Current Search: Self, William (x)
View All Items
(41 - 48 of 48)
Pages
- Title
- Biochemical Studies of ABCE1.
- Creator
-
Sims, Lynn, Igarashi, Robert, Ebert, Steven, Self, William, Moore, Sean, University of Central Florida
- Abstract / Description
-
The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and...
Show moreThe growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo-PabABCE1 increases (>)30 fold from ~30 (&)#181;M to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge to being dependent on energy charge with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1's ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.
Show less - Date Issued
- 2012
- Identifier
- CFE0004600, ucf:49204
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004600
- Title
- Defective Dynamics of Mitochondria in Amyotrophic Lateral Sclerosis and Huntington's Disease.
- Creator
-
Song, Wenjun, Bossy-Wetzel, Ella, Fernandez-Valle, Cristina, Cheng, Zixi, Self, William, University of Central Florida
- Abstract / Description
-
Mitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1),...
Show moreMitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), which are essential for mitochondrial function. Mutations in the mitochondrial fission and fusion machinery lead to neurodegeneration. Thus, whether defective mitochondrial dynamics participates in ALS and HD requires further investigation.ALS is a fatal neurodegenerative disease characterized by upper and lower motor neuron loss. Mutations in Cu/Zn superoxide dismutase (SOD1) cause the most common familiar form of ALS by mechanisms not fully understood. Here, a new motor neuron-astrocyte co-culture system was created and live-cell imaging was used to evaluate mitochondrial dynamics. Excessive mitochondrial fission was observed in mutant SOD1G93A motor neurons, correlating with impaired axonal transport and neuronal cell death. Inhibition of mitochondrial fission restored mitochondrial dynamics and protected neurons against SOD1G93A-induced mitochondrial fragmentation and neuronal cell death, implicating defects in mitochondrial dynamics in ALS pathogenesis.HD is an inherited neurodegenerative disorder caused by glutamine (Q) expansion in the polyQ region of the huntingtin (HTT) protein. In the current work, mutant HTT caused mitochondrial fragmentation in a polyQ-dependent manner in both primary cortical neurons and fibroblasts from human patients. An abnormal interaction between DRP1 and HTT was observed in mutant HTT mice and inhibition of mitochondrial fission or promotion of mitochondrial fusion restored mitochondrial dynamics and protected neurons against mutant HTT-induced cell death. Thus, mutant HTT may increase mitochondrial fission by elevating DRP1 GTPase activity, suggesting that mitochondrial dynamics plays a causal role in HD.In summary, rebalanced mitochondrial fission and fusion rescues neuronal cell death in ALS and HD, suggesting that mitochondrial dynamics could be the molecular mechanism underlying these diseases. Furthermore, DRP1 might be a therapeutic target to delay or prevent neurodegeneration.
Show less - Date Issued
- 2012
- Identifier
- CFE0004444, ucf:49356
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004444
- Title
- Delineating key genetic components on linear plasmid 36 that contribute to its essential role in Borrelia burgdorferi mammalian infectivity.
- Creator
-
Choudhury, Tisha, Jewett, Mollie, Khaled, Annette, Self, William, Cole, Alexander, University of Central Florida
- Abstract / Description
-
The spirochete Borrelia burgdorferi is the etiologic agent of Lyme disease. This pathogen has a complex enzootic life cycle that involves passage between the tick vector (Ixodes scapularis) and various vertebrate hosts with humans being inadvertent hosts. There is a pressing need to study the genetic aspects of the B. burgdorferi infectious cycle and particularly spirochete genes involved in mammalian infectivity so as to develop novel therapeutic and diagnostic strategies to combat Lyme...
Show moreThe spirochete Borrelia burgdorferi is the etiologic agent of Lyme disease. This pathogen has a complex enzootic life cycle that involves passage between the tick vector (Ixodes scapularis) and various vertebrate hosts with humans being inadvertent hosts. There is a pressing need to study the genetic aspects of the B. burgdorferi infectious cycle and particularly spirochete genes involved in mammalian infectivity so as to develop novel therapeutic and diagnostic strategies to combat Lyme disease. The B. burgdorferi genome is fragmented and comprised of a single 900 kb linear chromosome and multiple linear and circular plasmids. It has been observed that plasmids are lost during serial passage and manipulation in vitro and the loss of some of the plasmids has been shown to be related to the loss of infectivity and persistence in the host. One such plasmid is linear plasmid 36 (lp36). lp36 is approximately 36kb in size and carries 56 putative open reading frames a majority of which have no predicted function. B. burgdorferi lacking lp36 show no deficiency in survival in ticks; however, these mutant spirochetes are highly attenuated for mammalian infectivity. The genetic components of this plasmid that contribute to its function in mammalian infectivity have yet to be clearly defined. Using an in vivo expression technology (IVET) based genetic screen the lp36-encoded gene bbk46 was identified as a candidate B. burgdorferi gene that is expressed during mammalian infection. Herein we present evidence that bbk46 is required for B. burgdorferi persistent infection of immunocompetent mice. Our data support a molecular model of immune evasion by which bbk46 functions as an RNA to regulate expression of the antigenic variation protein VlsE. These data represent the first demonstration of a regulatory mechanism critical for controlling vlsE gene expression. Moreover these findings further define the critical role of linear plasmid 36 in Borrelia burgdorferi pathogenesis.
