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ItemRNA and Metals as a Window onto Ancient Biochemistry(Georgia Institute of Technology, 2023-04-30) Guth-Metzler, RebeccaRNA is one of life’s three essential biopolymers. RNA is so entrenched in biology that it is thought to have arisen near the origin of life itself. Work herein uses RNA’s ancient relationship with divalent metals (M²⁺) as a detective’s lens to peer back into time. RNA uses Mg²⁺ as its primary M²⁺ partner in modern life. However, RNA evolved when Fe²⁺ was more available, in a time before rising atmospheric oxygen caused Fe²⁺ to “rust out”. Moreover, Fe²⁺ exposed to oxygen forms free radicals that break down biomolecules including RNA. Our research shows that Fe²⁺ kept in anoxic conditions mimicking early Earth does not cause oxidation reactions, instead having the same reaction with RNA as does Mg²⁺. Reaction similarity of Fe²⁺ and Mg²⁺ adds to growing evidence that Fe²⁺ may have been an early binding partner of RNA, and that RNA adaption through swapping out M²⁺ to support RNA survival over billions of years. Moreover, Fe²⁺ may have accelerated early RNA evolution, allowing RNA to diversify and multiply. Yet, to RNA, M²⁺ is a double-edged sword. While M²⁺ ions catalyze RNA cleavage, shortening its lifetime, they also promote RNA folding, which in turn protects RNA from cleavage. We further combine these concepts into the following scheme: too little M²⁺ shortens RNA lifetime because there is no folding and therefore no cleavage protection, too much M²⁺ shortens RNA lifetime because cleavage overwhelms folding protection, but the in-between “Goldilocks peak” of moderate [M²⁺] is “just right”. We find RNA Goldilocks peaks that take on a variety of appearances, revealing unexpected complexity from the innate RNA-Mg²⁺ relationship. The average Goldilocks peak of modern RNA may reflect the metal conditions of its origin, giving a clearer picture of the environment where life emerged. The Goldilocks peak boost to RNA lifetime perhaps caused RNA to win out over competing polymers on early Earth, a possible explanation for why RNA is one of life’s universal biopolymers.
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ItemThe Saccharomyces cerevisiae Mitochondrial Ribosome as an Orthogonal Evolvable Translation System(Georgia Institute of Technology, 2023-04-27) Rothschild-Mancinelli, BrookeThe synthesis capabilities of the translation system provide engineering and directed evolution potential as a system for generating novel polymers. Orthogonal translation systems offer a parallel platform for translation engineering, enabling primary and secondary translation systems to operate independently without interfering with each other. Using a combination of indirect modifications through antibiotics, truncations of mito-rProteins mL50 and uL23, and replacement of mito-rProtein uL2, we develop and test the mitochondrial translation system in Saccharomyces cerevisiae as a fully orthogonal platform for in vivo directed evolution of translation. Our results show continuous, comprehensive, creative, directed-evolution of a fully orthogonal TS in vivo appears to be a useful approach for technical control of the TS. One of the drawbacks of the mito-translation system is the propensity for S. cerevisiae to lose mito-genomes in response to modifications to the mito-translation system. Loss of the mito-genome prevents recovery of mito-translation. Addition of the overexpression of RNR1 decreases petite formation rates further improve the platform for translation engineering. Decreased petite formation rates permits longer periods of evolution to recover from complex mito-translation edits. Using the mito-translation system, translation engineering can target any component of the translation system for modifications.
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ItemExperimental Predictions of Ribosomal Evolution(Georgia Institute of Technology, 2022-05-02) Haynes, Jay WilliamTranslation and the ribosome are universal and necessary components of biology. Testing the predictions of models that detail the evolution of translation and the ribosome can provide us with a fundamental understanding of the nature of life processes. This dissertation discusses work focused on some predictions based in evolutionary models and the tools used to test predictions. We develop a software tool for processing of melting data. This tool allows the user to see the effects of adjusting processing parameters in real time. Aims of this tool include aiding the experimentalist by shortening data processing timelines and enhancing the development of intuition regarding the effects of processing parameters on results. We survey possibility of mutualistic interactions between RNA and a plausible prebiotic protein ancestor, depsipeptides, is explored. We see that RNA and cationic depsipeptides can form direct interactions. We also see that these interactions result in increased thermal stability of folded RNA structure and increased lifetimes for depsipeptides. The findings imply that the interdependencies of RNA and protein extends to the earliest stages in the development of life. We observe the interchangeability of divalent metals within the translation system. Divalent metals, principally Mg2+, is a critical cofactor in many translation components. Other similar metals like Fe2+ and Mn2+ were once more abundant and may have played similar biological roles. We find that Fe2+¬ and Mn2+ can maintain translation structures and mediate translation itself in a manner comparable to Mg2+, implicating all three metals as cofactors in early stages of ribosomal evolution. We resurrect ancestral states of the ribosome that represent the earliest catalytic core, the PTC. Motifs of ribosomal evolution are used to design and recapitulate the rRNA fragments that make up the PTC. Structural characterization of these fragments provides evidence for an evolutionary trajectory characterized by stepwise growths in ribosomal structures with concomitant conservation of preexisting structure.
