Matching Items (7)
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- All Subjects: RNA
- All Subjects: Enzymes
- Creators: Ghirlanda, Giovanna
Description
Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, which retain function in trans. The in vitro reconstituted Neurospora telomerase is highly processive, synthesizing canonical TTAGGG repeats. Similar to Schizosaccharomycetes pombe, filamentous fungal TERs utilize the spliceosomal splicing machinery for 3' processing. Neurospora telomerase, while associating with the Est1 protein in vivo, does not bind homologous Ku or Sm proteins found in both budding and fission yeast telomerase holoenzyme, suggesting a unique biogenesis pathway. The development of Neurospora as a model organism to study telomeres and telomerase may shed light upon the evolution of the canonical TTAGGG telomeric repeat and telomerase processivity within fungal species.
ContributorsQi, Xiaodong (Author) / Chen, Julian (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
Telomerase is a specialized enzyme that adds telomeric DNA repeats to the chromosome ends to counterbalance the progressive telomere shortening over cell divisions. It has two essential core components, a catalytic telomerase reverse transcriptase protein (TERT), and a telomerase RNA (TR). TERT synthesizes telomeric DNA by reverse transcribing a short template sequence in TR. Unlike TERT, TR is extremely divergent in size, sequence and structure and has only been identified in three evolutionarily distant groups. The lack of knowledge on TR from important model organisms has been a roadblock for vigorous studies on telomerase regulation. To address this issue, a novel in vitro system combining deep-sequencing and bioinformatics search was developed to discover TR from new phylogenetic groups. The system has been validated by the successful identification of TR from echinoderm purple sea urchin Strongylocentrotus purpuratus. The sea urchin TR (spTR) is the first invertebrate TR that has been identified and can serve as a model for understanding how the vertebrate TR evolved with vertebrate-specific traits. By using phylogenetic comparative analysis, the secondary structure of spTR was determined. The spTR secondary structure reveals unique sea urchin specific structure elements as well as homologous structural features shared by TR from other organisms. This study enhanced the understanding of telomerase mechanism and the evolution of telomerase RNP. The system that was used to identity telomerase RNA can be employed for the discovery of other TR as well as the discovery of novel RNA from other RNP complex.
ContributorsLi, Yang (Author) / Chen, Julian Jl (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences were isolated from a pool of random TNA molecules. This implicates a possible chemical framework capable of crosstalk between TNA and RNA. Further, this shows that heredity and evolution are not limited to the natural genetic system based on ribofuranosyl nucleic acids. Another alternative genetic system, glycerol nucleic acid (GNA) undergoes intrasystem pairing with superior thermalstability compared to that of DNA. Inspired by this property, I demonstrated a minimal nanostructure composed of both left- and right-handed mirro image GNA. This work suggested that GNA could be useful as promising orthogonal material in structural DNA nanotechnology.
ContributorsZhang, Su (Author) / Chaut, John C (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2011
Description
Quercetin 2,3-dioxygenase from Bacillus subtilis has been identified and characterized as the first known prokaryotic quercetinase. This enzyme catalyzes the cleavage of the O-heteroaromatic ring of the flavonol quercetin to the corresponding depside and carbon monoxide. The first quercetinase was characterized from a species of Aspergillus genus, and was found to contain one Cu2+ per subunit. For many years, it was thought that the B. subtilis quercetinase contained two Fe2+ ions per subunit; however, it has since been discovered that Mn2+ is a much more likely cofactor. Studies of overexpressed bacterial enzyme in E. coli indicated that this enzyme may be active with other metal ions (e.g. Co2+); however, the production of enzyme with full metal incorporation has only been possible with Mn2+. This study explores the notion that metal manipulation after translation, by partially unfolding the enzyme, chelating the metal ions, and then refolding the protein in the presence of an excess of divalent metal ions, could generate enzyme with full metal occupancy. The protocols presented here included testing for activity after incubating purified quercetinase with EDTA, DDTC, imidazole and GndHCl. It was found that the metal chelators had little to no effect on quercetinase activity. Imidazole did appear to inhibit the enzyme at concentrations in the millimolar range. In addition, the quercetinase was denatured in GndHCl at concentrations above 1 M. Recovering an active enzyme after partial or complete unfolding proved difficult, if not impossible.
ContributorsKrojanker, Elan Daniel (Author) / Francisco, Wilson (Thesis director) / Allen, James P. (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry. Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis. Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and â-galactosidase (â-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays. Several peptides, selected from microarrays, were able to inhibit â-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation. PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry.
ContributorsFu, Jinglin (Author) / Woodbury, Neal W (Thesis advisor) / Johnston, Stephen A. (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
Enzymes keep life nicely humming along by catalyzing important reactions at relevant timescales. Despite their immediate importance, how enzymes recognize and bind their substrate in a sea of cytosolic small molecules, carry out the reaction, and release their product in microseconds is still relatively opaque. Methods to elucidate enzyme substrate specificity indicate that the shape of the active site and the amino acid residues therein play a major role. However, lessons from Directed Evolution experiments reveal the importance of residues far from the active site in modulating substrate specificity. Enzymes are dynamic macromolecules composed of networks of interactions integrating the active site, where the chemistry occurs, to the rest of the protein. The objective of this work is to develop computational methods to modify enzyme ligand specificity, either through molding the active site to accommodate a novel ligand, or by identifying distal mutations that can allosterically alter specificity. To this end, two homologues in the β-lactamase family of enzymes, TEM-1, and an ancestrally reconstructed variant, GNCA, were studied to identify whether the modulation of position-specific distal-residue flexibility could modify ligand specificity. RosettaDesign was used to create TEM-1 variants with altered dynamic patterns. Experimental characterization of ten designed proteins indicated that mutations to residues surrounding rigid, highly coupled residues substantially affected both enzymatic activity and stability. In contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Five of the TEM-1 variants were crystallized to see if the changes in function observed were due to architectural changes to the active site. In a second project, a computational platform using RosettaDesign was developed to remodel the firefly luciferase active site to accommodate novel luciferins. This platform resulted in the development of five luciferin-luciferase pairs with red-shifted emission maxima, ready for multicomponent bioluminescent imaging applications in tissues. Although the projects from this work focus on two classes of proteins, they provide insight into the structure-function relationship of ligand specificity in enzymes and are broadly applicable to other systems.
ContributorsKolbaba Kartchner, Bethany (Author) / Mills, Jeremy H (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2023
Description
Telomerase is a unique reverse transcriptase that has evolved specifically to extend the single stranded DNA at the 3' ends of chromosomes. To achieve this, telomerase uses a small section of its integral RNA subunit (TR) to reiteratively copy a short, canonically 6-nt, sequence repeatedly in a processive manner using a complex and currently poorly understood mechanism of template translocation to stop nucleotide addition, regenerate its template, and then synthesize a new repeat. In this study, several novel interactions between the telomerase protein and RNA components along with the DNA substrate are identified and characterized which come together to allow active telomerase repeat addition. First, this study shows that the sequence of the RNA/DNA duplex holds a unique, single nucleotide signal which pauses DNA synthesis at the end of the canonical template sequence. Further characterization of this sequence dependent pause signal reveals that the template sequence alone can produce telomerase products with the characteristic 6-nt pattern, but also works cooperatively with another RNA structural element for proper template boundary definition. Finally, mutational analysis is used on several regions of the protein and RNA components of telomerase to identify crucial determinates of telomerase assembly and processive repeat synthesis. Together, these results shed new light on how telomerase coordinates its complex catalytic cycle.
ContributorsBrown, Andrew F (Author) / Chen, Julian J. L. (Thesis advisor) / Jones, Anne (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014