Introduction: In recent years, there has been growing interest in regulation of gene expression by small non-coding RNA (sRNA). The first sRNA discovered in 1960s was 6S RNA from E. coli (length ~184 nt). It took ~ 30 years to obtain meaningful insights into its function. 6S RNA binds during stationary phase to RNA polymerase (RNAP) containing sigma factor 70 (primary sigma factor), thereby preventing transcription from σ70 - dependent promoters. In our laboratory we discovered a small RNA (length ~300 nt) in stationary phase of growht in Mycobacterium smegmatis. This sRNA was named Ms 1. The function of Ms 1 is uknown and preliminary experiments indicated that Ms 1may bind to RNAP that lacks σ factor (σA ). Goals: The aim of this Diploma project is to contribute to the characterization of Ms 1. Approaches: First, by molecular cloning, affinity chromatography and in vitro transcription I prepared the tools for subsequent experiments in vitro: RNAP, σA , Ms 1 and its mutated variants. Next, these tools were used for binding experiments on native gels and for transcription experiments. Results: RNAP, σA , Ms 1 and its variants were prepared. In vitro binding assays showed that wt Ms 1 but not a mutated variant of Ms 1 binds to RNAP. Using this assays were identified areas of Ms 1 that are important...
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:332209 |
| Date | January 2014 |
| Creators | Pospíšil, Jiří |
| Contributors | Krásný, Libor, Lichá, Irena |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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