6 items with the tag “resurrection

  • Molecular Resurrection of the Ancestral Peptidyl Transferase Center
    NAI 2009 Georgia Institute of Technology Annual Report

    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
  • Molecular Resurrection of the Ancestral Peptidyl Transferase Center
    NAI 2010 Georgia Institute of Technology Annual Report

    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
  • A Phylogenomic Approach to the Ur-Ribosome
    NAI 2011 Georgia Institute of Technology Annual Report

    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
  • Resurrection of an Ancestral Peptidyl Transferase
    NAI 2011 Georgia Institute of Technology Annual Report

    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
  • Experimental Evolution and Genomic Analysis of an E. Coli Containing a Resurrected Ancestral Gene
    NAI 2012 Georgia Institute of Technology Annual Report

    We have previously described a paleo-experimental evolution system that combines Ancestral Sequence Reconstruction (ASR) with experimental evolution in the laboratory. Briefly, we designed a system that is composed of an organism with a short generation time and a protein under strong selective constraints in the modern host but whose ancestral genotype and phenotype, if genomically integrated, causes the modern host to be less fit than a modern population hosting the modern form of the protein. The modern organism hosting the resurrected protein would obviously need to be viable, but sick. E. coli and a protein, whose ancestral sequences are available termed Elongation Factor-Tu (EF-Tu), turned out to be ideal for this type of experiment.

    ROADMAP OBJECTIVES: 3.4 4.1
  • Resurrection of an Ancestral Peptidyl Transferase
    NAI 2012 Georgia Institute of Technology Annual Report

    Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro, and in vivo. The resulting model of the ancestral ribosome presented here incorporates about 20% of the extant 23S rRNA and fragments of four ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure, and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

    ROADMAP OBJECTIVES: 3.2 4.2