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Project Reports

• An Atomic Level Description of the Specific Interactions Between Nascent Peptide and Ribosome Exit Tunnel

  The ribosome exit tunnel is 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.

  ROADMAP OBJECTIVES: 3.2

• In Vivo Deconstruction and Restoration of Ribosomal

  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.

  ROADMAP OBJECTIVES: 3.2 4.2

• Life on the Edge: Astrobiology Summer Learning Program

  In their 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.

  ROADMAP OBJECTIVES: None Selected

• Extremophile Ribosomes

  One of the biggest challenges facing eukaryote extremophiles is the loss of water leading to desiccation. Resistance to desiccation as adults, juveniles, seeds, or spores is found in species of five animal phyla and four divisions of plants. Little is understood about the biochemistry of desiccation tolerance in eukaryotes, but ribosomes are likely to figure prominently in this phenomenon. The model for our investigations are animals from the phylum Rotifera which are capable from going from completely desiccated to an active, swimming animal in minutes to hours. 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.

  ROADMAP OBJECTIVES: 3.2 4.2 5.3

• Ribosome Paleontology

  The origins of the translation machinery remain imprinted in the extant ribosome. The conformations of ribosomal RNA and protein components can be seen to change over time indicating clear molecular fossils. We are establishing methodology to determine chronologies of ancient ribosomal evolution. 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.

  ROADMAP OBJECTIVES: 3.2 4.2

• Domain III of the 23S rRNA: An Independent Domain

  The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize 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. 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.

  ROADMAP OBJECTIVES: 3.2 4.2

• Ironing Out the RNA World

  Life originated during the early Archean, which was characterized by a non-oxidative atmosphere and abundant soluble Fe²⁺. Current theories on the origin of life focus on RNA-based genetic and metabolic systems. Here we show, by theory and experiment, that critical roles of Mg²⁺ in extant RNA folding and function can be better served by Fe²⁺ in the absence of oxygen. 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.

  ROADMAP OBJECTIVES: 4.1 4.2

• A Phylogenomic Approach to the Ur-Ribosome

  A macromolecular complex emulating ancient ribosomal function would sharpen understanding of the peptide bond’s emergence in a biological context. Reconstructing evolutionary adaptive paths (1) offers a straightforward approach to building an ancient ribosome, in principle: 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.

  ROADMAP OBJECTIVES: 3.2

• Assessing the Role of Chance vs. Necessity in Evolution: Experimental Evolution of Ancient Proteins in E.coli

  The goal of our current work is to create a “Bacterial Jurassic Park” in the laboratory. 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 me to monitor evolution in action (1-6).

  ROADMAP OBJECTIVES: 4.2

• Astrobiology Research by Undergraduates and High School Students

  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.

  ROADMAP OBJECTIVES: None Selected

• Astrobiology Self Directed Short Course and the NASA Electronic Professional Development Network (Astrobiology ePDN)

  The Center for Ribosomal Origins and Evolution along with other contributors is working to bring Astrobiology content to teachers through the new NASA Electronic Professional Development Network (ePDN).

  ROADMAP OBJECTIVES: None Selected

• High Level Theory – the Role of Mg2+ in Ribosome Assembly

  Magnesium plays a special role in RNA function and folding. Although water is magnesium’s most common first-shell ligand, magnesium has significant affinity for the oxyanions of RNA phosphates. Here we provide 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. 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.

  ROADMAP OBJECTIVES: 3.2 4.2

• Resurrection of an Ancestral Peptidyl Transferase

  We have created and test both in silico and in vitro models of an ancestral pepidyl transerase center (PTC). Our most recent in silico and in vitro models contain a significantly reduced 23S rRNA (called a-rRNA-γ, Figure 1), 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.

  ROADMAP OBJECTIVES: 3.2 4.2