Date of Award
Summer 2019
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Chemistry
First Advisor
Reiter, Nicholas J.
Second Advisor
Yi, Chae
Third Advisor
Fiedler, Adam
Abstract
Transfer RNA (tRNA) maturation is an essential step that occurs after transcription and prior to the translation. In all domains of life, an RNA-based ribonuclease (RNase) P catalyzes the cleavage of the phosphodiester bond at the 5′ end of precursor tRNA. However, not all organelles utilize the RNA-based version of RNase P. A recent discovery altering the paradigm of RNase P has revealed that chloroplast organelles, parasitic trypanosomes, and the compact human mitochondrial (mt) genome rely solely on a protein-only 5′ tRNA processing enzyme. Structural and biochemical studies of the RNA-based and protein-based plant RNase P systems suggest that these distinct isoenzyme active centers are both comprised of conserved oxygen-rich, electronegative pockets that are stabilized by two catalytic metal ions. The similar mode of pre-tRNA substrate recognition and proposed similarities between the RNA-based and protein-only RNase P reaction mechanisms suggest an unprecedented example of convergent enzyme evolution. In the crystal structure of the PRoteinaceous Only Ribonuclease P 1 enzyme (PRORP1) (PDB: 4G24), at least four aspartate residues (D399D474D475D493) are involved in coordinating two catalytic Mg2+ metal ions. However, there is a lack of information about PRORP metal binding and the organization of the catalytic metal-ligand environment. In this study, we characterize the metal binding properties of the PRORP enzyme family found in Arabidopsis thaliana using Electron Paramagnetic Resonance (EPR) and Isothermal Titration Calorimetry (ITC). Results from these studies will help us to better understand the metal-ligand environment of the metallonuclease domain of PRORP enzymes. ITC results suggest that the PRORP2 homolog binds to Mg2+ with a stoichiometric mole ratio of 1:1.88. Endothermic binding of the (ΔH = 218.3 cal/mol) Mg2+ suggests non-favorable metal binding and this data is consistent with a high μM metal binding dissociation constant (Kd). Similarly, PRORP2 binds approximately 1.53 equivalents of Mn2+. Mn2+ binds 10-fold weaker than Mg2+ binding to PRORP2 and also undergoes non-favorable binding to PRORP2. Binding titrations of the PRORP3 homolog suggest that 1-2 metal ions are weakly associated with the active site. In addition to these metal binding experiments, analysis of EPR data suggest that PRORP2 bound Mn2+ exhibits monomeric binding, with Mn2+ oriented in a distorted octahedral geometry and a distinctive 90 G splitting pattern. In addition, a newly discovered form of PRORP from archaea (Aquifex aeolicus) was successfully purified in the soluble form and characterized by Thermofluor analysis and NMR spectroscopy. Structural studies of Aquifex aeolicus PRORP has been initiated and preliminary crystals have also been obtained. Aquifex aeolicus PRORP is smaller in size and is thought to resemble a minimal metallonuclease domain of RNase P. Modelling studies of the Aquifex aeolicus PRORP suggest that protein domain will be a useful candidate for structural and metal binding studies and serves as a valuable system to understanding the structural diversity of the PRORP enzyme active site.