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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...
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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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