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2009 Annual Science Report

Georgia Institute of Technology Reporting  |  JUL 2008 – AUG 2009

Executive Summary

The collective scientific goal of the Georgia Tech Center for Ribosomal Evolution and Adaptation is to rewind the “tape of life”: to shed light on the nature of protein synthesis prior to the last universal common ancestor of life. The Center focuses on the characteristics of ancient macromolecules and their assemblies, specifically on aboriginal mechanisms of peptide synthesis by ribonucleic acid (RNA). We aim to uncover clues about key steps in the transition from the RNA world to the protein world. Our work carries the potential of discovering and characterizing the oldest traceable macromolecules and machines of life, and the earliest discernable connection between RNA and protein.

Molecular time-travel, in the form of resurrecting extinct biological macromolecules, was first conceived by Linus Pauling and Emil Zuckerkandl.1,2 They introduced concepts such as paleo-genetics and paleo-biochemistry. They suggested that probable ancestral protein sequences can be inferred from extant sequences (an in silico process called reconstruction). Synthesis of reconstructed sequences (in vitro or in vivo) allows one to resurrect and determine physical-chemical and enzymatic properties of long vanished biopolymers.

The idea of molecular resurrection has been translated into practice.3 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 (serine proteases) 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
One of the goals of the Georgia Tech Center is to extend the time horizon of molecular resurrection to the most distant events in the evolution of life on earth, and to learn about life’s most ancient polymers and assemblies. To accomplish this we are incorporating three-dimensional (3D) structural information into the reconstruction/resurrection process. The recent availability of high resolution 3D structures of bacterial and archaeal ribosomes9-12

provides important new sources of information. We focus on two structures from lineages that diverged at the first branch point in the tree of life.

Conserved elements in these ribosomal structures provide information from the time of the last universal common ancestor, and before. We are developing methods to extend the input for molecular resurrection to information from structural biology, inorganic chemistry, etc.

The formation of the Peptidyl Transferase Center (PTC) marked the beginning of the translation machinery and the beginning of the end of the RNA world. Therefore, a primary focus of the Center is the Large Subunit (LSU) of the ribosome, which contains the PTC. We are working towards determination of LSU structures from uncharted areas of the tree of life, and on the resurrection of a-PTCs (ancestral Peptidyl Transferase Centers) and on sub-structures of the a-PTC.

The Georgia Tech Team has designed, and is resurrecting, a model of the a-PTC. The results of the resurrection, if successful, will allow one to test ideas about primitive living systems, including the origin of protein.

In gathering information for construction of the a-PCT, we discovered what we call “paleo-magnesium ions”.13 Paleo-Mg2+ ions are conserved over vast evolutionary timescales

and are characterized by their unusual coordination of RNA. Their importance in ancestral rRNA folding, stability and evolution is underscored by their conservation in 3D, their effect on 3D structure, their conservation in 1D and 2D structure, and their roles in function. Conservation in 3D – paleo-Mg2+ ions are highly conserved in position and mode of interaction in the LSUs of Haloarcula marismortui(archaea) and Thermus thermophilus (bacteria), species which are, as shown in Figure 2, separated by billions of years of evolution. Effect on 3D Structure – paleo-Mg2+ ions play a unique role in the structure of rRNA effecting rRNA rigidity, stability and forced dispositions of functional groups, effects that cannot be approximated by RNA alone or in conjunction with other ions. Conservation in 1D and 2D – paleo-Mg2+ ions associate with the most conserved sequence and secondary elements in rRNA, and link conserved 2D elements to each other. As shown in Figure 4,

the secondary elements linked by paleo-Mg2+ ions are conserved between bacteria and archaea, in the proposed secondary structures of eukaryotic14 and mitochondrial rRNAs,15 and in a proposed minimal 23S-rRNA.16 Role in Function – paleo-Mg2+ ions appear to form a scaffold surrounding the PTC (and the a-PTC).
Our current model of the a-PTC contains around 600 nucleotides of ancestral ribosomal rRNA (a-rRNA), three ancestral ribosomal peptides (a-rPeptides), and paleo-magnesium ions. We will characterize the assembly, structure and thermodynamics of the a-PTC by chemical mapping (including hydroxyl radical footprinting and SHAPE analysis), RNase H cleavage, temperature dependent hydrodynamics, and computational folding algorithms. It has been suggested, originally by Alex Rich, that peptide synthesis may not have been the function of the a-PTC.17 We intend to investigate the breadth of the catalytic potential of the a-PTC.

