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School of Biological Sciences

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School of Chemistry and Biochemistry

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School of Earth and Atmospheric Sciences

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RNA and Metals as a Window onto Ancient Biochemistry

(Georgia Institute of Technology, 2023-04-30) Guth-Metzler, Rebecca

RNA 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.
The Saccharomyces cerevisiae Mitochondrial Ribosome as an Orthogonal Evolvable Translation System

(Georgia Institute of Technology, 2023-04-27) Rothschild-Mancinelli, Brooke

The 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.
Experimental Predictions of Ribosomal Evolution

(Georgia Institute of Technology, 2022-05-02) Haynes, Jay William

Translation 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.
Ribosomal Structure, Function, and Trafficking

(Georgia Institute of Technology, 2022-04-28) Fakhretaha Aval, Sara

The 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.
Non-canonical structures and functions of the human ribosome: G-quadruplexes and heme appropriation

(Georgia Institute of Technology, 2020-07-14) Mestre Fos, Santiago

The 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.
Iron as an integral constituent of ancient metabolism and biochemistry

(Georgia Institute of Technology, 2019-05-21) Bray, Marcus Salvatore

Life on Earth evolved and proliferated for nearly 2 billion years in an environment devoid of molecular oxygen and replete with iron. Currently on Earth, iron has widespread uses in the biochemistry and metabolism of extant organisms. It is therefore likely that this metal filled a larger role at life’s inception and colonization across the planet. In this dissertation, I investigated the roles that iron could have played for early lifeforms and early biochemistry. I first studied iron’s ability to substitute for magnesium in life’s oldest macromolecular machine, the ribosome. I found that under conditions reminiscent of the ancient Earth, iron can mediate ribosomal structure and function in place of magnesium, both in vitro and in vivo. I then examined the mechanisms microorganisms use to respire iron, and the how this ancient metabolism interacts with others in the environment. I found that certain iron reducing strategies may me more phylogenetically and structurally diverse than previously realized, and that the competing interest of iron reducing organisms with methanogens in sediments could have constrained early planetary habitability. Collectively, my results deepen our knowledge of not only the past, but present and future of iron in life on this planet.
Green fluorescent protein inspired chromophore as estrogen receptor agonist-synthesis, biological evaluations and cellular application

(Georgia Institute of Technology, 2019-01-22) Walker, Christopher L.

Nuclear receptors are ligand activated transcription factors that are widely distributed throughout the mammalians. There are 48 known human nuclear receptors within the body located in various systems. While some nuclear receptors can be located wholly within certain regions and tissues the clear majority are widely distributed, overlapping expression in the same locations. The role of nuclear receptors as transcription factors has caused them to be implicated in a vast number of diseases including metabolic, cardiovascular and neurological. The role of nuclear receptors in diseases and the potential to promote ligand activated transcription makes nuclear receptors of pharmaceutical significance. Currently it is estimated that 33% of nuclear receptors are targeted by the pharmaceutical industry resulting in ~20% of pharmaceutical development worldwide. The potential to control physiological responses via introduction of a ligand to nuclear receptors has continued the interest in development of new ligands to further the understanding of nuclear receptor behavior. The challenge nuclear receptors present is to develop ligands that are selective in targeting within families and among different classes of nuclear receptors. At the core, ligand activated nuclear receptor modulation is chiefly centered around the relationship between the ligand binding pocket of the receptor and the ligand. Composed primarily of non-polar amino acid residues the ligand binding pocket is the cavity by which small hydrophobic molecules bind. Demonstrating large variance across classes of nuclear receptor and little divergence within families the ligand binding pocket serves as the focal point for targeting selectivity. Successful binding and thus receptor response is contingent upon the ligand meeting criteria established by the ligand binding pocket such as satisfactory size/volume of the ligand and key ligand-receptor amino acid residue interaction. Research conducted by Katzenellenbogen was paramount in understanding the relationship between the estrogen receptors and its ligands. His established pharmacophore unraveled features for potential ligands that are exchangeable from those that are indispensable. The commercial success of estrogen receptor ligands has fueled the interest in not only understanding ligand-receptor binding interactions but its subcellular movement. The Green Fluorescent Protein completely revolutionized the way in which cellular probing is conducted. The chromophore internally synthesized by the protein through a series of folding of amino acid residues afforded the opportunity to monitor cellular movements with the aid of fluorescence. Commonly utilized in visualization as a fusion protein, the GFP chromophore provided the ideal tool for understanding protein cellular movement and interaction. Simply put due to the chromophore that resides at the center, GFP provides the perfect technique for cellular probing. Here in we report the use of GFP-chromophore inspired ligands for utility as estrogen receptor agonist. By utilizing the GFP chromophore skeleton as a template for ligand development the potential arises for the molecule to co-function as a receptor binder and probe. The use of the GFP-chromophore skeleton boasts several advantages in addition to synthetic amenable features the arymethyleneimidazolone core maintains the same frame work as Katzenellenbogen’s proposed pharmacophore. Through the lens of Katzenellenbogen’s consideration and the use of a simple but elegant synthesis a small library of GFP chromophore inspired arylmethyleneimidazolone ligands were synthesized, screened for selective estrogen receptor activation and tested both in vivo and in vitro for cellular probing applications. Through this work we identified a set of 10 ligands that serve as agonist for the estrogen receptor. Although of the 10 ligands several demonstrate activation for both ERα and ERβ, a high degree of preference for ERα is observed. Of the ligands screened all estrogen receptor active ligands were nuclear receptor selective failing to activate other receptors such as RAR and RXR. Biological screening also uncovered a super agonist in CW32 that demonstrated the highest level of activation. Though a structure activity relationship model was established for top activators and additional generations synthesized no compound was found to be more active than CW32. While the majority of ligands displayed a preferential affinity for ERα ligand CW72 demonstrated complete specificity for ERα. All ligands were confirmed through TRFRET as binding in the same ligand binding pocket as estradiol. Computational modeling supports the rationale that the following three criteria governed the ligands ability to successfully bind: 1) hydrogen bonding network, 2) ligand size/volume and 3) molecular topology. Embracing the ligands skeleton originating from the Green fluorescent chromophore ligands that demonstrated ER activation were visualized under confocal both with in vivo and in vitro systems. Several ligands successfully demonstrated the ability to turn on fluorescence in responds to binding in vitro. While other ligands failed to display fluorescence in conjunction with binding. Despite all binders displaying fluorescence this represent a class of ligand that can serve as a activator and probe.
Data mining the structure of the ribosome to unravel the history of proteins

