National Repository of Grey Literature 46 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Molecular details of translation reinitiation in budding yeast
Mohammad, Mahabub Pasha ; Valášek, Leoš (advisor) ; Mašek, Tomáš (referee) ; Krásný, Libor (referee)
Eukaryotic translation initiation is a complex multi-step process. It begins with the formation of 43S pre-initiation complex (PIC), which binds to the 5' cap of the mRNA, scans downstream searching for an appropriate AUG start site. Upon AUG recognition by 48S PIC, most of the initiation factors dissociate. However, some initiation factors, such as eIF3 and may be eIF4G, remain transiently associated with the 48S PIC. Here, we have developed a yeast in vivo RNA-Protein Ni2+ -Pull down (Rap-Nip) assay for in vivo detection of eIF3 bound mRNA fragments. In Saccharomyces cerevisiae, we demonstrated showing that, after initiation, eIF3 persistently associated with the elongating ribosomes for few elongation cycles. Further, it stabilizes the post- termination 40S complex on the stop codon of both uORF1 and 2 by establishing a contact between the N-terminal domain of a/TIF32 subunit and the Reinitiation Promoting Elements (RPEs) of corresponding uORFs on GCN4. Furthermore, employing the β-galactosidase reporter assays we revealed that AU-rich motif (AU1-2A/ UUAU2 motif) that occurs immediately following the stop codon of reinitiation (REI)- permissive uORFs promotes REI in position-specific autonomous fashion. We also have shown that the exact length and the last coding triplet of the REI-permissive...
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...
Hybrid sigma factors of RNA polymerase in Corynebacterium glutamicum
Blumenstein, Jan ; Štěpánek, Václav (advisor) ; Krásný, Libor (referee)
Corynebacterium glutamicum is a Gram-positive non-sporulating soil bacterium which is used in biotechnology as a producer of amino acids, nucleotides, biofuels and alcohols. The aim of this thesis was to create a hybrid σ factor of RNA polymerase which would be able to recognize a matching hybrid promoter without effect on expression of the host genes. Based on the σD and σH amino acid sequence, two types of hybrid factors, σDH and σHD , were designed by the sequence combination of sigD and sigH. As an alternative approach, based on the in silico homology modeling, mutations of wild-type σH in the region recognizing the -35 promoter element of the σH -dependent promoter were introduced. Hybrid promoters were constructed by combining the -35 and -10 promoter regions that were derived from the σD - and σH - dependent promoters. Promoter activity was determined by using gfpuv reporter gene under the control of hybrid promoter. The expression of gfpuv in strains with hybrid sigma factors σDH / σHD and hybrid promoters was rather low compared to strains that carried wild-type σ factor and the respective promoter. The aim of the thesis was achieved by using one of the mutant σH factor (σmutH_6A ) with alterations in the region recognizing the -35 element of the σH -dependent promoter. This mutant σ...
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...
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...
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...
Mode of action and nature of different susceptibility of bacteria to antibacterial compounds lipophosphonoxins
Havlová, Noemi ; Seydlová, Gabriela (advisor) ; Krásný, Libor (referee)
Lipophosphonoxins (LPPO) are small synthetic antibacterial compounds targeting the cytoplasmic membrane. 1st generation of LPPO (LPPO I) displays an antimicrobial activity against Gram positive bacteria, however they do not show any activity against Gram negatives. After the modification of the iminosugar module (bearing the positive charge) the 2nd generation of LPPO (LPPO II) were synthetized. LPPO II exhibit broadened activity against Gram positive bacteria and also kill Gram negatives, including multiresistant strains. This work focuses on the mode of action of LPPO - the pore-forming activity of these substances is investigated on model membranes as well as in vivo. It also deals with the nature of different activity against Gram positive and Gram negative bacteria using model bacteria Bacillus subtilis and Escherichia coli. The results show that the insensitivity of Gram negative bacteria against LPPO I is probably caused by the different cell wall structure and the presence of the outer membrane that LPPO are almost unable to overcome. Also, the composition of phospholipids in the target membrane influences the antimicrobial activity of LPPO. Higher proportion of phospholipids with neutral charge reduces the LPPO pore-forming activity but is also responsible for low cytotoxicity in...
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...
Regulation of translation iniitiation in yeast saccharomyces cerevisiae
Mašek, Tomáš ; Pospíšek, Martin (advisor) ; Krásný, Libor (referee) ; Hašek, Jiří (referee)
IV. Shrnutí výsledků 63 64 Denaturační RNA elektroforéza v TAE agarózových gelech 60% koncentrace formamidu postačuje k dostatečné denaturaci RNA pro elektroforetickou separaci. Denaturační RNA elektroforéza v TAE pufru vykazuje stejné separační rozlišení RNA molekul jako nejčastěji používaná RNA elektroforéza v MOPSovém pufru a navíc je rychlejší. Denaturační RNA elektroforéza v TAE pufru je použitelná nejen k separaci čisté směsi RNA molekul, ale i směsných vzorků obsahujících také DNA a proteiny (např. buněčných lyzátů). Denaturační RNA elektroforézu v TAE pufru lze kombinovat s kapilárním přenosem a následnou hybridizací (pro blotování lze použít jak konveční 10xSSC pufr, tak levnější 8 mM NaOH). Tento elektroforetický protokol poskytuje levnější a rychlejší alternativu k RNA elektroforéze v MOPSovém pufru, snižuje expozici laboratorních pracovníků toxickým látkám a je vhodný i pro laboratoře, které s RNA běžně nepracují. Rck2 se zapojuje do reprogramování ribozómů během oxidativního stresu Oxidativní stres inhibuje translaci stejnou měrou jako stres osmotický. Lze pozorovat pokles množství aktivně translatujících ribozómů. Tento pokles je proporcionální ke zvětšení ploch 40S, 60S a 80S "vrcholů" v polyzomálním profilu. Aplikace t-BOOH vede k vyšší disociaci polyzomálních komplexů u rck2Δ...
Molecular principles of translation reinitiation in mammals
Hronová, Vladislava ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Translation initiation is a multistep process resulting in the formation of the elongation-competent 80S ribosome at the AUG start codon of the mRNA to be translated into a polypeptide chain. This process is orchestrated by numerous proteins called eukaryotic initiation factors (eIFs), out of which the most multitasking one is the eukaryotic initiation factor 3 (eIF3). The main focus of our laboratory aims at the complex characterization of the multisubunit protein eIF3 and the mechanisms of its contribution to various steps of translation initiation. Besides this, we also study one of the gene-specific translational control mechanisms called reinitiation which was, at least in yeast, also shown to be promoted by eIF3. Here I show that the N-terminal domain (NTD) of the largest subunit of yeast eIF3, a/Tif32, plays an important role not only in anchoring the eIF3 complex to the 40S small ribosomal subunit but it also critically contributes to mRNA recruitment to the 43S preinitiation complexes in vivo. The mRNA stabilization role of the a/Tif32-NTD at the mRNA exit channel of the 40S subunit was further confirmed in our following study by biophysical experiments. There, using in vivo approaches, we also demonstrated that mRNAs with longer 5'UTRs are more dependent on the stabilization role of the...

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