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Domain 1.1 of the primary sigma factor and a new expression system for Bacillus subtilis RNA polymerase.
Kálalová, Debora ; Krásný, Libor (advisor) ; Cvačková, Zuzana (referee)
RNA polymerase (RNAP) is a key multi-subunit enzyme of gene expression that, together with the σ factor, forms a holoenzyme and transcribes genetic information from DNA to RNA. RNAP from Bacillus subtilis and its primary factor σA were studied in this thesis. The σA factor determines the specificity for the promoters to which the holoenzyme binds. Part of its structure is domain 1.1, which is likely to prevent binding of σA to the promoter by itself (unless it is part of the holoenzyme) by binding to domains 2 and 4. The first part of the thesis verifies the hypothesis that domain 1.1 binds domains 2 and 4 and thus prevents binding of σA to the promoter. To this end, various domain constructs have been created and their interactions have been tested. Domain interaction was tested by Nitrocellulose Filter Binding Assay, EMSA, and in vitro transcription. The results did not show significant interaction between domains. The second part of the thesis deals with the creation of a tool for the study of the enzymatology of RNAP from B. subtilis - recombinant RNAP (rRNAP). First, a plasmid construct for expression of rRNAP in Escherichia coli was constructed by a series of cloning steps, followed by protein isolation and characterization. Isolation was achieved without contamination by σ factors (this...
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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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Regulatory network controlled by sigma factors of RNA polymerase in Corynebacterium glutamicum
Kučera, Tomáš ; Pátek, Miroslav (advisor) ; Roučová, Kristina (referee)
An important feature of bacteria is an ability to adapt to changing environment by regulating gene expression. Level of gene expression and its right timing depends mainly on activation or repression of the gene by transcriptional regulators and recognition of the respective promoter by the sigma factor which is a subunit of RNA polymerase. Transcription regulators with sigma factors and other control elements, form a complex regulatory network. The regulatory network in Corynebacterium glutamicum is one of the best studied networks among gram-positive bacteria owing to genome sequencing and application of a number of techniques at the genome level. There has been a lot of success in understanding the roles of individual regulators and interactions between regulators in response to changes in environment. This work summarizes currently known knowledge of mutual relationships between sigma factors, the influence of sigma factors on transcriptional regulators and their cooperative effect on the initiation of transcription. In the thesis, a regulatory network of sigma factors in C. glutamicum and a regulatory cascade in response to the stress situation is schematically created. Key words: sigma factor (FS), Corynebacterium glutamicum, transription regulator (TR), transcription, regulation
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The effect of 6S-like RNAs on physiological differentiation of Streptomyces coelicolor
Burýšková, Barbora ; Bobek, Jan (advisor) ; Branny, Pavel (referee)
The variety of bacteria and their genomes sometimes causes conservation of homologue molecules to be displayed not in sequence but in secondary and tertiary structures. In the case of the regulatory 6S RNA, sequence homologues have been found in over 100 bacterial species so far. However, none were found in the genus Streptomyces. The unique genome of these soil- dwelling bacteria, known for their capacity to produce antibiotics, has a high G/C content and diverges substantially from distantly related bacteria. Yet in the non-coding 6S RNA it is the secondary structure that is crucial for its function. The 6S RNAs trap sigma factors by mimicking target promoter sequences in order to help with switching sets of expressed genes during developmental transitions. 6S-like RNA genes in Streptomyces coelicolor have been computationally predicted by comparison of in silico modelled secondary structures of known 6S RNAs. The aim of this thesis was the verification of these 6S-like RNA predictions. The experimental approach was based on RNA co-immunoprecipitation (RNA CoIP), as well as RT- PCR from RNA samples. The outcomes of this project are the detection of six novel ncRNA transcripts with possible 6S-like RNA functions, which also served as the wet-lab verification of the in silico prediction technique...
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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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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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Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicus
Černý, Zbyněk ; Španová, Alena (referee) ; Pepeliaev,, Stanislav (advisor)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
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