<p><i>Staphylococcus aureus</i> (<i>S. aureus</i>) is a Gram-positive pathogen that causes a wide range of infections in both hospitals and communities, of which the total mortality rate is higher than AIDS, tuberculosis, and viral hepatitis combined. The drug resistant <i>S. aureus </i>is a member of the “ESKAPE” pathogens that require immediate and sustained actions of novel method to combat. However, the current antimicrobial development against <i>S. aureus</i> is in stagnation, which underscores the urgent need for novel antimicrobial scaffolds and targets. <i>S. aureus</i> Ribonuclease P protein (RnpA) is an essential protein that plays important roles in both tRNA maturation and mRNA degradation pathways. The goal of this research was to evaluate RnpA as an antimicrobial target using biophysical methods. The crystal structures of wild-type RnpA in three different constructs were determined, among which the tag-free RnpA construct has a structural model of 2.0 Å resolution and R_crys/R_free= 0.214/0.234, and its crystals are reproducible. This crystal structure of tag-free <i>S. aureus </i>RnpA shows a globular representation with key structural motifs, including the “RNR” Ribonuclease P RNA binding region and a substrate binding central cleft, which shares high similarity to previously solved RnpA structures from other species despite of their low sequence identity. Meanwhile, in a screen of <i>S. aureus </i>RnpA mutants performed by our collaborator, RnpA^P89A was found lacking the mRNA degradation activity while retaining the tRNA maturation function, and causing defects in cell viability. We therefore studied this mutant using differential scanning fluorimetry, crystallography, and circular dichroism. It was shown that RnpA^P89A is thermally less stable than wild-type RnpA by ~2.0 ˚C, but no secondary structural or 3D conformational differences were found between the two proteins. Although the mutant RnpA^P89A requires further characterization, the results of the studies in this thesis have begun to shed light on the relatively new role of <i>S. aureus </i>RnpA in mRNA degradation, and will serve as useful tools in future structure-based drug discovery for multi-drug resistant <i>S. aureus </i>treatment. </p>

<p>RNA is the only biological molecule that can function both as a repository of information and as a catalyst. This, together with the ability to self-replicate, led to recognition of RNA as ‘prelude to life’.</p><p>My work highlights some of the important features of RNA as a catalyst, exemplified by RNase P. It addresses questions of evolutionary preservations of residues and structure, involvement of metal ions and finally structure evolution towards minimal catalytically competent RNA motifs.</p><p>RNase P is the only enzyme involved in 5’ end processing of all pre-tRNAs. Until recently, it was believed that the RNA moiety of RNase P is responsible for mediating catalysis only in Bacteria. However, my recent study conclusively demonstrated that eukaryotic RNase P RNA is catalytically competent in vitro in absence of proteins. These findings evidenced evolutionary preservation of RNA-mediated catalysis in RNase P.</p><p>RNase P RNA is a metalloeznyme. In my studies I analyzed the contributions of individual chemical groups at the cleavage site to catalysis. My findings suggested that the 2’OH of N_-1 and the exocyclic amine of G₊₁ are involved in positioning of functionally important metal ions. Additionally, data appointed the function of Pb²⁺ as both structural metal ion and important in generating the nucleophile. My studies further indicate a conformational change upon RNase P RNA -substrate complex formation in keeping with an induced fit mechanism. </p><p>Studying the effects of reducing the ribozyme size upon dissection of bacterial RNase P RNAs, we defined the smallest catalytically competent domain i.e. P15-loop. Derivatives of this autonomous metal ion binding domain, (the smallest being 31nt-s), are able to cleave both whole-length pre-tRNAs as well as hairpin substrates, though with severely reduced rates relative to their parent ribozymes. The study has inferred that partite ES interactions at the cleavage site prove sufficient for catalysis.</p>

RNA is the only biological molecule that can function both as a repository of information and as a catalyst. This, together with the ability to self-replicate, led to recognition of RNA as ‘prelude to life’. My work highlights some of the important features of RNA as a catalyst, exemplified by RNase P. It addresses questions of evolutionary preservations of residues and structure, involvement of metal ions and finally structure evolution towards minimal catalytically competent RNA motifs. RNase P is the only enzyme involved in 5’ end processing of all pre-tRNAs. Until recently, it was believed that the RNA moiety of RNase P is responsible for mediating catalysis only in Bacteria. However, my recent study conclusively demonstrated that eukaryotic RNase P RNA is catalytically competent in vitro in absence of proteins. These findings evidenced evolutionary preservation of RNA-mediated catalysis in RNase P. RNase P RNA is a metalloeznyme. In my studies I analyzed the contributions of individual chemical groups at the cleavage site to catalysis. My findings suggested that the 2’OH of N-1 and the exocyclic amine of G+1 are involved in positioning of functionally important metal ions. Additionally, data appointed the function of Pb2+ as both structural metal ion and important in generating the nucleophile. My studies further indicate a conformational change upon RNase P RNA -substrate complex formation in keeping with an induced fit mechanism. Studying the effects of reducing the ribozyme size upon dissection of bacterial RNase P RNAs, we defined the smallest catalytically competent domain i.e. P15-loop. Derivatives of this autonomous metal ion binding domain, (the smallest being 31nt-s), are able to cleave both whole-length pre-tRNAs as well as hairpin substrates, though with severely reduced rates relative to their parent ribozymes. The study has inferred that partite ES interactions at the cleavage site prove sufficient for catalysis.