Show less - Date Issued
- 2013
- Identifier
- CFE0004982, ucf:49566
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004982
- Title
- Development of Molecular Diagnostic Tools for Mycobacterium Species.
- Creator
-
Bengtson, Hillary, Kolpashchikov, Dmitry, Rohde, Kyle, Self, William, Jewett, Travis, Masternak, Michal, University of Central Florida
- Abstract / Description
-
This dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and...
Show moreThis dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and the leading cause of infectious disease related deaths worldwide. The control of TB is limited by the lack of sensitive and specific diagnostic tools available at the point of care (POC). The studies presented here illustrate the advances in our technology for the detection and differentiation of M.tb and NTM. The use of BiDz sensors enables the selective recognition of DNA/RNA analytes containing single nucleotide polymorphisms associated with species-specific identification, drug susceptibility testing (DST) and strain typing. First, we developed a platform for the detection of M.tb and drug susceptibility using multiplex PCR and BiDz sensors. However, this method relies on the use of expensive instrumentation which is often not available in high TB burden countries. Therefore, additional studies focused on the development of tools for the detection of isothermal amplification products and the direct detection of rRNA. Based on these findings, we also developed an NTM species typing tool using BiDz sensors for species identification in ~1 hour. Despite the advantages of BiDz sensor technology, their use is limited to the detection of a few selected mutations. To address this limitation, we developed a 15-loci multiplex PCR followed by analysis with a custom microarray for high-throughput identification of SNPs. The work presented in this dissertation has the potential to enable the rapid, specific and sensitive identification of mycobacterial species necessary to reduce the diagnostic delay, ensure initiation of effective therapy, and prevent further transmission.
Show less - Date Issued
- 2017
- Identifier
- CFE0006856, ucf:51735
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006856
- Title
- Cerium oxide nanoparticles act as a unique catalyst and scavenge nitric oxide and peroxynitrite and decrease RNS in vitro and in vivo.
- Creator
-
Dowding, Janet, Self, William, Bossy-Wetzel, Ella, Zervos, Antonis, Seal, Sudipta, Santra, Swadeshmukul, University of Central Florida
- Abstract / Description
-
Cerium oxide nanoparticles (CeO2 NPs)(nanoceria) have been shown to possess a substantial oxygen storage capacity via the interchangeable surface reduction and oxidation of cerium atoms, cycling between the Ce4+ and Ce3+ redox states. Reduction of Ce4+ to Ce3+ causes oxygen vacancies or defects on the surface of the crystalline lattice structure of the particles, generating a cage for redox reactions to occur. The study of the chemical and biological properties of CeO2 NPs has expanded...
