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Executive Summary

The collective goal of the Georgia Tech Center for Ribosomal Origins and Evolution is to rewind the “tape of life”. We seek to understand and recapitulate macromolecular synthesis, folding, assembly and catalysis from far beyond the last universal common ancestor of life.

The GT Center uses in silico, in vitro and in vivo methods to study aboriginal macromolecules. We are excavating extant biochemistry for molecular fossils and relics of ancient biology. By following the noisy but traceable trails imprinted in biology and in the geological record, we are discovering the roots of the ancient symbiosis of protein and RNA. Our work focuses on discovering and characterizing the oldest macromolecules, enzymes and machines of life, revealing the interconnectedness of nucleotide and peptide. We are assembling a variety of data into a coherent timeline, forming specific models of ancestral assemblies, enzymes and evolutionary processes. We are testing and iterating various models.

Molecular Time Travel. One can travel in time by resurrecting extinct macromolecules from ancient evolutionary timepoints and environments. This method was first conceived by Linus Pauling and Emil Zuckerkandl [1,2]. Pauling and Zuckerkandl invented concepts such as paleo-genetics and paleo-biochemistry, and first suggested that probable ancestral sequences can be inferred from extant sequences (an in silico process called reconstruction). They were unable in the 1960s to resurrect molecules, because they lacked technology for synthesizing and expressing the reconstructed sequences.

With modern biotechnology one can readily synthesize and express reconstructed genes, allowing facile resurrection of long extinct macromolecules. Pauling and Zuckerkandl’s proposal has been put into practice [3]. A variety of investigators have determined the biological, physical-chemical and enzymatic properties of resurrected proteins. Benner demonstrated a fivefold decrease in the RNase activity over evolutionary time by resurrecting a lineage of RNases that initiated 40 million years ago [4]. Husain showed that resurrected chymases but not their modern orthologs efficiently and specifically form angiotensin II. They believe loss of angiotensin II forming activity occurred relatively late in their evolution [5]. Zhang and Rosenberg resurrected extinct RNase genes from primates to reveal the importance of complementary amino acid substitutions in functional divergence [6]. Thornton used molecular resurrection to demonstrate co-evolution of the steroid aldosterone and the mineralocorticoid receptor [7]. Eric Gaucher, a member of the Georgia Tech Team, resurrected ancestral elongation factors, and showed that their thermostabilities are consistent with temperatures of the ancient ocean [8].

Taking the Long View. The Georgia Tech Center is peering beyond the event horizon of previous resurrection efforts. We focus on ribosomal RNA and proteins, which are the oldest macromolecules in biology [9-14]. The translation system is a window to primeval events and molecular structures that profoundly influenced the evolution of all life on earth. The translation system maintains an interpretable molecular record of the deep evolutionary past, to biology before LUCA [10,15]. The ribosome contains primordial nucleotides, peptides, metal cations and even water molecules [16,17]. The core structure and catalytic center of the ribosome have been solidly frozen for around 4 billion years, since before the advent of coded protein [11,13,14]. We have clear guideposts in our path back in time in the form of high-resolution 3D structures of ribosomes from all three domains of life [18-23], a rapidly-expanding sequence database [24], and constraints on the timing of geochemical changes through earth history [25].

Extending the proposal of Pauling and Zuckerkandl to deep time in three dimensions, we incorporate information from 3D structures of macromolecules, which is more conserved over time and evolution than sequence, and can be much more ancient. And we incorporate information from geological models of the ancient earth, which provides important information on folding and catalytic cofactors, in particular iron. In sum, we are developing and exploiting new methods that allow us to travel back to the origin of life and to recapitulate the most remote events in the origin and evolution of life on earth.

New Methods in Ribosomal Analysis. To help our Center and others understand, deconstruct and manipulate the ribosome, which is extremely large and highly complex, we have developed a program/server called RiboVision [26]. RiboVision is a visualization and analysis tool for simultaneous display of multiple layers of diverse information on primary (1D), secondary (2D), and three-dimensional (3D) structures of ribosomes. RiboVision allows rapid retrieval, analysis, filtering, and display of a variety of ribosomal data. Preloaded information includes 1D, 2D, and 3D structures augmented by base-pairing, base-stacking and other molecular interactions. RiboVision is preloaded with rRNA secondary structures, rRNA domains and helical structures, phylogeny, crystallographic thermal factors, etc. RiboVision contains structures of ribosomal proteins and a database of their molecular interactions with rRNA. RiboVision contains pre-loaded structures and data for two bacterial ribosomes (Thermus thermophilus and Escherichia coli), one archaeal ribosome (Haloarcula marismortui), and three eukaryotic ribosomes (Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens). RiboVision has revealed to us several major discrepancies between 2D and 3D structures of 16S/18S and 23S/28S rRNAs. RiboVision is designed to allow users to distill complex data quickly and to easily generate publication-quality images of data mapped onto secondary structures. Users can readily import and analyze their own data in the context of other work. RiboVision has features in rough analogy with web-based map services capable of seamlessly switching the type of data displayed and the resolution or magnification of the display. RiboVision is available at http://apollo.chemistry.gatech.edu/RiboVision.

