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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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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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Regulation of bacterial transcription by alternative sigma factors.
Benda, Martin ; Krásný, Libor (advisor) ; Roučová, Kristina (referee)
An important feature of bacteria is their ability to respond to various environmental conditions by regulation of transcription. This thesis is focused on regulation of transcription initiation by different sigma factors. Sigma factor is a specific subunit of RNA polymerase, which ensures correct recognition of promotor sequences. During exponential growth phase under ideal conditions, the cell transcribes most of the genes under the control of the so-called housekeeping sigma factor. In the transition to stationary phase, during sporulation or upon exposure to different stresses, many genes are activated and transcribed under the control of alternative sigma factors. This work presents a list of these alternative sigma factors of Bacillus subtilis, focusing on conditions under which the individual sigma factor is used, methods of activation and repression of these factors and on regulons controled by these sigma factors.
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Characterization of Ms1, a newly identified small RNA from Mycobacterium smegmatis
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
Introduction: In recent years, there has been growing interest in regulation of gene expression by small non-coding RNA (sRNA). The first sRNA discovered in 1960s was 6S RNA from E. coli (length ~184 nt). It took ~ 30 years to obtain meaningful insights into its function. 6S RNA binds during stationary phase to RNA polymerase (RNAP) containing sigma factor 70 (primary sigma factor), thereby preventing transcription from σ70 - dependent promoters. In our laboratory we discovered a small RNA (length ~300 nt) in stationary phase of growht in Mycobacterium smegmatis. This sRNA was named Ms 1. The function of Ms 1 is uknown and preliminary experiments indicated that Ms 1may bind to RNAP that lacks σ factor (σA ). Goals: The aim of this Diploma project is to contribute to the characterization of Ms 1. Approaches: First, by molecular cloning, affinity chromatography and in vitro transcription I prepared the tools for subsequent experiments in vitro: RNAP, σA , Ms 1 and its mutated variants. Next, these tools were used for binding experiments on native gels and for transcription experiments. Results: RNAP, σA , Ms 1 and its variants were prepared. In vitro binding assays showed that wt Ms 1 but not a mutated variant of Ms 1 binds to RNAP. Using this assays were identified areas of Ms 1 that are important...
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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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Characterization of the HelD protein from Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
BACKGROUND: Bacterial RNA polymerase (RNAP) is an extensively studied enzyme required for gene expression. In our Laboratory we found a new protein named HelD. HelD copurifies with B. subtilis RNAP. HelD is a ~90 kDa protein from the UvrD/Rep helicase family, which contains protein with the 3'-5' DNA unwinding activity. The molecular role(s) HelD in cell are still unknown and its potential role in transcription has not been studied so far. OBJECTIVE: The main aim of this Diploma project was to describe HelD. APPROACHES: The characterization was carried out on three levels: (i) bioinformatics analysis in silico was used to identify HelD homologs in other bacteria; (ii) growth tests in vivo were used to determine the phenotype(s) of the HelD-null mutant strain compared to wt; and (iii) biochemical experiments in vitro were utilized to describe the effects of HelD on transcription, and to test whether HelD has DNA binding and DNA unwinding activities. RESULTS: The in silico analysis revealed that HelD is present in Firmicutes, an industrially and medicinally important group of G+ bacteria. The phenotypic experiments showed that HelD is required for rapid adaptations to nutritional changes in the environment. The biochemical experiments showed that HelD stimulates transcription despite the fact that it...
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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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The delta subunit of RNA polymerase from gram positive bacteria
Matějčková, Jitka ; Beranová, Jana (referee) ; Krásný, Libor (advisor)
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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