2012 Annual Science Report
Georgia Institute of Technology Reporting | SEP 2011 – AUG 2012
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 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 macromolecular folding and enzymology. We are excavating extant biochemistry for molecular fossils and relics of the ancient biology. By following the noisy but traceable data trails imprinted in biology and in the geological record, we are discovering the roots of protein synthesis by 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 the data into a coherent timeline, forming specific models of ancestral assemblies, enzymes and evolutionary processes. We are testing and iterating the models.
Molecular Time Travel. One can travel in time by resurrecting extinct macromolecules from predetermined 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). Previous 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 far 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 peptidyl transferase center (PTC), in particular, is thought to be even older than coded protein (15-18). In this view, the PTC emerged from some type of ‘RNA World’ (19-23) participating in the transformation to current biology in which information is transduced from nucleic acids to proteins. In essentially any reasonable model of origin of life and evolution of the ribosome, the PTC appears as one of our most direct biochemical links to the distant evolutionary past and to early life (24). Understanding the PTC is key to understanding ancient biology.
Extending the proposal of Pauling and Zuckerkandl, we incorporate information from three-dimensional (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.
The Data. 3D structures of ribosomes (25-29) 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 test both in silico and in vitro models of an ancestral pepidyl transerase center (aPTC) (30). Our most recent in silico and in vitro models contain a significantly reduced 23S rRNA (called a-rRNA-γ), retraining 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 have combined a-rRNA-γ with peptides derived from the ribosomal proteins. The results here indicate that the ribosome and its components are highly robust in folding and assembly. We have shaved around 2500 nucleotides from the 23S rRNA and the vast majority of amino acids from the protein components, excising the globular domains in toto. Yet, the remaining rRNA and peptides retain the ability to fold and specifically assemble.
We are employing the yeast-three hybrid system to investigate in vivo interactions between a-RNA and ribosomal proteins. Our results demonstrate that a-RNA-γ binds in vivo to L2, L3, L4, L15, and L22. L2 is an initial protein in ribosomal assembly and binds to the intact 23S rRNA independently of other r-proteins. We are currently examining the potential for greater binding in vivo of L3, L4, L15, and L22 to the a-RNA-γ with co-expression of L2 in a yeast hybrid system.
We have provided a quantum mechanical (QM) description of first shell RNA-magnesium and DNA-magnesium interactions, demonstrating unique features that appear to be required for folding of large RNAs (31,32). Our work focuses on multidentate chelation of magnesium by RNA and DNA, where multiple phosphate oxyanions enter the first coordination shell of magnesium. The results suggest that magnesium, compared to calcium and sodium, has enhanced ability to form bidentate chelation complexes with RNA. Sodium complexes, in particular, are unstable and spontaneously open. A magnesium cation is closer to the oxyanions of RNA than the other cations, and is stabilized not only by electrostatic interaction with the oxyanions but also by charge transfer and polarization interactions. Those interactions are quite substantial at close distances. The quantum effects are less pronounced for calcium due to its larger size, and for sodium due to its smaller charge. Additionally, we find that magnesium complexes with RNA are more stable than those with DNA. The nature of the additional stability is twofold: it is due to a slightly greater energetic penalty of ring closure to form chelation complexes for DNA, and elevated electrostatic interactions between the RNA and cations. In sum it can be seen that even at high concentration, sodium and calcium cannot replicate the structures or energetics of RNA-magnesium complexes.
We are investigating, by theory and experiment, whether critical roles of Mg2+ in extant RNA folding and function can be served by Fe2+ in the absence of oxygen (33). The results of our high-level quantum mechanical calculations show that the geometry of coordination of Fe2+ by RNA phosphates is similar to that of Mg2+. The conformation of Tetrahymena Group I intron P4-P6 domain is conserved between complexes of Fe2+ and Mg2+. Additionally, a ribozyme obtained previously by in vitro selection in the presence of Mg2+ and a natural ribozyme both have significantly greater catalytic competence in the presence of Fe2+ than in Mg2+. The combined biochemical and paleogeological data are consistent with an RNA-Fe2+ world that could have supported an array of RNA structures and catalytic functions far more diverse that of an RNA-Mg2+ world.
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 (34). 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.
We are developing and using novel bioinformatical methods to determine chronologies of ancient ribosomal evolution (35). It is hypothesized that substantial, though necessarily incomplete evidence, relating to the origins and early development of the translation machinery and its relation to other core cellular processes continues to exist in the primary sequences, three-dimensional folding and functional interactions of the various macromolecules involved in the modern versions of these processes. To this end, we are using ribosomal paleontology to determine the relative age of various ribosomal components and subsystems and thereby develop timelines for the history of the ribosome as a whole as well as various sub processes such as initiation, termination, translocation etc. The results of these studies will interface ribosomal history with other key relating to the origin of life including the emergence time of the genetic code, the origin of chirality and the nature of the last common ancestor. We have also been developing new tools of ribosomal paleontology, to visualize the changes, and to determine timelines for ribosomal origins.
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.
We are creating a “Bacterial Jurassic Park”(36-39). Our system 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 me 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.
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.
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 continues their week-long non-residential Summer Learning Program, called Life on the Edge: Astrobiology. The GT Team works with teachers and undergraduates, who are trained to run the camp, which is directed at high school students. Life on the Edge exposes participating teachers, undergraduates and high school students to the excitement of astrobiology, and offers teachers low cost and accessible methods and course materials for incorporating astrobiology into high school teaching programs. 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.
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