The first committed step in DNA biosynthesis occurs by direct reduction of ribonucleotides. This reduction is catalyzed by ribonucleotide reductase (RNR), an enzyme which uses a unique radical mechanism to facilitate the transformation. All four DNA precursors are synthesized by a single enzyme. Therefore, an intricate pattern of regulation has evolved to insure that RNR generates the proper quantity of each deoxyribonucleotide. It is this regulation, and conditions that influence this regulation, that are the central focal points of this dissertation. The studies described in this thesis have been aided by the development of a novel RNR assay. Unlike the traditional assay, this new procedure permits the simultaneous monitoring of all four RNR activities. This four-substrate assay was used to investigate whether the four enzyme activities of RNR were differentially sensitive to inhibition by the radical scavenger, hydroxyurea. The assay results, along with the results of a technique that measured enzyme inhibition as a function of radical decay, suggest that all activities of RNR are equally inhibited by hydroxyurea. Instead of differential inhibition, it appears that the activity level of RNR determines the relative sensitivity to hydroxyurea. The effects of nucleotide effectors and substrates on the relative turnover rates of the vaccinia virus and T4 phage RNR were also investigated by use of the four-substrate assay. When physiological concentrations of the allosteric effectors and substrates were added to the reaction mixtures, both enzyme forms produced dNDPs in ratios that approximate the nucleotide composition of their respective genomes. Non-physiological nucleotide concentrations generated significantly different product profiles, indicating that RNR has evolved to function within a defined nucleotide environment. Interestingly, the substrate component of the nucleotide environment proved to be as important as the allosteric effectors in modulating the reaction rates. Although the allosteric effects of nucleoside triphosphates have been known for some time, little attention has been given to the potential role that substrates play in the regulation of RNR. The results from my research suggest that the regulation of RNR in vivo results from a complex interplay between the enzyme and its substrates, products, and allosteric effectors. / Graduation date: 1998

The function of biomolecules is very sensitive to structure. Folding in proteins and nucleic acids is a hierarchical process progressing from primary to secondary, then tertiary, and finally, quaternary structures. RNA in its folded form performs a variety of biological activities. Obtaining intramolecular distance measurements makes it possible to generate structural models along the folding pathway that may be related to the overall function of the molecule. Distances can be measured by Site-Directed Spin-Labeling (SDSL), in which nitroxyl spin-label probes are attached and observed by EPR spectroscopy. Spin-labels can provide information concerning structure and conformational changes because they are particularly sensitive to molecular motion and interspin distances. Continuous-wave EPR spectroscopy has been commonly applied to detect and monitor nitroxide spin-label probes within biological systems. A previous published SDSL study from this laboratory investigated a 10-mer RNA duplex model system with spin-label probe succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate; however, an increased spin-labeling efficiency was observed with an isocyanate derivative of tetramethylpiperidyl-N-oxy (TEMPO). In this thesis, a 4-isocyano TEMPO spin-label probe replaced the previously used succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate in 10-mer SDSL studies. The influence of labeling with the 4-iscocyano TEMPO spin-label in a 10-mer RNA model system was investigated with thermal denaturation, Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF-MS), Electron Paramagnetic Resonance (EPR) spectroscopy, and reverse phase high performance liquid chromatography (RP-HPLC). In the 10-mer RNA duplex model system a 4-isocyano TEMPO spin-label is individually attached to one strand and two strands are annealed to measure distances. This methodology is limited to systems in which two oligonucleotides are annealed together. To circumvent this limitation and also to explore single-strand dynamics a new methodology was implemented, double spin-labeling. Double spin-labeled single-stranded RNA was investigated as a single-strand and within a duplex via MALDI-TOF-MS, EPR spectroscopy and RP-HPLC. A double spin-labeling strategy in this work will be applicable to large complex RNAs like Group I intron of Tetrahymena thermophilia.

The Varkud Satellite (VS) ribozyme is the largest of the “small” nucleolytic ribozymes and is the only one for which there are no high resolution crystal structures available. The VS ribozyme comprises a catalytic domain and a substrate domain. The catalytic domain includes five helices that interact with the stem-loop substrate. The substrate is docked within a cleft that is formed by helices II and VI. This naturally brings the cleavage site in close proximity to the A730 loop in helix VI. The adenines within the A730 loop are very crucial to the cleavage reaction and any substitution causes a major decrease in the cleavage activity of the ribozyme. This study is aimed at designing and producing a variant of the Varkud Satellite ribozyme that consists of multiple parts that can be used for detailed studies of ribozyme kinetics and assembly.

The Varkud Satellite (VS) ribozyme is the largest of the “small” nucleolytic ribozymes and is the only one for which there are no high resolution crystal structures available. The VS ribozyme comprises a catalytic domain and a substrate domain. The catalytic domain includes five helices that interact with the stem-loop substrate. The substrate is docked within a cleft that is formed by helices II and VI. This naturally brings the cleavage site in close proximity to the A730 loop in helix VI. The adenines within the A730 loop are very crucial to the cleavage reaction and any substitution causes a major decrease in the cleavage activity of the ribozyme. This study is aimed at designing and producing a variant of the Varkud Satellite ribozyme that consists of multiple parts that can be used for detailed studies of ribozyme kinetics and assembly.