(Georgia Institute of Technology, 2018-11-09) Kovacs, Nicholas Attila

Contained within every cell of every organism on Earth is a molecular fossil that has recorded the evolution of life since its origin approximately 4 billion years ago. Composed of both RNA and protein, the ribosome is the molecular tape-recorder that has logged the evolution of life within the sequences and structures of its RNA and protein. The structure of ribosomal RNA contains its evolution. We extend the model of ribosomal evolution to include ribosomal protein. The evolution of protein was guided by the ribosome, on the ribosome, and for the ribosome. Ribosomal proteins that are present in all of life today present a molecular chronology of the origins and evolution of protein. We show that partitions of ribosomal proteins reveal the history of protein folding which stretches back since the origin of life. The data support a model in which (i) short, random coil polypeptides accreted onto the surface of the ribosome, and (ii) lengthened over time and coalesced into β-structures. (iii) These β-structures then collapsed, primarily into β-domains. (iv) Domains accumulated and gained complex super-secondary structures composed of both α-helix and β-strands. Life then diversified into its 3 domains and ribosomal proteins accrued elaborations on their structures. (v) In Archaea and Eukarya, insertions in ribosomal proteins gave rise to internal loop protrusions from their globular domains composed of unstructured segments as well as α-helices which buried into ribosomal RNA structure and made contacts with other ribosomal proteins. (vi) In Eukarya, α-helical extensions on the termini of ribosomal proteins gave rise to intricate ribosomal protein-ribosomal protein interactions on the surface of the ribosome.
Engineering elongation factor Tu and tRNAs to better accommodate non-canonical amino acids during translation

(Georgia Institute of Technology, 2018-07-26) DeLey Cox, Vanessa E.

Non-canonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proof-reading aa-tRNAs that substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Herein, we describe our efforts to transform how EF-Tu is utilized to incorporate ncAAs. Leveraging computational methods, we present data on four EF-Tu libraries and two tRNA libraries used in both in vitro and in vivo translation. Ultimately, the in vivo assay led to the identification of EF-Tu variants which accept non-native substrates. By mass spectrometry, we demonstrate two variants have expanded substrate acceptance. Fitness tests show a polyspecific EF-Tu is an accepted addition to the translation machinery and improves host organism fitness relative to an ncAA-specific engineered EF-Tu. These results lend credence to our choice of computational method (in this case REAP) and also suggest that EF-Tu and SelB may have a shared, substrate-promiscuous ancestor. Overall, we believe this approach complements current research highlighting the advantages of improved OTSs and promotes a more comprehensive approach critical to achieving future goals.
Development and molecular understanding of plasmonic photothermal therapy (PPTT) in combating cancer

(Georgia Institute of Technology, 2017-04-14) Ali, Moustafa Ragab

In cancer plasmonic photothermal therapy (PPTT), gold nanoparticles (AuNPs) are used to convert light energy into localized heat leading to cancer cell death. Among plasmonic nanoparticles, gold nanorods (AuNRs) have been widely used as they absorb near-infrared (NIR) laser light (safe to biological system). Detailed background information was introduced in Chapter 1. Herein, 1) we developed new AuNRs formulations techniques, yielding better generation of AuNRs, for enhancing PPTT (Chapter 2). 2) We studied PPTT in vitro (Chapter 3). We developed novel surface conjugations, greatly enhance the endocytosis of gold nanoparticles and the PPTT efficacy in cells. We explored its molecular mechanism. We also studied the AuNPs’ effect on inhibiting cancer cells migration. 3) Moving from cells to animals (Chapter 4). We studied the efficacy, molecular mechanism and long-term toxicity of AuNRs in mice. Furthermore, the feasibility of PPTT has been verified on natural tumors in dogs and cats without any relapse or toxicity effects even after 1 year of treatment. In conclusion, together these data on cells, mice, cats and dogs demonstrated that our AuNR-PPTT inducing apoptosis is effective and safe for cancer therapy provides a strong framework for translation this approach to the clinics.