Show moreCerium oxide nanoparticles (CeO2 NPs)(nanoceria) have been shown to possess a substantial oxygen storage capacity via the interchangeable surface reduction and oxidation of cerium atoms, cycling between the Ce4+ and Ce3+ redox states. Reduction of Ce4+ to Ce3+ causes oxygen vacancies or defects on the surface of the crystalline lattice structure of the particles, generating a cage for redox reactions to occur. The study of the chemical and biological properties of CeO2 NPs has expanded recently, and the methods used to synthesize these materials are also quite diverse. This has led to a plethora of studies describing various preparations of CeO2 NPs for potential use in both industry and for biomedical research. Our own work has centered on studies that measure the ability of water-based CeO2 NPs materials to reduce reactive oxygen and nitrogen species in biological systems, and correlating changes in surface chemistry and charge to the catalytic nature of the particles. The application in experimental and biomedical research of CeO2 NPs began with the discovery that water-based cerium oxide nanoparticles could act as superoxide dismutase mimetics followed by their ability to reduce hydrogen dioxide similar to catalase. While their ROS scavenging ability was well established, their ability to interact with specific RNS species, specifically nitric oxide (NO) or peroxynitrite (ONOO-) was not known. The studies described in this dissertation focus on the study of RNS and cerium oxide nanoparticles.Our in vitro work revealed that CeO2 NPs that have higher levels of reduced cerium sites (3+) at the surface (which are effective SOD mimetics) are also capable of accelerating the decay of peroxynitrite in vitro. In contrast, CeO2 NPs that have fewer reduced cerium sites at the particle surface (which also exhibit better catalase mimetic activity) have NO scavenging capabilities as well as some reactivity with peroxynitrite. Our studies and many others have shown cerium oxide nanoparticles can reduce ROS and RNS in cell culture or animal models. The accumulation of ROS and RNS is a common feature of many diseases including Alzheimer's disease (AD). Testing our CeO2 NPS in cortical neurons, we used addition of A? peptide as an AD model system. CeO2 NPs delayed A?-induced mitochondrial fragmentation and neuronal cell death. When mitochondrial ROS levels are increased, mitochondrial fission is activated by DRP1 S616 phosphorylation. Specifically, our studies showed the reduction of phosphorylated DRP1 S616 in the presence of CeO2 NPs. Results from our studies have begun to unravel the molecule mechanism behind the catalytic nature of how CeO2 NPs reduce ROS/RNS in biological systems and represents an important step forward to test the potential neuroprotective effects of CeO2 NPs in model systems of AD.A plethora of studies describing various preparations of CeO2 NPs for potential use in both industry and for biomedical research have been described in the past five years. It has become apparent that the outcomes of CeO2 NPs exposure can vary as much as the synthesis methods and cell types tested. In an effort to understand the disparity in reports describing the toxicity or protective effects of exposure to CeO2 NPs, we compared CeO2 NPs synthesized by three different methods; H2O2 (CNP1), NH4OH (CNP2) or hexamethylenetetramine (HMT-CNP1). Exposure to HMT-CNP1 led to reduced metabolic activity (MTT) at a 10-fold lower concentration than CNP1 or CNP2 and surprisingly, exposure to HMT-CNP1 led to substantial decreases in the ATP levels. Mechanistic studies revealed that HMT-CNP1 and CNP2 exhibited robust ATPase (phosphatase) activity, whereas CNP1 lacked ATPase activity. HMT-CNP1 were taken up into HUVECs far more efficiently than the other preparations of CeO2 NPs. Taken together, these results suggest the combination of increased uptake and ATPase activity of HMT-CNP1 may underlie the mechanism of the toxicity of this preparation of CeO2 NPs, and may suggest ATPase activity should be considered when synthesizing CeO2 NPs for use in biomedical applications. Overall the studies have uncovered two new catalytic activities for water-based CeO2 NPs (NO scavenging and accelerated decay of peroxynitrite), demonstrated their ability to reduce RNS in an AD cell culture model as well as identifying a catalytic activity (phosphatase) that may underlie the observed toxicity of CeO2 NPs reported in other studies.
Show less - Date Issued
- 2012
- Identifier
- CFE0004782, ucf:49783
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004782
- Title
- The Role of Mitochondrial Omi/HtrA2 Protease in Protein Quality Control and Mitophagy.
- Creator
-
Ambivero, Camilla, Zervos, Antonis, Teter, Kenneth, Siddiqi, Shadab, Self, William, University of Central Florida
- Abstract / Description
-
Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors...
Show moreOmi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a Lys-63 deubiquitinating (DUB) complex. In addition, we found the mechanism by which Omi/HtrA2 protease participates in mitophagy by directly regulating the protein level of Mulan E3 ubiquitin ligase, especially during mitochondrial stress.Abro1 is a scaffold protein of the DUB complex known as BRISC (BRCC36 isopeptidase complex). In addition, Abro1 is involved in a cytoprotective pathway and is regulated by Omi/HtrA2. Three specific interactors of Abro1 protein were identified, ATF4, ATF5 and JunD, all members of the activating protein 1 (AP-1) family. We focused our studies on ATF4 since, like Abro1, it is ubiquitously expressed and is important in cell cycle regulation and survival. Abro1's interaction with ATF4 was specific and occurred only when cells were stressed. The significance of this interaction was the translocation of Abro1 from the cytoplasm to the cell nucleus. These results establish a new cytoprotective function of cytoplasmic Omi/HtrA2 as a regulator of the BRISC DUB complex.Furthermore, we have recently identified the mitochondrial Mulan E3 ubiquitin ligase as a substrate of Omi/HtrA2 protease. Mulan, along with MARCH5/MITOL and RNF185, are the only three mitochondrial E3 ubiquitin ligases identified thus far. The function of Mulan has been linked to cell growth, cell death, and autophagy/mitophagy. To investigate Mulan's function and its control by Omi/HtrA2, E2 conjugating enzymes that form a complex with Mulan E3 ligase were identified. Four specific interacting E2s were isolated, namely Ube2E2, Ube2E3, Ube2G2, and Ube2L3. To identify substrates for each unique Mulan-E2 complex, fusion baits were used in a modified yeast two-hybrid screen. Our results suggest that Mulan participates in various pathways, depending on the nature of its E2 conjugating enzyme partner. One of the interactors isolated against the Mulan-Ube2E3 bait was the GABARAP (GABAA receptor-associated protein), a member of the Atg8 family. We characterized this interaction both in vitro and in vivo and its potential role in mitophagy. Our studies defined a new pathway by which Mulan participates in mitophagy by recruiting GABARAP to the mitochondria.