2D Data. Accurate secondary structures are important for understanding ribosomes. Using 3D structures of ribosomes as input, we have revised and corrected traditional secondary (2°) structures of rRNAs [27,28]. We identify helices by specific geometric and molecular interaction criteria, not by co-variation. The resulting rRNA 2° structures are up-to-date and consistent with three-dimensional structures, and are information-rich. These 2° structures are relatively simple to understand and are amenable to reproduction and modification by end-users. The 2° structures made available here broadly sample the phylogenetic tree and are mapped with a variety of data related to molecular interactions and geometry, phylogeny and evolution.

First we presented a de novo re-determination of the 2° structure and domain architecture of the 23S and 5S rRNAs [28]. In historical co-variation 2° structures, the center of the 23S rRNA is an extended single-stranded region, which in three-dimensions is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. The revised 2° structure also reveals a clear relationship with the three-dimensional structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us reconsider the domain architecture of the LSU. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities, and compactness. A proposed Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted.

Next we have generated accurate 2° structures for small subunit (SSU) 16S/18S rRNAs of Escherichia coli, Thermus thermophilus, Haloarcula marismortui (LSU rRNA only), Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens [27]. For the SSU rRNA, the 2° structures use an intuitive representation of the central pseudoknot where base triples are presented as pairs of base pairs. Both LSU and SSU secondary maps are available (http://apollo.chemistry.gatech.edu/RibosomeGallery).

Resurrection. 3D structures of ribosomes [18-23] are treasure troves of information about the origin of life. The availability of 3D structures of ribosomes from bacteria, archaea and eukarya allows us formulate models of the LUCA ribosome. We have created and tested both in silico and in vitro models of a resurrected ancestral pepidyl transerase center (aPTC) [29]. Our in silico and in vitro models contain a significantly reduced 23S rRNA (called a-rRNA-γ), retaining the rRNA that forms and surrounds the PTC. To complete the in silico and in vitro models of the ancestral PTC (a-PTC-γ in silico and a-PTC-γ in vitro), we combined a-rRNA-γ with peptides derived from the ribosomal proteins. a-rRNA-γ and peptides contain intrinsic ability to fold and specifically assemble. We are currently investigating the catalytic activity of a-PTC-γ. In addition we are continuing to develop the yeast-three hybrid system to investigate in vivo interactions between a-RNA and ribosomal proteins and their fragments.

In addition we have developed new methods for determining chronologies of ribosomal evolution. It is known that rRNA growth and evolution are hierarchical. We have recently observed deeply buried insertion fingerprints within the core of the LSU that appear identical in form to modern insertion fingerprints of eukaryotic expansions. These buried insertion fingerprints suggest the mechanism of modern rRNA expansion follows a pattern established in biological antiquity. The buried insertion fingerprints point to some of the oldest imaginable evolutionary events, and imply a method to work backwards in time, to identify pathways of expansion during formation of the ribosomal core.

Iron and RNA. We have proposed that Fe²⁺ was an RNA cofactor on the ancient earth when iron was benign and abundant, and that Fe²⁺ was replaced by Mg²⁺ during the great oxidation. Our hypothesis is supported by our observations [30,31] that (i) RNA folding is conserved between complexes with Fe²⁺ and Mg²⁺ and (ii) at least some phosphoryl transfer ribozymes are more active in the presence of Fe²⁺ than Mg²⁺. We have shown that reversing the putative metal substitution in an anoxic environment, by removing Mg²⁺ and adding Fe²⁺, expands the catalytic repertoire of some RNAs. Over the last year we have shown that Fe²⁺ can confer on RNA a previously uncharacterized ability to catalyze single electron transfer [31]. Catalysis is specific, in that it is dependent on the type of RNA. The 23S rRNA and tRNA, some of the most abundant and ancient RNAs [14], are found to be efficient electron transfer ribozymes in the presence of Fe²⁺. Therefore, the catalytic competence of ancient RNAs may have been greater in early earth conditions than in extant conditions. The Center is currently testing the hypothesis that replacement of Fe²⁺ by Mg²⁺ in RNA assemblies has not been universal.

