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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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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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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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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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Development of high-throughput screening assay for the identification of inhibitors targeting influenza A polymerase
Karlukova, Elena ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus A circulates in birds and mammals and causes severe infectious disease that affects from 3 to 5 million people each year. There are two classes of anti-influenza drugs currently available: neuraminidase and M2 channel inhibitors. However, increasing resistance against these two types of inhibitors along with the potential emergence of new viral strains and unpredictability of pandemic outbreaks emphasize an unmet need for new types of inhibitors. RNA-dependent influenza polymerase serves as a novel promising target for the development of anti-influenza medications. The aim of this master thesis is to develop in vitro high-throughput assays for screening of compounds targeting influenza RNA polymerase, particularly, its cap binding and endonuclease domains. For cap-binding domain the screening is based on DIANA (DNA-linked Inhibitor ANtibody Assay) method that was recently developed in our laboratory; for endonuclease domain, the method is based on AlphaScreen technology. For the purposes of the methods development, recombinant cap binding domain of PB2 subunit and N-terminal endonuclease domain of PA subunit of influenza polymerase were expressed with appropriate fusion tags and purified using affinity and gel permeation chromatography. The probes for the screening assays were...
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Computational modeling of complexes consisting of the Zika virus RNA-dependent RNA polymerase and NTP analogs
Ronovský, Michal ; Barvík, Ivan (advisor) ; Pospíšil, Miroslav (referee)
The recently published crystallographic structure of the Zika virus RNA-dependent RNA polymerase (RdRp) allows to produce molecular dyna- mics (MD) simulations of this enzyme that can help in the rational design of its inhibitors - potential chemotherapeutics. In the theoretical part of the thesis are first described basic principles of MD simulations and the method of calculation of binding free energy. This is followed by a brief introduction into the structure of nucleic acids, proteins, RNA polymerases and into mechanisms of expression of genetic information. In the practical part are presented the alchemical trans- formations of ADP to its analogues, which were recently synthesized at ÚOCHB AV ČR. Transformations are performed first in the water envelope and then in the active site of HCV RdRp. Additionally, the conformation of the Zika virus RdRp is modified to match the hepatitis C virus polymerase conformation that can accommodate nucleic acids. Finally, MD simulations of the ADP analogues in the active site of the Zika virus polymerase are produced.
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Intrinsic transcription termination by multisubunit RNA polymerases
Vojáčková, Jitka ; Sýkora, Michal (advisor) ; Kuthan, Martin (referee)
Transcription is a process of genetic information rewriting from DNA sequence to RNA copy by DNA dependent RNA polymerase. Two mechanisms of transcription termination are known for bacteria: intrinsic transcription termination, independent of any accessory factors, and transcription termination dependent on proten factor called Rho. Intrinsic transcription termination is common mainly for bacteria, yet different, but partially similar forms of intrinsic transcription termination occur also in eukaryotes and archaea. This thesis includes a brief summary of RNA polymerase structure and elongation complex stability, describes individual steps of intrinsic transcription termination mechanism in bacteria including all disscused models and gives examples of intrinsic transcription termination in eukaryotes and archaea and their comparison with bacterial model of intrinsic transcription termination. Key words: RNA polymerase, elongation complex, intrinsic transcription termination, hairpin RNA structures
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Domain structure and function of primary bacterial sigma factors
Kálalová, Debora ; Krásný, Libor (advisor) ; Roučová, Kristina (referee)
Transcription initiation is one of the crucial steps of gene expression. A multisubunit enzyme RNA polymerase (RNAP) transcribes the genetic information from DNA to RNA. However, RNAP itself is unable to recognize a specific promoter and initiate transcription. For this purpose, bacteria have a protein called σ factor, which binds to RNAP and together form the RNAP holoenzyme. In this thesis I describe the mechanism of bacterial transcription and the structure, function and regulation of σ factors. I focus mainly on the primary σ factors of two important model species, namely gramnegative Escherichia coli and grampositive Bacillus subtilis. I describe them in the context of alternative σ factors, and I point out their differences in structure, function and regulation. Key words: RNA polymerase, primary σ factors, transcription, bacteria, Bacillus subtilis, Escherichia coli
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Transcription regulation by sigma factors in Bacillus subtils
Benda, Martin ; Krásný, Libor (advisor) ; Seydlová, Gabriela (referee)
RNA polymerase (RNAP) is a key enzyme in regulation of bacterial gene expression. RNAP is multi-subunit enzyme and its σ subunits (factors) are used for DNA recognition. Regulation of RNAP complexed with the major σ factor has been thoroughly studied; in contrast, mechanisms of regulation of RNAP containing alternative σ factors are much less understood. This thesis is focused mainly on the model organism Bacillus subtilis and its alternative σ factors σF , σG , σI a σK . We studied the ability of RNAP in complex with these factors to recognize promoter sequences, to initiate transcription in dependence on the concentration of the initiating nucleoside triphosphate (iNTP), and to interact with selected proteins. For σF , a promoter regulated by the concentration of iNTP was discovered; for σI , to the contrary, this mechanism was not observed. In the case of σG -dependent transcription we were not able to examine regulation by the concentration of iNTP. Nevertheless, stimulation of σG -dependent transcription by a protein called YlyA, previously described in the literature, was confirmed. This stimulation was newly identified also for σF -dependent transcription. Further, we examined possible functional interaction between HelD and σK , but this link was not confirmed. Finally, this thesis...
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Regulation of expression of Ms1, a sRNA from Mycobacterium smegmatis
Páleníková, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...
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