Aufreinigung und Charakterisierung der RNase P in Aquifex aeolicus

5‘ end maturation of precursor tRNAs is catalyzed by the enzyme Ribonuclease P (RNase P) in all kingdoms of life. RNase P enzymes characterized in bacteria, archaea, yeast nuclei / mitochondria and animal cell nuclei are ribozymes composed of one catalytic RNA subunit and one to ten protein subunits. Recently, human mitochondrial RNase P was identified to be composed of three polypeptides but to lack any RNA subunit. Thereupon, the RNase P in land plants and kinetoplastids was found to consist of only one single polypeptide and was hence named “PRORP” (proteinaceous RNase P). In the hyperthermophilic bacterium Aquifex aeolicus, neither a gene for the RNA nor the protein component of bacterial RNase P has been identified in its sequenced genome. However, RNase P activity can be detected in A. aeolicus cell lysates. This PhD thesis aimed at developing a robust and reproducible method for the purification and enrichment of A. aeolicus RNase P activity. Mass spectrometry analysis identified one A. aeolicus protein in highly purified fractions. According to homology structure modeling, this protein might well be involved in A. aeolicus RNase P activity. Furthermore and for the first time, a protocol for successful in vitro reconstitution of RNase P activity from RNA and protein preparations from A. aeolicus fractions was established. The reconstitution experiments provide evidence that RNA as well as protein components are involved in A. aeolicus RNase P activity. Protein and RNA analysis also suggest an association of A. aeolicus RNase P with ribosomes. Additionally, roughly 100 noncoding RNA (ncRNA) candidates, many represented by sense and antisense transcripts, were identified bioinformatically and by deep sequencing (RNAseq); selected ncRNA candidates were verified via Northern blot detection in total RNA of A. aeolicus.