Show less - Date Issued
- 2013
- Identifier
- CFE0004805, ucf:49752
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004805
- Title
- Preparation, Characterization, and Delivery of Antibodies Binding to a Model Oncogenic RNA, Human Initiator tRNA.
- Creator
-
Archer, Jennifer, Santra, Swadeshmukul, Ye, Jingdong, Ye, Jingdong, Self, William, Khaled, Annette, University of Central Florida
- Abstract / Description
-
Non-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for...
Show moreNon-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for gene regulation and other functional roles and have also found to contribute to pathogenesis in humans. The increased size and structural complexity require novel tools to study their interactions beyond RNA interference. Synthetic antibodies are classic tools and therapeutics utilized to study and treat proteins involved in human disease. Likewise we hypothesize that structured RNAs can also take advantage of synthetic antibodies to probe their functions and be utilized as therapeutics.Currently, antibodies have been raised against microbial riboswitches and other structured RNAs of single-celled organisms, and only one human structured RNA to the best of our knowledge. However, no one has yet to create a synthetic antibody capable of behaving as a therapeutic against a structured RNA. We therefore sought to raise an antibody Fab against a structured RNA, human initiator tRNA, a model oncogenic non-coding RNA and demonstrate its efficacy in vitro. We then characterized the antibody and explored delivery options in cancer cells including the use of nanoparticle delivery systems. With the emerging transcriptome revealing new ncRNAs implicated in human disease, our research has begun to address a new therapeutic strategy, laying down the foundation for the future of structured RNA-targeted therapies.
Show less - Date Issued
- 2014
- Identifier
- CFE0005756, ucf:50072
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005756
- Title
- Structural and Functional Studies of Glycine Riboswitches and Development of Fab Chaperone Assisted RNA Crystallography.
- Creator
-
Sherman, Eileen, Ye, Jingdong, Kolpashchikov, Dmitry, Koculi, Eda, Harper, James, Self, William, University of Central Florida
- Abstract / Description
-
The glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few...
Show moreThe glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few nucleotides upstream of the established glycine riboswitch sequence which changed its ligand binding characteristics (Chapter 1). Previously, the two aptamers of the riboswitch were thought to cooperatively bind glycine, but with the inclusion of this leader sequence which forms a kink turn motif with the linker between the two aptamers, glycine binding in one aptamer no longer requires glycine binding in the other. Furthermore, the Kd from three species tested are now a similar, lower value of about 5 (&)#181;M, indicating authenticity of this new consensus sequence. Glycine binding and interaptamer interaction both enhanced one another in trans aptamer assays. Another discovery from this was a shortened construct including all of aptamer II but only part of aptamer I in which a few specific nucleotides prevented glycine binding in aptamer II (Chapter 2). This may provide insight into the nature of interaptamer interactions in the full switch; addition of an oligonucleotide complimentary to these nucleotides restored glycine binding ability to aptamer II. With future development, this could also be a useful molecular biology tool, using two signals, glycine and an oligonucleotide, to allow gene expression.To precisely understand how any macromolecule functions, a 3D structure, obtainable by x-ray crystallography, is vital. A new technique to accomplish that for RNA, precedented in the protein world, is Fab chaperoned crystallography, which has advantages compared to RNA alone. A phage displayed library of Fabs with reduced codon diversity designed for RNA was created, the YSGR Min library (Chapter 3). Its Fabs had specificities and affinities equal to or greater than previous libraries which were originally created for phage displayed selection against proteins. Fab chaperoned RNA crystallography is currently in progress for the glycine riboswitch; the best resolution thus far is 5.3 (&)#197; (Chapter 4). In addition to providing molecular insight into its gene regulation mechanism, a structure of the glycine riboswitch could be applied for use in structure based drug design of novel antibiotics targeting the riboswitch to disrupt important downstream carbon cycle genes in pathogenic bacteria.
Show less - Date Issued
- 2014
- Identifier
- CFE0005549, ucf:50285
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005549
