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The omega subunit of Bacillus subtilis RNA polymerase.
Mikesková, Klára ; Krásný, Libor (advisor) ; Nešvera, Jan (referee)
Transcription is catalysed by the enzyme RNA polymerase (RNAP). RNAP contains a core made up of two α subunits, one of each β, β'and ω. These subunits are conserved in all bacteria. The ω subunit is a small subunit with a molecular weight of 7.6 kDa that binds β'. ω is important for the folding and integrity of RNAP and promoter selection. This was shown by experiments performed with Gram-negative bacteria but the knowledge about in Gram-positive bacteria is minimal. In my Diploma Thesis, I characterized from the model Gram-positive bacterium from the phylum Firmicutes, Bacillus subtilis. First, I prepared various expression strains for isolation of Bacillus subtilis ω. Then, I successfully isolated the ω subunit, which was the main initial aim of this Diploma Thesis. Subsequently, I tested the influence of the ω subunit on in vitro transcription by RNAP associated with the primary σA factor and alternative σF and σE factors that regulate sporulation in Bacillus subtilis. I also evaluated the effect of , a small RNAP subunit found in Firmicutes, both alone and in combination with . The experiments revealed that ω stimulated transcription both from vegetative promoters and sporulation-related promoters. Moreover, this stimulation was synergistically amplified by the δ subunit. This nicely...
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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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