We will develop small model systems in which the interactions of a-rPeptides, paleo-Mg2+ ions and a-rRNA can be studied by nuclear magnetic resonance, X-ray diffraction, calorimetry, molecular dynamics simulations, and other 'high resolution’ biophysical techniques. Within the large subunit of the extant ribosome, one can observe a tail of ribosomal protein L2 (see Figure 3), which we call a-rPeptideL2. a-rPeptideL2 interacts with ribosomal helices rRNA 65 and 66 (conserved in a-rRNA), which in turn combines with Mg2+ to form a “Mg2+-microcluster”. The Georgia Tech Team will define the smallest a-rRNA and peptide segments (of L2) sufficient for assembly of this complex, and will characterize the assembly by a variety of experimental and computational methods.

The Georgia Tech Team has embarked on a computational evaluation of the role of the Mg2+ microclusters observed to form a 'scaffold’ for the extant and a-PTC. The interaction energies of ribosomal RNA with single and multiple Mg2+ cations are computed, and decomposed. The results will be compared to those with other metals to determine why Mg2+ plays a special role in RNA folding.

The Georgia Tech Team is establishing methods to determine chronologies of ancient ribosomal evolution. One method, just published,18 uses structure-based and sequence-based comparisons of the LSUs of H. marismortui and T. thermophilus, along with an “onion approximation”, in which superimposed LSUs are sectioned into concentric shells. A shell-by-shell comparison of the ribosomal onion captures significant information about ribosomal evolution, suggesting that the conformation and interactions of both RNA and protein have changed over evolutionary time. Similarly, we will compare biochemistry and the three-dimensional structures of ribosomes from modern organisms on particular lineages of the tree of life. Extremophiles are of special interest due to their ability to thrive in environments reminiscent of early biotic earth. In addition, the Georgia Tech Team will investigate the effect of freezing on assemblies of a-rRNA, paleo-magnesiums and a-rPeptides in aqueous solution. Freezing nucleic acid solutions concentrates non-water molecules into small liquid pockets in the ice. This promotes the assembly of small nucleic acids into larger complexes.

The RNA World hypothesis suggests an RNA molecule capable both of encoding information and replicating it. In essence, this model predicts an RNA-based RNA polymerase. Since there are no extant RNA-based RNA polymerases, we are searching the molecular-fossil record for hints about the origin and disposition of this activity. Our primary goal here is to test the hypothesis of Poole19 that the small subunit (SSU) of the ribosome may have evolved from an RNA-dependent RNA polymerase ribozyme. We will test the plausibility of an RNA polymerase origin of the SSU with in vitro reverse evolution: if we can reverse-evolve the SSU into an RNA polymerase, we can demonstrate a possible evolutionary pathway between a putative primordial ribozyme polymerase and modern ribosomes.