Transcription and translation of mammalian mitochondrial DNA (mtDNA) occurs within the mitochondrial matrix to produce oxidative phosphorylation subunits required for efficient energy production. These mtDNA-encoded subunits complex with mitochondrial-localized, nuclear-encoded subunits to form the respiratory chain, and aberrant production or function of these subunits can cause devastating human disease. In addition to 13 oxidative phosphorylation subunits, mtDNA encodes 2 rRNAs and 22 tRNAs. All proteins required for mitochondrial RNA transcription, processing, and translation are encoded in the nucleus and translocated into the mitochondria. Here, I characterize over 100 nuclear-encoded mitochondrial proteins with predicted RNA-binding domains. Using RNAi and an RNA profiling approach, MitoString, we further characterize previously identified RNA processing factors and identify the novel regulator FASTKD4, which influences the abundance of a subset of mitochondrial mRNAs. Next, we apply knowledge of the RNA degradation component SUPV3L1 gleaned from our RNAi studies and previous research to test whether a specific set of variants influence the function of this gene in patient fibroblasts. Using MitoString, we find no evidence of pathogenicity of these variants in our fibroblast model. Our approach highlights the value of a thorough understanding of mitochondrial proteins and the necessity of experimental techniques to validate the effect of variants found in exome-sequencing studies. Finally, we take an unbiased approach to characterizing the mitochondrial transcriptome of mouse liver by sequencing RNA from sequentially enriched mitochondrial fractions. Although we find an abundance of nuclear-encoded 5S rRNA, consistent with previous research, we fail to identify any imported nuclear-encoded tRNAs. Uniting genomics, biochemistry, and medicine, these findings advance our understanding of mitochondrial RNA biology.

Η ριβονουκλεάση Ρ (RNase P) είναι μια πανταχού παρούσα ενδονουκλεάση, και σε πολλές περιπτώσεις αποτελεί ένα ριβοένζυμο, η οποία συμμετέχει στον μηχανισμό ωρίμανσης των πρόδρομων tRNAs. Στην πλειοψηφία των περιπτώσεων είναι ένα ριβονουκλεοπρωτεϊνικό σύμπλοκο που αποτελείται από μια RNA υπομονάδα και τουλάχιστον μια πρωτεϊνική υπομονάδα. Όσον αφορά τα ευκαρυωτικά κύτταρα, πιστεύεται πως υπάρχουν δυο διακριτές μορφές του ολοενζύμου, μια πυρηνική και μια μιτοχονδριακή. Στο Saccharomyces cerevisiae η μιτοχονδριακή RNase P διαθέτει μια RNA και μια πρωτεϊνική υπομονάδα που κωδικοποιούνται από ένα μιτοχονδριακό (rnpB) και ένα πυρηνικό γονίδιο, αντίστοιχα. Σε αυτήν την εργασία απομονώσαμε και μερικώς καθαρίσαμε την μιτοχονδριακή RNase P από τον Schizosaccharomyces pombe. Κλωνοποιήθηκε, επίσης, το γονίδιο που κωδικοποιεί την RNA υπομονάδα της μιτοχονδριακής RNase P. Αυτό το ένζυμο παρουσιάζει διαφορετική εξειδίκευση για τα υποστρώματα SupS1 (pre-tRNASer) και pre-tRNATyr και δεν απενεργοποιείται από την μικροκοκκική νουκλεάση. / Ribonuclease P is a universally conserved ribozyme that it is involved in the 5΄ maturation of precursors tRNAs. It is in most cases a ribonucleoprotein complex which comprises an RNA subunit and at least one protein subunit. Concerning the eukaruotic cells, it is expected that distinctive nuclear and mitochondrial RNase P activities exist. In Saccharomyces cerevisiae the mitochondrial RNase P consists of an RNA and a protein subunit encoded by a mitochondrial (rnpB) and a nuclear gene, respectively. In the present study we isolated and partially purified mitochondrial RNase P from Schizosaccharomyces pombe and we cloned the gene that encodes the mitochondrial RNase P RNA subunit. This enzyme exhibits different specificity on SupS1 and pre-tRNATyr substrates and is not inactivated by micrococcal nuclease.

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