Struktur- und Funktionsanalyse der Protein-basierten RNase P des hyperthermophilen Bakteriums Aquifex aeolicus und homologer Enzyme

Die Reifung einer transfer-RNA (tRNA) ist ein essenzieller Schritt in der Proteinbiosynthese in allen drei Domänen des Lebens. Dabei sind mehrere Schritte notwendig, um von einer Vorläufer- zu einer maturen tRNA zu gelangen. Die Spaltung der 5‘-Flanke wird einzig durch die Ribonuklease P (RNase P) v...

Full description

Saved in:
Bibliographic Details
Main Author: Wäber, Nadine Bianca
Contributors: Hartmann, Roland K. (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2019
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!

The transfer RNA (tRNA) maturation is an essential step in the biosynthesis of proteins in all domains of life. It is a multistep process to get the precursor tRNA (pre-tRNA) into a mature form. The cleavage of the 5’-leader and the 3’-trailer is performed by different ribonucleases (RNases). Furthermore, single nucleotides are modified by a number of various enzymes. Whereas processing of the 3’-trailer can occur by diverse RNases, RNase P is the unique enzyme for cleavage of the 5’-leader. The bacterial RNase P is composed of a catalytic RNA and a small protein subunit. The RNA-based RNase P exists in all domains of life and the protein content in the ribonucleoprotein increases with the complexity of the organism. In eukaryotes a protein-based form of RNase P was identified which can be either a complex of three different proteins (e.g. in human mitochondria) or a singular protein subunit called PRORP (e.g. in Arabidopsis thaliana). In the present thesis a novel single subunit RNase P enzyme was characterized in the hyperthermophilic bacterium Aquifex aeolicus, called AaRP. It is the smallest form known so far (~23 kDa) and consists of only a PIN-like nuclease domain. In contrast PRORP enzymes consists of three domains: (i) a pentatricopeptide-repeat (PPR) motif domain, (ii) a zinc finger binding motif domain and (iii) a NYN-metallonuclease domain. The active site of AaRP was identified by point mutation analysis. AaRP was able to replace the endogenous RNase P of E. coli (one RNA plus one protein-subunit) as well as the complex nuclear RNase P of yeast (one RNA plus nine protein-subunits) in vivo. By bioinformatic analyses a small number of homologs of AaRP (HARP) could be identified in some bacteria and archaea. All these organisms also contain the genes for the classical RNA-based RNase P. The HARPs from three archaea (Methanosarcina mazei, Methanothermobacter thermautotrophicus, Haloferax volcanii) and two bacteria (Thermodesulfatator indicus and Methylacidiphilum infernorum) were selected for further biochemical characterization. All analyzed archaeal and bacterial HARPs were able to remove the 5’-leader of pre-tRNAs by side-specific endonucleolytic cleavage. Deletion mutants of H. volcanii HARP, as well as M. mazei HARP were constructed and characterized. Further, the bacterial RNA-based RNase Ps from T. indicus and M. infernorum were analyzed for tRNA-processing functionality in vitro and in vivo. Substrate specificity and recognition by AaRP is still unclear since no RNA-binding motif could be identified. Therefore, biochemical studies were designed to identify the substrate determinants of AaRP/HARP. The studies showed that interaction with the 5´-leader seems to be important for tRNA recognition by AaRP/HARP since the length of the 5´ flank has an influence on cleavage efficiency. This is similar to bacterial RNA-based RNase P where the protein subunit also interacts with positions in the 5´ leader of the precursor tRNA. In contrast, as described for PRORP, the AaRP/HARP enzymes obviously make no interactions with extensions of the substrate 3´-end (CCA or trailer). Structure analysis could give further informations to substrate recognition. While analysis of AaRP/HARP by nuclear magnetic resonance spectroscopy was unsuccessful, dynamic light scattering experiments indicated that AaRP assembles in a large complex (~420 kDa). This finding was corroborated by gel filtration experiments. Results of a circular dichroism spectroscopy analysis indicate that AaRP has a nearly complete α-helical structure. In contrast H. volcanii HARP contains more β-sheets and assembles in a smaller complex (~90 kDa). Analytical ultra-centrifugation and cryo-electron microscopy showed a heterogenous distribution of AaRP even after gel filtration indicating a heterogenous complex formation. Crystallization screens (manual and automatically) yielded only small crystals. A more detailed analysis of those crystals and an optimization of the crystallization process was not able in the available time frame of the present work.