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Browsing Theses and Dissertations by Subject "Archaea"
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Item Open Access Archaeal isoprenoid biosynthesis(Colorado State University. Libraries, 2018) Liman, Lie Stefanus Geraldy, author; Santangelo, Thomas, advisor; Laybourn, Paul, committee member; Peebles, Christie, committee memberMany high value natural products - including artemisinin, squalene, and farnesene – are isoprenoids. Efforts to commercially produce isoprenoids are often complicated by low concentrations of isoprenoid precursors and the toxicity of isoprenoids in common production platforms (i.e. bacteria and yeasts). Archaeal-based production platforms provide a potential solution to the precursor toxicity problems as archaea produce isoprenoids in large quantities to generate their unique membrane hydrocarbon chains. One roadblock to commercial archaeal isoprenoid production platforms is the uncharacterized pathway leading to isoprenoid precursor synthesis. This project details, genetically and biochemical, the first three steps in the proposed pathway of archaeal isoprenoid biosynthesis - from acetyl-CoA to mevalonate - in Thermococcus kodakarensis.Item Open Access Archaeal transcription and replication: new insights into transcription-coupled DNA repair and origin-independent DNA replication(Colorado State University. Libraries, 2017) Gehring, Alexandra Marie, author; Santangelo, Thomas, advisor; Argueso, J. Lucas, committee member; Nyborg, Jennifer K., committee member; Peersen, Olve B., committee memberThe three Domains of extant life use similar mechanisms for information processing systems. Although many aspects of replication, transcription and translation are universally conserved, the evolutionary history of the enzymes involved is not always clear and domain-specific differences are known. The transcription apparatus, especially the multi-subunit RNA polymerase (RNAP), has a clear evolutionary conservation across all Domains. Elucidating the mechanisms of the transcription apparatus in Archaea will help further understanding of underlying transcription mechanisms and regulation of those mechanisms, not only in Archaea but also in Bacteria and Eukarya. Conversely, the DNA replication machinery, most notably the replicative DNA polymerases, are distinct for each Domain. Any demonstration of the activities of the replication proteins, and especially discovery of unique pathways and mechanisms underlying replication helps to improve the understanding of the larger evolutionary questions surrounding DNA replication. The compact nature of archaeal genomes necessitates timely termination of transcription to prevent continued transcription of neighboring genes while ensuring complete transcription of the gene of interest. Transcription elongation is processive, and the transcription elongation complex is exceptionally stable. The disruption of this transcription elongation process, transcription termination, is an essential step in the transcription cycle. The presence of DNA lesions causes early termination of transcription in Bacteria and Eukarya. The results of this dissertation demonstrate this is also true in Archaea. Archaeal RNAP arrests transcription at DNA lesions and likely initiates transcription-coupled DNA repair (TCR) as will be soon demonstrated using in vivo techniques developed during this dissertation work. DNA replication is a highly regulated cellular process, particularly initiation of DNA replication. The long-standing replicon hypothesis states a trans-acting replication initiation protein must recognize a cis-acting DNA element, the origin of replication. For the 50 years after the replicon hypothesis was first posited, the replication hypothesis was supported in phages, Bacteria, Archaea, and Eukarya. The work presented in this dissertation describes the non-essentiality of Cdc6 and the origin of replication, and further demonstrates that origin-independent DNA replication is the mechanism by which Thermococcus kodakarensis replicates its genome. The results of this study and others in the field brings forward questions about the evolutionary history of DNA replication in all three Domains of extant life.Item Embargo DNA replication in the environmental extremes(Colorado State University. Libraries, 2024) Liman, Geraldy Lie Stefanus, author; Santangelo, Thomas J., advisor; Markus, Steven, committee member; Schauer, Grant, committee member; Sloan, Daniel, committee memberDNA replication is an essential biological process across all life on Earth. For the prokaryotic Archaea