Ribosomal Deconstruction. We have developed a hypothesis that Domain III is a truly independent structural domain that can fold to a near-native state in the absence of the remainder of the LSU [32]. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained within the intact 23S rRNA using SHAPE (selective 2’-hydroxyl acylation analyzed by primer extension), in the absence and presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with secondary structure, intra-domain tertiary interactions and inter-domain interactions that are independent of whether or not it is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.

Ribosomes in the Extreme. We are examining how their ribosomes are capable of tolerating near complete dehydration, then rehydrate and engage in translation within minutes. Our hypothesis is that specific proteins associate with ribosomes during desiccation, protecting them from damage and then dissociate upon rehydration. We want to enumerate these proteins and discover the underlying genes. In the future, this knowledge could be used to engineer desiccation tolerance into organisms that currently lack this ability.

A Bacterial Jurassic Park. Our system [33-36] combines synthetic biology (paleogenetics) with experimental evolution whereby we insert ancient genes into a modern bacterial genome. Replacing an essential bacterial gene with its ancient counterpart allows one to initiate a struggle for existence in these microbial populations since the ancient gene is maladapted to modern environments. Observing the real-time evolution of these resurrected genes as they adapt to the conditions of modern bacteria therefore allows us to monitor evolution in action.

We are reconstructing evolutionary adaptive paths with the objective of building the last common bacterial ribosome. The sequences of the ribosomal proteins and ribosomal RNA are aligned, and subjected to likelihood-based phylogenetic reconstruction, followed by over-expression and purification of the ancestral components. In practice, sequence retrieval is complicated by inconsistent annotation, gene absence, and database redundancy. One accomplishment of the past year was development and implementation of a novel algorithm for large-scale protein sequence retrieval and family discernment.

The Tunnel. We are investigating the ribosome exit tunnel, an ancient path that must be traveled by all peptides/proteins synthesized by the ribosome. We have synthesized peptolides and demonstrated their potential as probes to decipher the interaction between the nascent peptide and the exit tunnel. This study is yielding vital information about attributes that confer nucleic acids with selective advantage as building blocks for exit tunnel construction.

EPO Our EPO program the Georgia Tech team leverages resources and assets including (a) CEISMC (Georgia Tech’s Center for Education Integrating Science, Mathematics, and Computing), (b) the Georgia’s GIFT program (Georgia Intern-Fellowships for Teachers), (c) the NSF-sponsored SURE program, which provides funding for undergraduates interested in teaching, (d) the NSF-sponsored Research Experiences for Undergraduates Program, (e) the Siemens-sponsored Research, Experiment, Analyze and Learn (REAL) Program, and (f) Center members’ laboratories and research groups. This year the Georgia Tech Team discontinued their week-long non-residential Summer Learning Program, called Life on the Edge: Astrobiology. Instead they instituted a program in which 10th grade students (from under-represented groups) spent the entire summer working along side Center researchers in the laboratory. These 10th graders will maintain a long-term relationship with our center and will be mentored and guided through their college application processes. In addition the Center, along with other contributors, is working to bring Astrobiology content to teachers through the new NASA Electronic Professional Development Network (ePDN). In addition, many undergraduates and high school students work in research labs run by the Co-PIs of the Georgia Tech Center. These students participate in all aspects of astrobiology research, including presentation their work at local and regional meetings.

