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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 ribosomes^9-12 (Figure 1) 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 (Figure 2). 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 ancestral Peptidyl Transferase Centers (a-PTCs) 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-PTC, we discovered what we call “magnesium-microclusters.”¹³ Paleo-magnesium ions are conserved over vast evolutionary timescales (Figure 3) 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 – magnesium-microclusters 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 – magnesium-microclusters 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 available ions. Conservation in 1D and 2D – magnesium-microclusters 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 magnesium-microclusters 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 – magnesium-microclusters appear to form a scaffold surrounding the a-PTC.

Our current model of the a-PTC contains around 600 nucleotides of ancestral ribosomal rRNA (a-rRNA), five ancestral ribosomal peptides (a-rPeptides), and two magnesium-microclusters. We have made significant progress in characterizing the assembly, structure and thermodynamics of the a-PTC by chemical mapping, band shift assays and activity assays. Our RNase H cleavage mapping indicates that our first model of the a-PTC folds essentially as predicted, with the exception of one tetraloop and an additional secondary structural element. We are currently reiterating the design and rRNA synthesis to increase the stability and decrease the conformational restraints on this tetraloop. In addition, we have implemented SHAPE technology ¹⁷ on model systems and will soon have a highly detailed secondary structural analysis of the a-PTC.

It has been suggested, originally by Alex Rich,¹⁸ that peptide synthesis may not have been the aboriginal function of the a-PTC, which might instead have performed non-specific condensations yielding racimates, esters, thiolesters, peptides, etc. We are investigating the breadth of the catalytic potential of the a-PTC and other minimal systems such as the intact 23S rRNA in the absence of ribosomal proteins. Thus far we have seen weak catalytic function, although full and careful characterization of the reaction products is still in progress.

We are developing small model systems in which the interactions of a-rPeptides, r-Proteins, magnesium ions, a-rRNA and intact 23S rRNA can be studied by nuclear magnetic resonance, X-ray diffraction, calorimetry, fluorescence, molecular dynamics simulations, footprinting, band shifts and other biophysical techniques. Using the P4-P6 domain of the group 1 intron from tetrahymena, we have developed a method for footprinting magnesium-microclusters. A magnesium-microcluster is a binuclear magnesium complex, as we have described previously.¹⁹ Over the next year we will apply this footprinting method to the a-PTC, domains of the 23S rRNA, and to the full length 23S rRNA. We will test our hypothesis that domain III of the LSU can fold independently. We have recently shown that this footprinting method allows us to obtain Kd’s of individual magnesium ions, and can allow us to dissect the effects of ribosomal proteins on binding of individual magnesium ions.
Within the large subunit (LSU) of the extant ribosome, one can observe a ‘tail’ of ribosomal protein L2 (see Figure 3), which we call a-rPeptide^L2 (ancestral peptide derived from protein L2). a-rPeptide^L2 does not engage in protein secondary structural interactions. Based on our recent alignment results, we hypothesize that a-rPeptide^L2 is the ancestral peptide. That is to say, a-rPeptide^L2 is the oldest protein fragment in biology, and is a fossil of a proto-protein. a-rPeptide^L2 interacts with ribosomal helices rRNA 65 and 66 (conserved in a-rRNA), which in turn combines with Mg2+ to form a magnesium-microcluster. The Georgia Tech Team is in the process of investigating the structure and interactions of a-rPeptide^L2.

The Georgia Tech Team has embarked on a computational evaluation of the magnesium-microclusters that form a ‘scaffold’ for the PTC. The interaction energies of ribosomal RNA with single and multiple Mg2+ cations, in complexes extracted from the ribosome, are computed, and decomposed. The goal is determine why Mg2+ plays a special role in RNA folding, and whether other metals such as iron (II) could have been ancestral binding partners for RNA. Our recently accepted paper in RNA Journal describes RNA “clamps” with sodium, calcium and magnesium.²⁰ In that manuscript we report that RNA forms more stable clamps with magnesium than with calcium, and clamps with sodium are unstable and spontaneously open. We have also observed manganese (II) and iron (II) form clamps. Iron (II) clamps are slightly more stable than magnesium clamps. Manganese clamps are slightly less stable. As noted above we have developed a method to footprint ions in RNA microclusters. Therefore we can directly link theory and experiment. Our footprinting and computation give the same order of affinity of sodium < calcium < manganese < magnesium.
The Georgia Tech Team is developing methods to determine chronologies of ancient ribosomal evolution. We described a 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.²¹ 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. We have developed a hypothesis that the amino terminus of ribosomal protein L2 derives from the oldest protein in biology (above) and use the interactions of L2 to map the age of elements of the rRNA. 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 Poole²² 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.

References
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