeras are used: RNA1, Pveg, lac UV5 and rrnB P1. Also, I tried to compare RNA polymerase from E. coli and B. subtilis in terms of transcription initiation substrate usage. Lastly, I describe here isolation of NudC, enzyme that cleaves NAD+ to nicotinamide mononucleotide (NMN) and adenosine monophosphate (AMP). NudC will be used for upcoming...
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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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Regulatory mechanisms of CD47 surface expression
Jakubec, Martin ; Drbal, Karel (advisor) ; Dibus, Michal (referee)
CD47 glycoprotein can be found on the surface of all healthy cells in our body. The interaction of CD47 with inhibitory receptor SIRPα on the macrophage leads to the inhibition of phagocytosis. This makes CD47 irreplaceable for the safe recognition of own cells and removal of aged or apoptotic cells. Apart from this, CD47 plays a major role in several essential signalling pathways, such as cell adhesion and motility or calcium homeostasis. The level of CD47 expression and its presence on the cell membrane depends not only on the type of tissue, but also on the age of a cell. An increased expression of CD47 protein has also been observed in the cells undergoing tumorigenic transformation, allowing them to escape from tumour immunosurveillance. Spontaneous regulation of the CD47 gene expression is achieved via regulatory transcription factors, such as NF-κB or HIF-1. Another mechanism of CD47 regulation includes the 3'UTR of CD47 mRNA, which serves as a binding site for either regulatory proteins, such as HuR, or miRNAs. CD47 expression can thus be regulated on both transcriptional, as well as translational level. However, appropriate topological CD47 localization within the cell and on the cell surface has also an important effect of its physiological function. Our in depth understanding of key regulatory...
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Characteristics of expression vectors for Corynebacterium glutamicum and their use for studies of sigma factors of RNA polymerase
Dvořáková, Pavla ; Pátek, Miroslav (advisor) ; Konopásek, Ivo (referee)
The aim of the thesis was to characterize chosen expression vectors used in biotechnologically important bacterial species, Corynebacterium glutamicum, and to test their use in studies of promoter activity control by sigma factors of RNA polymerase. Different properties of these vectors (level of expression of the cloned gene, leaky expression without inducer, dependence of expression level on inducer concentration and cell population homogeneity) were found by determination of expression level of the model gfpuv gene by fluorescence intensity assay of the produced protein and by gfpuv-expressing C. glutamicum cell population analysis using flow cytometry. The vector pEC-XT99A was chosen for testing the bi-plasmid system for assignment of a sigma factor to the chosen promoter. Although the level of expression provided by pEC-XT99A was not high, the vector showed no leaky expression, expression from the vector was comparable for a wide range of IPTG concentrations and the cell population was homogenous concerning the gene expression. Using pEC-XT99A from which individual stress sig genes were expressed, the σD factor was clearly assigned to the up-to-now unknown Pcg0420 promoter. Another vector for isolation and purification of C. glutamicum proteins was used to express the C. glutamicum sigM gene and to...
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The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav ; Nešvera, Jan (advisor) ; Branny, Pavel (referee) ; Lichá, Irena (referee)
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
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Functions of sigma factors of RNA polymerase in Corynebacterium glutamicum
Dvořáková, Pavla ; Pátek, Miroslav (advisor) ; Dvořáček, Lukáš (referee)
The aim of this thesis was to characterize the function of sigma factors of the bacterium Corynebacterium glutamicum and to analyze the promoter sequences which are recognized by individual sigma factors. Sigma (σ) factors are the subunits of RNA polymerase, which allow recognizing the sequences of specific promoter regions of the gene and initiating its transcription. The C. glutamicum genome carries genes encoding the primary sigma factor σA and six alternative sigma factors, σB , σC , σD , σE , σH and σM , whose expression is changed depending on the growth conditions and in response to the stimuli from the surrounding environment. Regulation of gene expression at the level of transcription is one of the mechanisms of the adaptation of cells to changes of living conditions. At the conclusion of this work, a model of the regulatory network of sigma factors, which is a core of the complex regulatory network controlling all processes in the cell, is proposed. Key words: sigma factor (SF), RNA polymerase, Corynebacterium glutamicum, transcription, promoter
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Role of promoter in the regulation of alternative splicing
Kozáková, Eva ; Staněk, David (advisor) ; Půta, František (referee) ; Blažek, Dalibor (referee)
It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...
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