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Arbeitsgruppe Hartmann
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Research topics

Ribonuclease P

The enzyme ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein particle that cleaves primary tRNA transcripts endonucleolytically at the 5´-end of the tRNA domain. The discovery of a variety of non-tRNA substrates of RNase P in Escherichia coli suggest a more general function of RNase P in RNA metabolism. The RNA component of the bacterial RNase P enzyme is catalytically active in the absence of the single protein cofactor. However, in vivo both the RNA and protein components of RNase P are essential. Beside the ribosome, bacterial RNase P RNA is the only known in trans acting ribozyme in nature. In Archaea, Eukarya and organelles, the loss of robust RNA-alone (ribozyme) activity of RNase P RNA correlates with an increase in the number of protein subunits (usually 4 in Archaea and 9-10 in Eukarya). All RNase P RNAs seem to stem from a common progenitor, as they share a conserved core structure. This qualifies RNase P as an interesting model system to study the evolutionary transition from an almost true RNA enzyme to a protein-dominated ribonucleoprotein. Another advantage of RNase P as a model system is the availability of a relatively straightforward in vitro activity assay (tRNA processing). Since RNase P is an essential enzyme, data from in vitro experiments can be tested in vivo by complementation analyses in bacterial and archaeal RNase P mutant strains.

The topic of our research activities is the understanding of the structure, function, dynamics and evolution of this ribonucleoprotein enzyme at the molecular level. This includes studies on the cleavage mechanism, enzyme-substrate interaction, RNP assembly and on the architectural / functional role of the protein component(s) in bacterial, archaeal and eukaryotic RNase P enzymes.

Our current studies focus on the identification and characterization of RNase P in Aquificales strains (particularly Aquifex aeolicus), the in vivo function of RNase P in different bacterial mutant strains, restoration of archaeal RNA-alone activity and isolation of functional ribonucleoprotein complexes after recombinant overexpression for structural/ functional studies, as well as the identification of cellular components associated with RNase P.

For RNase P research we apply biomolecular and biochemical methods (RNomics, structure probing, RACE, modification interference studies, crosslinking, enzyme kinetics, UV- and fluorescence spectroscopy), genetic studies or NMR to study metal-dependent catalysis.

Structural and functional aspects of the small riboregulator 6S RNA

6S RNA is a ubiquitous small non-coding bacterial RNA. The RNA was recently identified as a growth-phase dependent riboregulator of transcription in E. coli, which forms a complex with the RNA polymerase holoenzyme and thereby controls its function.

The conserved secondary structure of 6S RNA, a hairpin with an internal loop, suggests that the RNA might mimic an open promoter, thereby acting as a competitive inhibitor of RNA polymerase. Indeed it was shown that 6S RNA represents a template for the RNA polymerase from which short transcripts of 14-22 nucleotides are synthesized. In E. coli, 6S RNA increases cell viability by acting in stationary phase, where it prepares cells for the outgrowth from stationary phase.

In contrast to the single 6S RNA species found in most bacteria, Bacillus subtilis expresses two different kinds of 6S RNAs which are nonetheless structurally similar. Both interact with the housekeeping RNA polymerase, but they substantially differ in their expression profiles. Our research efforts aim at understanding the structural basis of 6S RNA function, at understanding why B. subtilis possesses two types of 6S RNA, and at elucidating the 6S RNA-dependent regulatory mechanisms.

Our approaches include crystallography and cryo-electron microscopy for the 3D structure (in cooperation with IGBMC, Strasbourg, and Charité, Berlin), RNA-protein crosslinking and related biochemical/biophysical techniques for analysis of 6S RNA interaction with RNA polymerase, and transcriptome and proteome analyses of B. subtilis 6S RNA knockout mutants for the role of 6S RNAs in cellular stress responses.

miRNA- and c-Myc-mediated pathogenesis of B cell lymphomas - regulation by Pim kinases

Pim kinases, normally activated by cytokine and growth receptors, phophorylate several target proteins that are involved in cell cycle regulation and apoptosis. The Pim-1 kinase appears to be essential for proliferation of several types of tumor cells. For B cell lymphoma, a synergistic effect between overexpression of Pim-1 and transcription factor c-Myc has been observed. Interestingly, c-Myc stimulates expression of the miRNA-Clusters miR-17-92, possibly suggesting a link between Pim-1 and miR-17-92 expression. The role of Pim-1 and miR-17-92 in the development of tumors and their potential as anti-tumor targets are at the center of our research interests. At present, we are investigating the mechanisms of transcriptional regulation of the miR-17-92 cluster using chromatin immunoprecipitation assays (ChIP assays) and 5’-RACE experiments. Here, Pim-1 phosphorylation of its target HP1gamma may enhance miR-17-92 expression. Knockdown of Pim-1 by RNAi is employed to identify additional, so far unknow targets of Pim-1; LNA-AntimiRs directed against miRNAs encoded in the miR-17-92 cluster are used to reveal mRNA targets of the cluster. The techniques applied to several tumor cell lines include Western- und Northern blots, qRT-PCR, proliferations and apoptosis assays, luminiscence (luciferase)-based quantification of reporter gene expression as well as transcriptome and proteome analyses. Animal experiments for the therapeutic application of siRNAs and LNA-modified antisense oligonucleotides are performed in collaboration with the Institute of Pharmacology and Toxicology at the Medical Faculty of this university.


Fb. 16 - Pharmazie

AG Hartmann, Marbacher Weg 6, Gebäude C, D-35032 Marburg
Tel. 06421 28-25553, Fax 06421 28-25854, E-Mail: huette@staff

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