Center Publications
1. Bernier C, Petrov AS, Waterbury C, Jett J, Li F, Freil LE, Xiong b, Wang L, Le A, Milhouse BL, Hershkovitz E, Grover M, Xue Y, Hsiao C, Bowman JC, Harvey SC, Wartel JZ, Williams LD (2014) Ribovision: Visualization and analysis of ribosomes. Discussions of the Faraday Society: in press.
2. Cacan E, Kratzer JT, Cole MF, Gaucher EA (2013) Interchanging functionality among homologous elongation factors using signatures of heterotachy. J Mol Evol 76: 4-12.
3. Cole MF, Cox VE, Gratton KL, Gaucher EA (2013) Reconstructing evolutionary adaptive paths for protein engineering. Methods Mol Biol 978: 115-125.
4. Fox GE (2013) Carl r. Woese, 1928-2012. Astrobiology 13: 1201-1202.
5. Gromiha MM, Pathak MC, Saraboji K, Ortlund EA, Gaucher EA (2013) Hydrophobic environment is a key factor for the stability of thermophilic proteins. Proteins 81: 715-721.
6. Harris LA, Watkins D, Williams LD, Koudelka GB (2013) Indirect readout of DNA sequence by p22 repressor: Roles of DNA and protein functional groups in modulating DNA conformation. J Mol Biol 425: 133-143.
7. Hsiao C, Chou I-C, Okafor CD, Bowman JC, O’Neill EB, Athavale SS, Petrov AS, Hud NV, Wartell RM, Harvey SC, Williams LD (2013) Iron(II) plus RNA can catalyze electron transfer. Nature Chemistry 5: 525-528.
8. Hsiao C, Lenz TK, Peters JK, Fang PY, Schneider DM, Anderson EJ, Preeprem T, Bowman JC, O’Neill EB, Lie L, Athavale SS, Gossett JJ, Trippe C, Murray J, Petrov AS, Wartell RM, Harvey SC, Hud NV, Williams LD (2013) Molecular paleontology: A biochemical model of the ancestral ribosome. Nucleic Acids Res 41: 3373-3385.
9. Hud NV, Cafferty BJ, Krishnamurthy R, Williams LD (2013) The origin of RNA and “my grandfather’s axe”. Chem Biol 20: 466-474.
10. Ingles-Prieto A, Ibarra-Molero B, Delgado-Delgado A, Perez-Jimenez R, Fernandez JM, Gaucher EA, Sanchez-Ruiz JM, Gavira JA (2013) Conservation of protein structure over four billion years. Structure 21: 1690-1697.
11. Jones BL, VanLoozen J, Kim MH, Miles SJ, Dunham CM, Williams LD, Snell TW (2013) Stress granules form in brachionus manjavacas (rotifera) in response to a variety of stressors. Comparative biochemistry and physiology Part A, Molecular & integrative physiology 166: 375-384.
12. Kacar B, Gaucher EA (2013) Experimental evolution of protein-protein interaction networks. Biochem J 453: 311-319.
13. Laos R, Shaw R, Leal NA, Gaucher E, Benner S (2013) Directed evolution of polymerases to accept nucleotides with nonstandard hydrogen bond patterns. Biochemistry 52: 5288-5294.
14. Petrov AS, Bernier CR, Gulen B, Waterbury CC, Hershkovitz E, Hsiao C, Harvey SC, Hud NV, Fox GE, Wartell RM, Williams LD (2014) Secondary structures of rRNAs from all three domains of life. PLoS One: in press.
15. Petrov AS, Bernier CR, Hershkovitz E, Xue Y, Waterbury CC, Grover MA, C. HS, Hud NV, Wartell RM, Williams LD (2013) Secondary structure and domain architecture of the 23S rRNA. Nucleic Acids Res 41: 7522-7535.
16. Petrov AS, Bernier CR, Hsiao C, Norris AM, Kovacs NA, Waterbury CC, Stepanov VG, Harvey SC, Fox GE, Wartell RM, Hud NV, Williams LD (2014) Evolution of the ribosome at atomic resolution. Proc Natl Acad Sci USA: submitted.
17. Risso VA, Gavira JA, Mejia-Carmona DF, Gaucher EA, Sanchez-Ruiz JM (2013) Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian beta-lactamases. J Am Chem Soc 135: 2899-2902.
18. Rivas M, Tran Q, Fox GE (2013) Nanometer scale pores similar in size to the entrance of the ribosomal exit cavity are a common feature of large RNAs. RNA 19: 1349-1354.
19. Sapp J, Fox GE (2013) The singular quest for a universal tree of life. Microbiol Mol Biol Rev 77: 541-550.
20. Tirumalai MR, Fox GE (2013) An icebs1-like element may be associated with the extreme radiation and desiccation resistance of bacillus pumilus safr-032 spores. Extremophiles 17: 767-774.
21. Tirumalai MR, Rastogi R, Zamani N, O’Bryant Williams E, Allen S, Diouf F, Kwende S, Weinstock GM, Venkateswaran KJ, Fox GE (2013) Candidate genes that may be responsible for the unusual resistances exhibited by bacillus pumilus safr-032 spores. PLoS One 8: e66012.

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