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Project Reports

• Extremophile Ribosomes

  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 that reminiscent of early biotic earth.

  ROADMAP OBJECTIVES: 5.3

• Reverse-Evolution of an RNA-based RNA Polymerase

  The RNA World Hypothesis suggests an RNA molecule is capable both of encoding information and replicating it. In essence, the RNA World Hypothesis predicts an RNA polymerase ribozyme. Since there are no extant RNA-based RNA polymerases, we must instead search the evolutionary fossil record for hints. Our primary goal is to test the hypothesis of Poole that the Small Subunit (SSU) of the ribosome may have evolved from an RNA-dependent RNA polymerase ribozyme.1 We will test the plausibility of an RNA polymerase origin of the SSU by using in vitro reverse evolution; If we can reverse-evolve the SSU into an RNA polymerase, we can demonstrate the a possible evolutionary pathway between a putative primordial ribozyme polymerase and modern ribosomes.

  ROADMAP OBJECTIVES: 3.2

• Fostering Synergetic Interactions Among NAI Teams Reconstructing Early Life on Earth, and Attaching a Time Scale to the Genomic Record of Life

  Today there is a nearly universal consensus that a tree cannot describe the early evolution of life, but there is not yet a consensus about how to describe life’s early evolution. Our lab is developing new methods to incorporate symbioses and endosymbioses into reconstructions of early life on Earth and thereby represent life as a combination of tree like- and symbiotic like- evolution. Using these improved methods, we are attaching a time scale to the rings that represent this early evolution, in order to better understand significant early events in Earth’s history like the origin of oxygenic photosynthesis.

  ROADMAP OBJECTIVES: 3.2

• Molecular Resurrection of the Ancestral Peptidyl Transferase Center

  We have resurrected, reconstructed, and are currently reconstituting a model of the a-PTC (ancestral Peptidyl Transferase Center), which we believe to have evolved around 4 billion years ago. The proposed a-PTC contains 644 nucleotides of ancestral ribosomal RNA (a-rRNA), five ancestral ribosomal peptides (a-rPeptides), and inorganic cations. Here we show data of the a-rRNA folding with Mg2+ and a-rPeptides

  ROADMAP OBJECTIVES: 3.2

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

  We investigated a unique role of Mg2+ ions to form stable complexes with ribosomal RNA, and specifically their role in a formation of ancestral peptidyl transferase center using modern quantum mechanics methods. The interaction energies of ribosomal RNA with single and multiple Mg2+ cations are computed in the gas phase and water, and partitioned into specific tems. RNA-Mg interactions are compared to those with other metals, to determine why Mg2+ plays a special role in RNA folding. Additionally, we hypothesize a possible unique role of Fe2+ in a formation of ribosomal catalytic centers during early stages of life. The project is performed using NASA’s HEC supercomputer recourses.

  ROADMAP OBJECTIVES: 3.2 5.3

• Experimental Model System – an Ancestral Magnesium-RNA-Peptide Complex

  We are developing small model systems in which the interactions of rPeptides, Mg2+ ions and rRNA can be studied by NMR, X-ray diffraction, calorimetry, molecular dynamics simulations, and other ‘high resolution’ biophysical techniques. Within the large subunit of the extant ribosome, one observes an autonomous rRNA:Mg2+-mc complex in Domain III, which appears to fold independent of the rest of the LSU. Ribosomal protein L23 associates closely with this rRNA: Mg2+-mc complex in both bacteria and archaea, suggesting the possibility of distinct evolutionary origin. We will define the smallest Domain III rRNA and associated peptide segments sufficient for assembly of this complex, and will characterize their assembly and interactions with a-PTC and 23S lacking Domain III by a variety of experimental and computational methods.

  ROADMAP OBJECTIVES: 3.2

• RNA Folding and Assembly

  We will characterize the assembly, structure and thermodynamics of the a-PTC by chemical mapping, including hydroxyl radical footprinting1,2 and SHAPE analysis,3 RNase H cleavage, temperature dependent hydrodynamics,4 and computational folding algorithms. In addition we will investigate the effect of freezing aqueous solutions of RNA and DNA molecules on their ability to assemble into larger more complex structures. Freezing nucleic acid solutions concentrates non-water molecules into small liquid pockets in the ice. This enables reactions that can promote the assembly of small segments of nucleic acids into larger complexes.

  ROADMAP OBJECTIVES: 3.2 5.3

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

  Ribosome peptide exit tunnel plays a crucial role in the functioning of ribosomes across all domains of life.1 2 3 Before the transition of nascent peptides to mature functional proteins, they must travel through the functionally conserved peptide exit tunnel. 4 Additionally, the latent chaperone activity of the exit tunnel 5 6 suggests its role in ribosomal evolution, in the transition from short non-structured peptides to extant globular proteins. The wall of the tunnel is constructed mostly from RNA. As high as 80% of the tunnel is RNA in some species. 4 Our objective is to gain an understanding of the molecular basis of the latent chaperone activity and the preferential construction of the ribosome exit tunnel from the RNA component of the ribosome. Toward this end we have designed ketolide-peptide compounds (peptolides) to probe the mechanisms employed by the ribosome to, (i) facilitate in-tunnel folding of nascent peptides and (ii) distinguish between some peptide sequences while facilitating unhindered passage of the vast majority of peptides through the peptide exit tunnel.

  ROADMAP OBJECTIVES: 3.2

• Ribosome Paleontology

  We are inventing methodologies to determine the chronology of ribosomal origin and evolution. Our premise, which is generally accepted, is that substantial information relating to the origins and early development of the translation machinery remains imprinted in the ribosome: in the sequences, folding, assembly, molecular interactions, and functions of the ribosome’s various macromolecules and small molecule affectors. To this end, we are developing new methods for ribosomal paleontology. We are using these methods to determine the relative ages of ribosomal components and subsystems, and to understand fundamental aspects of the folding and assembly of RNA and protein. We will develop timelines for the history of the ribosome as a whole, as well as for various sub-processes such as initiation, termination, and translocation. The results of these studies will interface ribosomal history with other keys relating to the origin of life, including the origin of proteins and RNA, the emergence of the genetic code, the origin of chirality, and the nature of the last common ancestor.

  ROADMAP OBJECTIVES: 3.2