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Project Reports

• Ribosome Paleontology

  We are establishing method to determine chronologies of ancient ribosomal evolution. One method, just published, uses structure-based and sequence-based comparisons of the LSUs of Haloarcula marismortui and Thermus thermophilus, along with an “onion approximation”. The results suggest that the conformation and interactions of both RNA and protein change, in an observable manner, over evolutionary time.

  ROADMAP OBJECTIVES: 3.2

• 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

• 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

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

  We have embarked on a computational evaluation of the role of Mg2+ microclusters observed to form a scaffold for the extant and ancestral peptidyl transferase center. The interaction energies of ribosomal RNA with single and multiple Mg2+ cations are computed, and deconvolved. The results will be compared to those with other metals, to determine why Mg2+ plays a special role in RNA folding.

  ROADMAP OBJECTIVES: 3.2 5.3

• 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

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

  We will develop small model systems in which the interactions of a-rPeptides, Mg2+ ions and a-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 can observe a tail of ribosomal protein L2 (which we call a-rPeptideL2) that interacts with ribosomal helices rRNA 65 and 66 (which are conserved in a-rRNA), which in turn combines with Mg2+ to form a Mg2+-mc. We will define the smallest a-rRNA and peptide segments (of L2) that are sufficient for assembly of this complex and will characterize the assembly by a variety of experimental and computational methods.

  ROADMAP OBJECTIVES: 3.2

• Molecular Resurrection of the Ancestral Peptidyl Transferase Center

  We have designed, and are resurrecting, a model of the a-PTC (ancestral Peptidyl Transferase Center), which we believe to be around 4 billion years old. The proposed a-PTC contains around 600 nucleotides of ancestral ribosomal RNA (a-rRNA), three ancestral ribosomal peptides (a-rPeptides), and inorganic cations, all of which are relatively straightforward to obtain or produce. The results of our molecular resurrection will allow one to test ideas about primitive living systems, including the origin of protein.

  ROADMAP OBJECTIVES: 3.2