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Regulation of mycobacterial transcription
Kafka, Vojtěch ; Krásný, Libor (advisor) ; Dostálová, Hana (referee)
RNA polymerase (RNAP) is the enzyme that catalyzes synthesis of RNA. Mycobacterial RNAP significantly differs from RNAPs from other bacterial species. It requires special transcription factors such as RbpA or CarD. Another difference is the presence of a small RNA (sRNA), Ms1, that binds to mycobacterial RNAP. Ms1 regulates the amount of RNAP in the cell. In our laboratory we recently discovered MoaB2, a new binding partner of mycobacterial A (encoded by sigA), an RNAP subunit, which is essential for recognition of the initial promoter sequence and initiation of transcription. The function of MoaB2 in the regulation of transcription and gene expression is still unknown. The first aim of this Thesis is contribute to elucidation of the mechanism by which Ms1 affects the amount of RNAP. The experiments revealed that this regulation occurs at the level of transcription; Ms1 affects the activity of promoter(s) that drive the transcription of rpoB- rpoC that encode the two catalytic subunits of RNAP. The second aim of this Thesis is to characterize the interactions of MoaB2 with protein of the transcription apparatus. The results confirmed the interaction of MoaB2 with A and showed that neither RNAP nor transcription factors RbpA and CarD are required for this interaction. Finally, a role of the...
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Regulation of transcription in Gram-positive bacteria
Rabatinová, Alžběta ; Krásný, Libor (advisor) ; Bobek, Jan (referee) ; Valášek, Leoš (referee)
Bacteria are the most abundant organisms on the planet. They live almost in all environments, including those that are most extreme. All land and water ecosystems depend heavily upon their activity. Bacteria play essential roles in cycling of nutrients such as carbon, nitrogen, and sulphur. Due to their short cell cycle, they must be able to swiftly adapt to the conditions of their habitat to survive. Microbial growth itself is an autocatalytic process. There are three distinct phases of the growth curve: lag, exponential (log), and stationary. Bacterial cells must change their gene expression between these phases in order to adapt to the new conditions. The first stage of gene expression is transcription. The key enzyme of this stage is RNA polymerase (RNAP) that transcribes DNA into RNA. RNAP is regulated by a number of accessory proteins and also small molecule effectors. Understanding how RNAP functions is essential for understanding how bacteria cope with changing environments. This Thesis presents studies of selected aspects of bacterial gene expression regulation at the level of transcription, using Bacillus subtilis as the model organism. The first part of this Thesis focuses on protein determinants of the ability of RNAP to be regulated by the concentration of the initiating nucleoside...
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Regulation of mycobacterial transcription
Kafka, Vojtěch ; Krásný, Libor (advisor) ; Dostálová, Hana (referee)
RNA polymerase (RNAP) is the enzyme that catalyzes synthesis of RNA. Mycobacterial RNAP significantly differs from RNAPs from other bacterial species. It requires special transcription factors such as RbpA or CarD. Another difference is the presence of a small RNA (sRNA), Ms1, that binds to mycobacterial RNAP. Ms1 regulates the amount of RNAP in the cell. In our laboratory we recently discovered MoaB2, a new binding partner of mycobacterial A (encoded by sigA), an RNAP subunit, which is essential for recognition of the initial promoter sequence and initiation of transcription. The function of MoaB2 in the regulation of transcription and gene expression is still unknown. The first aim of this Thesis is contribute to elucidation of the mechanism by which Ms1 affects the amount of RNAP. The experiments revealed that this regulation occurs at the level of transcription; Ms1 affects the activity of promoter(s) that drive the transcription of rpoB- rpoC that encode the two catalytic subunits of RNAP. The second aim of this Thesis is to characterize the interactions of MoaB2 with protein of the transcription apparatus. The results confirmed the interaction of MoaB2 with A and showed that neither RNAP nor transcription factors RbpA and CarD are required for this interaction. Finally, a role of the...
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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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5' end modification of bacterial RNA
Pinkas, Daniel ; Krásný, Libor (advisor) ; Schierová, Michaela (referee)
Regulation of gene expression is a key feature of all organisms and can occur at several levels ranging from transcription initiation to protein degradation. An important mechanism of this process is regulation of mRNA stability by various modifications. The best known modification is eukaryotic 7mG cap, which protects RNA from RNase degradation. Recently, several new prokaryotic modifications have been discovered thanks to advances in liquid chromatography and mass spectrometry methods. One such a modification is nicotinamide adenine dinucleotide at the 5' end of some RNA. Nicotinamide adenine dinucleotide is analogous to 7mG cap. This study describes this phenomenon in context of bacterial transcription. Powered by TCPDF (www.tcpdf.org)
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