Plant luteoviral RNA viruses employ -1 frameshifting for the production of P1 and P1-P2 fusion proteins important for viral replication. Luteoviral pseudoknots are characterized by three adenosines in the 3' side of loop L2 known to be important for maintaining frameshifting efficiency and pseudoknot stability. A proposed P1-P2 mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) was of interest since it contained two adenosine to cytidine substitutions in L2. Functional analysis shows that the in vitro frameshifting efficiency is greater (~15%) than any other luteoviral pseudoknot. The NMR-derived solution structure of the ScYLV RNA pseudoknot shows that C25 is looped out of the triplex structure and the 3' most L2 cytidine (C27) and A24 form cis Watson-Crick/sugar-edge interactions with C14 and C15 in stem S1, respectively. Thus, the ScYLV pseudoknot maintains a similar triple helical architecture as other luteoviral pseudoknots. Surprisingly, the frameshifting efficiency of the C27A ScYLV pseudoknot is decreased by ~8 fold relative to wild-type ScYLV. The solution structure of the C27A ScYLV RNA exhibits a global fold similar to the wild-type RNA; however, distinct hydrogen bonding interactions at the helical junction are observed. Specifically, C8+ in the C8+ major groove base triple moves ~2.3 relative to the accepting (G12-C28) base pair relative to the WT RNA. New NMR experiments have been developed and/or applied to confirm Watson-Crick base pairs and tertiary structural interactions in the PEMV-1 and ScYLV pseudoknots by direct observation of trans hydrogen bond scalar couplings. In addition, intrabase couplings in cytidine and adenosine have been measured, providing a valuable tool for the assignment of amino and N3/N1 resonances in RNA. Finally, thermodynamic analysis of the pairwise coupling between the major groove and minor groove tertiary structural hydrogen bonds at the helical junction have been investigated by monitoring the thermal unfolding of WT, dC14, C27A, and dC14/C27A RNAs as a function of pH. Favorable pairwise coupling characterized the WT ScYLV and BWYV RNAs, while unfavorable coupling characterized the poorly functional C27A ScYLV RNA. The implications of these structural, functional, and thermodynamic findings on the mechanism of frameshift stimulation is discussed.

The function of biomolecules is very sensitive to structure. Folding in proteins and nucleic acids is a hierarchical process progressing from primary to secondary, then tertiary, and finally, quaternary structures. RNA in its folded form performs a variety of biological activities. Obtaining intramolecular distance measurements makes it possible to generate structural models along the folding pathway that may be related to the overall function of the molecule. Distances can be measured by Site-Directed Spin-Labeling (SDSL), in which nitroxyl spin-label probes are attached and observed by EPR spectroscopy. Spin-labels can provide information concerning structure and conformational changes because they are particularly sensitive to molecular motion and interspin distances. Continuous-wave EPR spectroscopy has been commonly applied to detect and monitor nitroxide spin-label probes within biological systems. A previous published SDSL study from this laboratory investigated a 10-mer RNA duplex model system with spin-label probe succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate; however, an increased spin-labeling efficiency was observed with an isocyanate derivative of tetramethylpiperidyl-N-oxy (TEMPO). In this thesis, a 4-isocyano TEMPO spin-label probe replaced the previously used succinimdyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-carboxylate in 10-mer SDSL studies. The influence of labeling with the 4-iscocyano TEMPO spin-label in a 10-mer RNA model system was investigated with thermal denaturation, Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF-MS), Electron Paramagnetic Resonance (EPR) spectroscopy, and reverse phase high performance liquid chromatography (RP-HPLC). In the 10-mer RNA duplex model system a 4-isocyano TEMPO spin-label is individually attached to one strand and two strands are annealed to measure distances. This methodology is limited to systems in which two oligonucleotides are annealed together. To circumvent this limitation and also to explore single-strand dynamics a new methodology was implemented, double spin-labeling. Double spin-labeled single-stranded RNA was investigated as a single-strand and within a duplex via MALDI-TOF-MS, EPR spectroscopy and RP-HPLC. A double spin-labeling strategy in this work will be applicable to large complex RNAs like Group I intron of Tetrahymena thermophilia.

Telomeres are critical for the integrity of eukaryotic genomes. They function to protect chromosome ends from DNA damage surveillance and inappropriate repair. Telomeres are maintained by the specialized ribonucleoprotein complex telomerase. Without telomerase, telomere shortening would ultimately lead to compromised genome stability and cellular senescence. Therefore, telomerase function is necessary for extension of the proliferative capacity of the cell. In this dissertation, we describe the characterization of core components of telomerase ribonucleoprotein complex in the flowering plant, Arabidopsis thaliana. We find that dyskerin, one of the core telomerase components in humans is also conserved in Arabidopsis telomerase. Arabidopsis dyskerin associates with the telomerase RNP in an RNA-dependent manner and is required for telomere length maintenance in this organism. We also describe the characterization of another core telomerase component, the telomerase RNA subunits (TERs). Unexpectedly, we uncovered two distinct TER subunits that share a region of high identity. The two TERs named TER1G7 and TER5G2, based on their chromosomal positions, display differences in their expression levels and their association with telomere-related proteins. Both TERs can serve as templates for telomerase in vitro. Through genetic analyses, we show a templating function for TER1G7 in vivo and a novel role for TER5G2 as a negative regulator of telomerase. Finally, the presence of TER genes in other plant species was investigated and evidence for duplication of TER genes in plants closely related to Arabidopsis was obtained. We also show evidence for a template mutation in Asparagus TER that could lead to variant repeats in this organism. In summary, the studies presented in this dissertation reveal that Arabidopsis telomerase shares both similarities and differences with other telomerase RNPs, making it an exciting model system for study of telomere biology.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 71-76). Available also in a digital version from Dissertation Abstracts.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 99-101).

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.