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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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SigN from Bacillus subtilis: Functional characterization.
Kambová, Milada ; Krásný, Libor (advisor) ; Nešvera, Jan (referee)
Bacillus subtilis strain 3610 is an ancestral undomesticated strain. It diers from the laboratory strain 168 in many aspects. One dierence in strain 3610 is the presence of plasmid pBS32 encoding the sigma factor N (σN). This σ factor is activated when DNA damage occurs and induces the bacteria's cell death. The aim of the Thesis was a systematic characterisation of σN-dependent transcription. First, I showed that plasmid-borne but not chromosome-borne predicted σN-dependent promoters were ac- tive in transcription in vitro. Next, the anities of RNAP with σN for DNA, initiating NTP (iNTP) were determined for both relaxed and supercoiled DNA templates. Sur- prisingly, the activity of RNAP on relaxed σN-dependent promoters was higher than on their supercoiled versions, an opposite trend than displayed by RNAP associated with other σ factors. This property of σN-dependent promoters was not encoded by the core promoter sequence. In summary, this Thesis contributed to our understanding of the bacterial transcription apparatus. 1
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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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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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Intracellular and intercellular regulation of gene expression in Gram-positive bacteria.
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Malínský, Jan (referee)
Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....
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The effect of selected endogenous and exogenous factors on bacterial growth
Šiková, Michaela ; Krásný, Libor (advisor) ; Valášek, Leoš (referee) ; Vopálenský, Václav (referee)
The growth of bacteria by binary division is a key characteristic of these organisms. This growth depends on two types of factors: endogenous and exogenous. Endogenous factors make up the molecular apparatus of cells. Among important endogenous factors belong also those involved in gene expression and its regulation. Exogenous factors are external conditions such as nutrient availability, temperature, pH, various stresses or the presence of antibacterial agents. The main aim of my Thesis was to study the effects of selected endogenous and exogenous factors on bacterial growth. As endogenous factors I studied RNase J1 in Bacillus subtilis and a small RNA called Ms1 in Mycobacterium smegmatis, which are involved in regulation of gene expression at the transcriptional level. I showed that RNase J1 can, besides its role in RNA degradation, play a role in genome integrity by removing stalled RNA polymerase (RNAP) complexes from DNA. I further showed that Ms1 binds to the RNAP core and affects the level of RNAP in the cell. The results revealed new mechanistic aspects of the transcription apparatus and show how individual components or their combinations affect bacterial growth. As exogenous factors I studied the recently discovered antibacterial compounds, called lipophosphonoxins, their interaction...
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The effect of the environment on bacterial DNA topology and gene expression.
Mikesková, Klára ; Krásný, Libor (advisor) ; Večerek, Branislav (referee)
Biological processes in the cell are affected by DNA topology, i. e. by DNA structure and shape. An important topological parameter is the level of supercoiling - additional twisting of DNA is relieved by positive (twisting in the same direction as the helix turns) or negative (twisting in the opposite direction) supercoils. In this Thesis I review the role of supercoiling in gene expression regulation. I describe how supercoiling is involved in homeostatic mechanisms that control the transcriptional output from some genes. Environmental changes such as shifts in temperature, oxidative stress, extreme pH and antibiotics and other inhibitors affect the level of DNA supercoiling. DNA supercoiling then affects the expression of enzymes, which influence DNA topology, as well as some other genes/proteins. In summary, this Thesis describes how changes in the environment influence bacterial DNA topology and gene expression with a brief mention of this type of regulation in eukaryotes.
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Interaction of nucleic acids with RNA polymerase
Janoušková, Martina ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Knejzlík, Zdeněk (referee)
Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...
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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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