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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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Factors affecting gene expression in Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Vohradský, Jiří (referee)
Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...
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Function of sigma factors in strains of genus Rhodococcus
Nový, David ; Štěpánek, Václav (advisor) ; Sudzinová, Petra (referee)
Thanks to their large genome sizes, rhodococci share considerable biotechnological potential, however, there is still little known about the regulation of gene expression during the initiation of transcription process. The knowledge of this process might help to optimize biotechnological application. A large number of sigma factors are involved in the transcriptional regulation of rhodococci and this number often positively correlates with their genome sizes. Sigma factors can be potentially divided into two groups: the first, conserved factors that may in some variants occur in many different species among bacteria, and the second, specific sigma factors, that have been evolutionarily selected in individual strains due to environmental and lifestyle conditions. All sigma factors present in rhodococci belong to the σ70 family and can also be divided into four groups according to the presence of conserved structural domains (groups 1 to 4). A special type of sigma factors with extracytoplasmic function (ECF, group 4) are factors carrying specific domains at their C-terminus, namely NTF- 2-like or TPR, whose functions are still not fully understood. Very little information about the physiological functions of individual sigma factors in rhodococci has been published but it can be supplemented by...
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Overlaps of sigma factors regulons of RNA polymerase in Corynebacterium glutamicum
Zíková, Jaroslava ; Pátek, Miroslav (advisor) ; Sudzinová, Petra (referee)
Sigma factor (σ) is a part of the RNA polymerase enzyme complex. This complex (referred to as a holoenzyme) ensures the recognition of the consensus promoter sequences of the individual genes and the initiation of transcription. Seven sigma factors were found in Corynebacterium glutamicum. The genome of this bacterium encodes one primary factor σA and another six alternative sigma factors: σB , σC , σD , σE , σH a σM . These alternative sigma factors are expressed in response to changes in the internal and external environment and ensure the adaption of the bacterium to growth conditions. They are also one of many ways to regulate gene expression at the transcriptional level. In specific cases, the regulation of gene expression is caused by alternative sigma factors that recognize corresponding dual (recognized alternatively by two sigma factors) or overlapping promoters. Thus, the genes controlled by these promoters are classified into overlapping regulons. Key words: Corynebacterium glutamicum, sigma factor, RNA polymerase, transcription, promoter, regulons, RNA-seq, in vitro transcription, in vivo two-plasmid system
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Factors affecting gene expression in Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Vohradský, Jiří (referee)
Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...
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Applications of flow cytometry in the study of microbial subpopulations.
Hřebíček, Ondřej ; Lichá, Irena (advisor) ; Sudzinová, Petra (referee)
This work reviews common flow cytometric methods and applications for the study of bacterial organisms. Flow cytometry is fluorescent method capable of both quantitative and qualitative analysis at the single cell level. It can offer insights about bacterial population dynamics, phenotypic heterogeneity and more. This work features a basic introduction to flow cytometry and presents some of the commonly measured variables, such as viability or membrane potential with an emphasis on the fluorescent probes used to visualize them. The difficulties of adapting flow cytometry to bacterial physiology are discussed, as well as the advantages and disadvantages of the particular probes and methods. Finally, this work seeks to demonstrate the flexibility as well as the shortcomings of flow cytometry using examples of practical applications in basic research, environmental microbiology, biotechnology, clinical practice. Keywords: flow cytometry, microbial subpopulations, fluorescent labelling, bacterial physiology, bacterial viability
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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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