Die hoch abundante sRNA 6S RNA reguliert in Prokaryoten durch Bildung eines stabilen Riboproteinkomplexes mit der RNA Polymerase maßgeblich die globale Genexpression zur Anpassung an wechselnde Umweltbedingungen. In der vorliegenden Arbeit wurde die regulative Funktion von 6S RNA in Cyanobakterien am Beispiel des Modellorganismus Synechocystis sp. PCC 6803 beleuchtet. Die Analysen zeigten, dass die 6S RNA-Transkriptmenge sowohl unter phototrophen als auch unter photoheterotrophen Bedingungen in der stationären Wachstumsphase abnahm. Zudem wurden physiologische Untersuchungen einer 6S RNA-Deletionsmutante (delta_ssaA) und einer Überexpressionsmutante unter diversen Stressbedingungen durchgeführt. Während für delta_ssaA eine erhöhte Sensitivität gegenüber oxidativem Stress gemessen wurde, war die Thermotoleranz in den Mutanten nicht beeinträchtigt. Ferner wurde für delta_ssaA eine verlangsamte Regeneration nach Stickstoffmangel ermittelt, die sich phänotypisch durch eine verzögerte Reassemblierung der Phycobilisomen und des Glykogenabbaus sowie durch eine verminderte photosynthetische Aktivität äußerte. Vergleichende Microarray-Analysen zeigten, dass sich die Genexpression in delta_ssaA unter Stickstoffmangel kaum vom Wildtyp unterschied. Hingegen waren während der Regeneration Gene kodierend für die ATP-Synthase, ribosomale Proteine, Photosynthese-Komplexe und Phycobilisomen in delta_ssaA signifikant negativ exprimiert. Zudem wurde eine charakteristische Expressionskinetik für die sRNA SyR11 ermittelt. In vivo-pulldown-Analysen der RNA Polymerase zeigten, dass 6S RNA während der Regeneration die Rekrutierung des Hauptsigmafaktors SigA begünstigt und die Dissoziation von Sigmafaktoren der Gruppe 2 beschleunigt. Derweil blieb das 6S RNA-Transkriptlevel unter Stickstoffstress nahezu konstant. Ein Nachweis von in vivo-synthetisierten pRNA-Transkripten blieb negativ. Die Ergebnisse werden in der vorliegenden Arbeit hinsichtlich der funktionellen Bedeutung von 6S RNA für die Adaptation an Stressbedingungen in Cyanobakterien diskutiert. / The highly abundant sRNA 6S RNA extensively regulates the global gene expression for adaptation to changing environmental conditions in many prokaryotes by forming stable complexes with the RNA polymerase. In this work, Synechocystis sp. PCC 6803 was used as a model organism to study the regulative function of 6S RNA in cyanobacteria. A decline in 6S RNA transcript levels during stationary phase was measured under phototrophic and photoheterotrophic conditions. Physiological studies were carried out under various stress conditions using a 6S RNA deletion mutant (delta_ssaA) and an overexpression mutant. Delta_ssaA exhibited increased sensitivity toward oxidative stress, whereas the thermo-tolerance of the cells was not affected in the mutant strains. The recovery from nitrogen depletion was considerably delayed in delta_ssaA compared to the wild type. This phenotype was physiologically characterized by a decelerated phycobilisome reassembly and glycogen degradation as well as reduced photosynthetic activity in delta_ssaA. Comparative transcriptome analysis verified these observations. While under nitrogen depletion similar gene expression patterns were measured in delta_ssaA and wild type, genes encoding ATP synthase, ribosomal proteins, photosystem components and phycobilisomes were significantly negatively affected in the mutant strain during recovery. Furthermore, a distinctive accumulation kinetics was found for the sRNA SyR11. In vivo pulldown analyses of the RNA polymerase revealed a promoting effect of 6S RNA on the recruitment of the housekeeping sigma factor SigA as well as on the complex dissociation of group 2 sigma factors. Meanwhile, the 6S RNA transcript level remained nearly constant during nitrogen deficiency and the detection of in vivo synthesized pRNA transcripts was negative. The results are discussed regarding the functional role of 6S RNA for the adaptation to stress conditions in cyanobacteria.

Bacterial RNA polymerase (RNAP) is a multisubunit complex essential for transcription of DNA into RNA. As a key enzyme responsible for regulation of gene expression it interprets regulatory signals from the cell and based on these cues RNAP adjusts transcription level of particular genes. This process is affected both by the regular subunits of RNAP as well as other transcription factors (TFs) directly or indirectly interacting with RNAP. The general focus of this Thesis was to extend the knowledge about the complex transcriptional regulatory networks and about the connections between individual pathways. The main specific topic and the main publication of the thesis are focused on the HelD protein, a novel binding partner of RNAP in Bacillus subtilis. We showed that HelD binds between the secondary channel of RNAP and alpha subunits of the core form of the enzyme. We proved that HelD stimulates transcription in an ATP dependent manner by enhancing transcriptional cycling and elongation. We revealed a new connection in the transcription regulatory machinery when we demonstrated that the stimulatory effect of HelD can be amplified by delta, a small subunit of RNAP specific for gram positive (G+) bacteria. Two other publications of the thesis are dealing with the delta subunit. We solved the 3D...

The variety of bacteria and their genomes sometimes causes conservation of homologue molecules to be displayed not in sequence but in secondary and tertiary structures. In the case of the regulatory 6S RNA, sequence homologues have been found in over 100 bacterial species so far. However, none were found in the genus Streptomyces. The unique genome of these soil- dwelling bacteria, known for their capacity to produce antibiotics, has a high G/C content and diverges substantially from distantly related bacteria. Yet in the non-coding 6S RNA it is the secondary structure that is crucial for its function. The 6S RNAs trap sigma factors by mimicking target promoter sequences in order to help with switching sets of expressed genes during developmental transitions. 6S-like RNA genes in Streptomyces coelicolor have been computationally predicted by comparison of in silico modelled secondary structures of known 6S RNAs. The aim of this thesis was the verification of these 6S-like RNA predictions. The experimental approach was based on RNA co-immunoprecipitation (RNA CoIP), as well as RT- PCR from RNA samples. The outcomes of this project are the detection of six novel ncRNA transcripts with possible 6S-like RNA functions, which also served as the wet-lab verification of the in silico prediction technique...