domain, which contains organisms that can thrive in inhospitable environments like hydrothermal vents or salt deposits in the Dead Sea, the cell machinery for these conserved processes have acclimated over the course of evolution to encourage survival. While the origin of replication (ori), a predetermined position within the genome where DNA replication starts, is conserved in all Domains, its significance is not equal between them. Surprisingly, the model hyperthermophilic archaeon, T. kodakarensis, replicates its genome without relying on origin-dependent replication (ODR), and instead, relies mostly on recombination-dependent replication (RDR). In fact, the ori in T. kodakarensis is dispensable from the organism without much phenotypic consequence. Although dispensable, ori persists after millions of years of evolution in this organism, suggesting some functional significance under certain conditions. Not to mention, archaeal replisomes are comprised of unique components that are distinct from the other two domains of life, though surprisingly more similar to those found in Eukarya. Central to all replisomes is the activity of the DNA polymerase (DNAP). Most archaeal organisms, except for the Creanarchaea, encode two main replicative DNAPs, the eukaryotic-like B-family DNAP (PolB) and the archaeal-specific D-family DNAP (PolD). In T. kodakarensis, PolD is the essential replicative DNAP while PolB is dispensable. This thesis aims to (1) characterize the activity and regulation of RadA, the main recombinase in Archaea, (2) characterize the exaptation of inteins to regulate DNA replication, (3) delineate the in vivo function(s) of PolB. Furthermore, I hope to further characterize DNA replication in the context of evolutionary biology and how it relates to the three Domains of life.Item Open Access Factor dependent archaeal transcription termination(Colorado State University. Libraries, 2017) Walker, Julie, author; Santangelo, Thomas J., advisor; Montgomery, Tai, committee member; Stargell, Laurie, committee member; Yao, Tingting, committee memberRNA polymerase activity is regulated by nascent RNA sequences, DNA template sequences and conserved transcription factors. Transcription factors regulate the activities of RNA polymerase (RNAP) at each stage of the transcription cycle: initiation, elongation, and termination. Many basal transcription factors with common ancestry are employed in eukaryotic and archaeal systems that directly bind to RNAP and influence intramolecular movements of RNAP and modulate DNA or RNA interactions. We describe and employ a flexible methodology to directly probe and quantify the binding of transcription factors to the archaeal RNAP in vivo. We demonstrate that binding of the conserved and essential archaeal transcription factor TFE to the archaeal RNAP is directed, in part, by interactions with the RpoE subunit of RNAP. As the surfaces involved are conserved in many eukaryotic and archaeal systems, the identified TFE-RNAP interactions are likely conserved in archaeal-eukaryal systems and represent an important point of contact that can influence the efficiency of transcription initiation. While many studies in archaea have focused on elucidating the mechanism of transcription initiation and elongation, studies on termination were slower to emerge. Transcription factors promoting initiation and elongation have been characterized in each Domain but transcription termination factors have only been identified in bacteria and eukarya. Here we characterize the first archaeal termination factor (termed Eta) capable of disrupting the transcription elongation complex, detail the rate of and requirements for Eta-mediated transcription termination and describe a role for Eta in transcription termination in vivo. Eta-mediated transcription termination is energy-dependent, requires upstream DNA sequences and disrupts transcription elongation complexes to release the nascent RNA to solution. Deletion of TK0566 (encoding Eta) is possible, but results in slow growth and renders cells sensitive to DNA damaging agents. Structure-function studies reveal that the N-terminal domain of Eta is not necessary for Eta-mediated termination in vitro, but Thermococcus kodakarensis cells lacking the N-terminal domain exhibit slow growth compared to parental strains. We report the first crystal structure of Eta that will undoubtedly lead to further structure-function analyses. The results obtained argue that the mechanisms employed by termination factors in archaea, eukarya, and bacteria to disrupt the transcription elongation complex may be conserved and that Eta stimulates release