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ItemRibosomal Structure, Function, and Trafficking(Georgia Institute of Technology, 2022-04-28) Fakhretaha Aval, SaraThe ribosome is the most abundant assembly within cells on the earth. The mRNA biological codes are translated to proteins by ribosomes for approximately 4 billion years. All ribosomes are composed of ribosomal RNA and ribosomal proteins. Sequence and structural analysis of ribosomal RNA show that all cytoplasmic ribosomes share a conserved common core. However, eukaryotic ribosomal RNAs obtain more structural complexities by accretion of RNA helices onto the ribosomal common core. The inserted RNA helices are called ribosomal RNA expansion segments. As a result, ribosomal RNA structural complexity develops functional complexity in the ribosome, particularly the human ribosome, which possesses the longest expansion segments among all living organisms. In this dissertation, we investigate the structure and function of rRNA expansion segments in three domains of life. Here, we first show that elongated expansion segments were present in ancient ribosomes of the last Asgard and Eukarya common ancestor. We predicted and validated the secondary structures of Asgard ribosomal expansion segments using covariation analysis and chemical footprinting technique. We then explored the structures and functions of expansion segments in mammals. We showed human expansion segments interact with proteins known to interact with G-quadruplexes, and ribonucleoprotein granules, suggesting a critical role in the molecular transport system. The results suggest that human ribosomes can form ribonucleoprotein granules through expansion segments. In addition, we demonstrated that expansion segments form liquid condensates through G-quadruplexes multivalent interactions in vitro. Moreover, we showed that ribosomes can traffic between human cells which is not mediated by mRNA-ribosome association. In addition, we explored the correlation between the complexity of brain and ribosomal RNA in Eukarya. The length of ribosomal RNA, expansion segments 7, and expansion segment 27 correlate with the number of neurons in brains. Furthermore, we investigated the correlation between the presence of G-Quadruplexes and number of neurons in brains. The results show that organisms with more complex brains have more G-quadruplexes on the surface of ribosomal RNA. In summary, we proposed a model for ribosomal trafficking. In this model, the tentacles of expansion segments interact with granule-associated proteins, promote liquid granule formation through phase separation, and mediate ribosome trafficking. This model is applicable to neuronal axons, nanotubes between cells, and probably in other circumstances. Finally, we discussed in detail the preparation of an RT-qPCR assay to diagnose SARS-CoV-2 in academic laboratories and describe the implementation of environmental testing across campus.
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ItemAnalysis and illustration of primary and secondary structures of ribosomal RNA and ribosomal proteins(Georgia Institute of Technology, 2020-08) Meade, Caeden DanielRiboVision is a collection of applications housed on servers at the Georgia Institute of Technology which serves to facilitate the development of publication-quality diagrams of ribosomal RNA (rRNA) and ribosomal protein (rProtein) structures (Petrov et. al, 2014). In particular, RiboVision seeks to promote analysis of key properties of rRNA and rProteins in primary, secondary, and tertiary structures. As key semantides (ubiquitous macromolecules which carry genetic equivalent to the information intrinsic to DNA molecules and may be used by comparison to inform phylogenetic relationships), comparison of the primary and secondary structures of 16S and 18S RNA allows for the phylogenetic comparison of prokaryotic species and eukaryotic species, respectively (Fuerst, 2001). Sequence alignments are housed on the RiboVision server and stored in a MySQL database. Over the next two semesters, major improvements will be made to the server resulting in the newest edition, RiboVision3, which will feature improvements over the preceding RiboVision2 including the integration of XRNA, a program responsible for the generation of rRNA secondary structures and their exportation of their data into common computer-file formats (CSV, SVG, PDF, etc.) and the PDB Topology Viewer, a program responsible for production of protein secondary structures and their exportation into SVG image files. The core functionality of XRNA - demonstration and editing tools of rRNA secondary structures needs to be iterated upon to allow for a more diverse set of purposes, including processing of high-quality hand-edited images into formats which are compatible with on-server management and conversion into formats native to web browsers.
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ItemNon-canonical structures and functions of the human ribosome: G-quadruplexes and heme appropriation(Georgia Institute of Technology, 2020-07-14) Mestre Fos, SantiagoThe ribosome is a macromolecular ribonucleoprotein machine that is responsible for the synthesis of all proteins in cells. Mammalian ribosomal RNAs (rRNAs) are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments (ESs), which are double-stranded RNA ramifications that protrude from the ribosomal surface. Here we show that numerous human rRNA ESs are capable of forming stable G-quadruplexes (G4s) in vitro and in vivo. G4s are non-canonical nucleic acid secondary structures that are thought to play key regulatory roles in cells. In addition, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomal G4s appropriate heme and regulate its cytosolic bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Overall, these results indicate that the RNA G-quadruplexome is ribosome-centric and suggest ribosomes are hubs of heme metabolism.