1. Pauling, L. & Zuckerkandl, E. (1963). Chemical Paleogenetics Molecular Restoration Studies of Extinct Forms of Life. Acta Chemica Scandinavica, 17, 9-16.
2. Zuckerkandl, E. & Pauling, L. (1965). Molecules as Documents of Evolutionary History. J Theor Biol, 8, 357-366.
3. Benner, S. A., Sassi, S. O. & Gaucher, E. A. (2007). Molecular Paleoscience: Systems Biology from the Past. Adv Enzymol Relat Areas Mol Biol, 75, 1-132.
4. Jermann, T. M., Opitz, J. G., Stackhouse, J. & Benner, S. A. (1995). Reconstructing the Evolutionary History of the Artiodactyl Ribonuclease Superfamily. Nature, 374, 57-59.
5. Chandrasekharan, U. M., Sanker, S., Glynias, M. J., Karnik, S. S. & Husain, A. (1996). Angiotensin II-Forming Activity in a Reconstructed Ancestral Chymase. Science, 271, 502-505.
6. Zhang, J. & Rosenberg, H. F. (2002). Complementary Advantageous Substitutions in the Evolution of an Antiviral RNase of Higher Primates. Proc Natl Acad Sci U S A, 99, 5486-5491.
7. Bridgham, J. T., Carroll, S. M. & Thornton, J. W. (2006). Evolution of Hormone-Receptor Complexity by Molecular Exploitation. Science, 312, 97-101.
8. Gaucher, E. A., Govindarajan, S. & Ganesh, O. K. (2008). Palaeotemperature Trend for Precambrian Life Inferred from Resurrected Proteins. Nature, 451, 704-707.
9. Selmer, M., Dunham, C. M., Murphy, F. V. t., Weixlbaumer, A., Petry, S., Kelley, A. C., Weir, J. R. & Ramakrishnan, V. (2006). Structure of the 70s Ribosome Complexed with mRNA and tRNA. Science, 313, 1935-1942.
10. Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F. & Yonath, A. (2001). High Resolution Structure of the Large Ribosomal Subunit from a Mesophilic Eubacterium. Cell, 107, 679-688.
11. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000). The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution. Science, 289, 905-920.
12. Cate, J. H., Yusupov, M. M., Yusupova, G. Z., Earnest, T. N. & Noller, H. F. (1999). X-Ray Crystal Structures of 70s Ribosome Functional Complexes. Science, 285, 2095-2104.
13. Hsiao, C., Tannenbaum, M., VanDeusen, H., Hershkovitz, E., Perng, G., Tannenbaum, A. & Williams, L. D. (2008). Complexes of Nucleic Acids with Group I and II Cations. In Nucleic Acid Metal Ion Interactions (Hud, N., ed.), pp. 1-35. The Royal Society of Chemistry, London.
14. Cannone, J. J., Subramanian, S., Schnare, M. N., Collett, J. R., D’Souza, L. M., Du, Y., Feng, B., Lin, N., Madabusi, L. V., Muller, K. M., Pande, N., Shang, Z., Yu, N. & Gutell, R. R. (2002). The Comparative RNA Web (Crw) Site: An Online Database of Comparative Sequence and Structure Information for Ribosomal, Intron, and Other RNAs. BMC Bioinformatics, 3, 2.
15. Mears, J. A., Sharma, M. R., Gutell, R. R., McCook, A. S., Richardson, P. E., Caulfield, T. R., Agrawal, R. K. & Harvey, S. C. (2006). A Structural Model for the Large Subunit of the Mammalian Mitochondrial Ribosome. J Mol Biol, 358, 193-212.
16. Mears, J. A., Cannone, J. J., Stagg, S. M., Gutell, R. R., Agrawal, R. K. & Harvey, S. C. (2002). Modeling a Minimal Ribosome Based on Comparative Sequence Analysis. J Mol Biol, 321, 215-234.
17. Rich, A. (1971). The Possible Participation of Esters as Well as Amides in Prebiotic Polymers. In Chemical Evoltuion and the Origin of Life (Buvet, R. & Ponnamperuma, C., eds.). North-Holland Publishing Company, Amsterdam.
18. Hsiao, C., Mohan, S., Kalahar, B. K. & Williams, L. D. (2009). Peeling the Onion: Ribosomes Are Ancient Molecular Fossils. Mol Biol Evol, 26, 2415-2425.
19. Jeffares, D. C., Poole, A. M. & Penny, D. (1998). Relics from the RNA World. J Mol Evol, 46, 18-36.