of stalled or arrested transcription elongation complexes.Item Open Access Post-initiation activities of the archaeal RNA polymerase in a chromatin landscape(Colorado State University. Libraries, 2020) Sanders, Travis James, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Peersen, Olve B., committee member; Ben-Hur, Asa, committee memberThe machineries that control transcription initiation and elongation in Archaea and Eukarya are highly homologous. These similarities support the prevailing evolutionary theory of Archaea being the progenitor of Eukarya. Due to the retention of a core transcription apparatus, while lacking complexities of the eukaryotic counterpart, archaeal systems offer the unique potential to study and characterize the basic protein components necessary for transcription. Transcription termination was less well understood in both Archaea and Eukarya. Shared homology of the initiation and elongation phases argued for a homologous method of termination in Archaea and Eukarya. Additionally, both the archaeal and eukaryotic transcription apparatuses are frequently impeded by histone proteins bound to DNA. Like the transcription complex, archaeal histones are a simplified mirror to eukaryotic histones, permitting evaluation of all steps in the transcription cycle in the context of a chromatin landscape. This thesis summarizes the core molecular machineries involved in the regulation of archaeal transcription during elongation and termination in the greater context of archaeal histone-based chromatin. Thus, the discoveries made have contributed to both the transcription and chromatin fields by providing mechanistic details of the core, conserved transcription apparatus in the framework of evolution.Item Open Access The role of Ferredoxin 3 in hydrogen metabolism in the hyperthermophilic archaeon Thermococcus kodakarensis(Colorado State University. Libraries, 2022) Stettler, Meghan, author; Santangelo, Thomas, advisor; Hansen, Jeffrey, committee member; Peers, Graham, committee memberLife faces innumerable challenges to cellular maintenance and reproduction, including access to sufficient energy. As such, all domains of life ubiquitously utilize energetically conservative mechanisms to maximize energy gains from the environment. Use of proteinaceous electron carriers, like ferredoxins, allows cells to harness energy from catabolic reactions that would otherwise be lost to the system as entropy or enthalpy. The hyperthermophilic, anaerobic archaeon Thermococcus kodakarensis is of particular interest as a target for bioengineering to maximize total energy gains, as it natively produces hydrogen gas resulting from terminal electron transport through a Membrane Bound Hydrogenase. T. kodakarensis encodes for three physiologically distinct ferredoxins. Prior to this thesis, only the sequence and molecular weight of the T. kodakarensis ferredoxins were known. Efforts in this thesis laid the groundwork for the biophysical characterization of each ferredoxin isoform via protein-film voltammetry and x-ray crystallography by the development of a reliable recombinant expression and purification scheme. Preliminary biophysical assay trials resulted in a Ferredoxin 1 crystal capable of diffracting to 1.1 Ångstroms, and midpoint reduction potentials for Ferredoxin 1 and Ferredoxin 3 confirming the predicted redox center geometry, demonstrating the efficacy of the developed protein expression and purification scheme for producing high-quality samples. Further investigation into the activity of the ferredoxins resulted in the generation of T. kodakarensis strains encoding for a tether protein between Ferredoxin 3 and its presumed sole electron acceptor Membrane Bound Hydrogenase at two respective locations. The parent strain includes a deletion of Ferredoxin 3, resulting in a deficient phenotype during sulfur-independent growth. The tethered strains of T. kodakarensis demonstrates a full recovery of sulfur-independent growth. Additionally, western-blotting revealed retention of the tethered protein in-vivo, and headspace measurements demonstrated restoration of hydrogen gas production compared to the parent deletion strain, and a reduction in total hydrogen gas output per cell compared to the lab parent strain. These findings implicate the importance of Ferredoxin 3 in hydrogen metabolism in T. kodakarensis and indicate Ferredoxin 3 as a potential target for bioengineering. Furthermore, this thesis is the foundation for further characterization of the T. kodakarensis ferredoxins as proteinaceous electron carriers with potential applications outside of this model organism.