Date of Award
Summer 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Reiter, Nicholas
Second Advisor
Reid, Scott
Third Advisor
Timerghazin, Qadir
Abstract
RNA structures and RNA-protein interactions are studied as potential drug targets, biomarkers in cancer, and can be administered as vaccines. The cancer associated HOTAIR (HOX transcript antisense RNA) exists in higher vertebrates and interacts with chromatin remodeling enzymes. We examined the thermodynamic folding properties and structural propensity of the exonic regions of HOTAIR using biophysical methods and NMR spectroscopy. Different exons of HOTAIR contain variable degrees of structural heterogeneity. We identify one exonic region, exon 4, that adopts a stable and compact fold under low magnesium concentrations. Close agreement of NMR spectroscopy and chemical probing confirm conserved base pair interactions within helix 10 of exon 4 of the human HOTAIR long non-coding RNA (lncRNA). Unlike HOTAIR, the ribonuclease P (RNase P) exists in bacteria, archaea and eukarya. RNase P is a universal RNA-protein endonuclease that catalyzes 5′ precursor-tRNA (ptRNA) processing. Protein concentration and temperature dependent NMR studies were performed on a thermostable RNase P protein from Thermatoga maritima to understand its oligomerization properties. The identification of a monomeric P protein conformer from NMR relaxation data and chemical shift information provided new insight into the conformational dynamics of the P protein. Taken together, local structural changes of the P protein and the 5′ leader RNA facilitate optimal substrate alignment and catalytic activation of the RNase P holoenzyme. As RNase P is an essential enzyme in life, knowledge of the structural differences between pathogenic bacterial and human RNase P may help in the development of new antibiotic therapeutics that target RNase P. The enzyme activity of Mycobacterium tuberculosis RNase P was examined through 32P radioactivity assays, and multidimensional 2D/3D NMR spectroscopy was implemented to study the solution structure of the M. tuberculosis RNase P protein. A comparative analysis of the pathogenic and non-pathogenic RNase P proteins brings important structural insight into the development of antibiotics that target tuberculosis RNase P.