nai

Project Reports

• Ribosome Palentology

  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 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

• Experimental Evolution and Genomic Analysis of an E. Coli Containing a Resurrected Ancestral Gene

  In order to study the historical pathways and modern mechanisms of protein evolution in a complex cellular environment, we combined ancestral sequence reconstruction with experimental evolution. Our first goal was to identify how ancestral states of a protein effect cellular behavior by directly engineering an ancient gene inside a modern genome. We could then identify the evolutionary steps of this organism harboring the ancient gene by subjecting it to laboratory evolution, and directly monitoring the resulting changes within the integrated ancient gene as well as the rest of the host genome.

  ROADMAP OBJECTIVES: 3.4 4.1 5.1 5.2 6.1 6.2

• Life on the Edge: Astrobiology Summer Learning Program

  The Ribo Evo Center changed their focus this year and hosted 10 high school students and undergraduates in research labs over the summer. These students prepared posters and presented them at the 2013 so Max meeting in Atlanta. Currently, RiboEvo members are mentoring this cohort of high school students for their local science fair.

  ROADMAP OBJECTIVES: None Selected

• 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 has furnished vital information about the path of travel of peptides attached to the flag-pole moiety of a ketolide. In continuation of our study, we have designed and synthesized a second generation of hybrid molecules taking inspiration from peptide sequences that are known to naturally stall translation. We will characterize the interactions of these stall peptolides with the ribosome exit tunnel using the tools we have developed during our investigation of the peptolides.

  ROADMAP OBJECTIVES: 3.2

• Resurrection of an Ancestral Peptidyl Transferase

  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 incorporates about 20% of the extant 23S rRNA and fragments of four ribosomal proteins. We confirmed that the 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. We call the assembled a-RNA and rPeptide fragments the aPTC. We are currently focusing on characterizing the catalytic activity of the a-PTC.

  ROADMAP OBJECTIVES: 3.2 4.2

• Deconstruction of the Ribosome

  In this Project we are investigating the folding and interactions of a fragment of rRNA with a fragment of a ribosomal protein (rProtein), both derived from T. thermophilus. The goal is to examine the granularity of rRNA-rProtein recognition, to determine if small RNA and protein components of the ribosome can recapitulate interactions observed in the native ribosome. We have assayed the in vitro and in vivo folding and interactions of an isolated subdomain of rRNA with an rProtein and with a peptide fragment of the rProtein. Chemical mapping shows that a 199-nucleotide fragment of Domain III of the 23S rRNA (defined here as Domain IIIcore) folds to a near-native state. This rRNA fragment binds to ribosomal protein L23 in a yeast three-hybrid assay, as predicted from interactions in the native ribosome. A peptide was designed based on the segment of the rProtein that penetrates deep into the core of the native ribosome and associates primarily with Domain IIIcore. A spectroscopic assay shows that the peptide forms a 1:1 complex with both Domain III and Domain IIIcore. The results indicate that rRNA-rProtein recognition is fine-grained, and can be directed by specific interactions between small rRNA and rProtein fragments.

  ROADMAP OBJECTIVES: 3.2 4.1 4.2

• Extremophile Ribosomes

  Many animals share a common response to environmental stresses. The responses include reorganization of cellular organelles and proteins. Similar stress responses between divergent species suggest that these protective mechanisms may have evolved early and been retained from the earliest eukaryotic ancestors. Many eukaryotic cells have the capacity to sequester proteins and mRNAs into transient stress granules (SGs) that protect most cellular mRNAs. Our observations extend the phylogenetic range of SGs from trypanosomatids, insects, yeast and mammalian cells, where they were first described, to a species of the lophotrochozoan animal phylum Rotifera. We focus on the distribution of three proteins known to be associated with both ribosomes and SG formation: eukaryotic initiation factors eIF3B, eIF4E and T-cell-restricted intracellular antigen 1. We found that these three proteins co-localize to SGs in rotifers in response to temperature stress, osmotic stress and nutrient deprivation as has been described in other eukaryotes. We have also found that the large ribosomal subunit fails to localize to the SGs in rotifers. Furthermore, the SGs in rotifers disperse once the environmental stress is removed as demonstrated in yeast and mammalian cells. These results are consistent with SG formation in trypanosomatids, insects, yeast and mammalian cells, further supporting the presence of this protective mechanism early in the evolution of eukaryotes.

  ROADMAP OBJECTIVES: 3.2 4.2 5.3

• Ironing Out the RNA World

  We have proposed hypothesize that Fe²⁺ was an RNA cofactor on the ancient earth when iron was benign and abundant, and that Fe²⁺ was replaced by Mg²⁺ during the great oxidation. Our hypothesis is supported by our observations (1,2) that (i) RNA folding is conserved between complexes with Fe²⁺ and Mg²⁺ and (ii) at least some phosphoryl transfer ribozymes are more active in the presence of Fe²⁺ than Mg²⁺. We have shown that reversing the putative metal substitution in an anoxic environment, by removing Mg²⁺ and adding Fe²⁺, expands the catalytic repertoire of some RNAs. Fe²⁺ can confer on RNA a previously uncharacterized ability to catalyze single electron transfer. Catalysis is specific, in that it is dependent on the type of RNA. The 23S rRNA and tRNA, some of the most abundant and ancient RNAs (3), are found to be efficient electron transfer ribozymes in the presence of Fe²⁺. Therefore, the catalytic competence of ancient RNAs may have been greater in early earth conditions than in extant conditions, and the experiments described here may be reviving latent function. The Center is currently testing the hypothesis that replacement of Fe²⁺ by Mg²⁺ in RNA assemblies has not been universal.

  ROADMAP OBJECTIVES: 4.1 4.2

• RiboVision: Visualization and Analysis of Ribosomes

  Ribosomes present special problems and opportunities related to visualization and analysis because they are exceeding complex and information-rich. Many structures have determined at near-atomic resolution, a large number of rRNAs have been sequenced, and each is a large macromolecular assembly with many components and highly complex function. We are devising visualization and analysis methods in analogy with Google Maps, but applied to the ribosome. We have used these tools to make important discoveries relevant to ribosomal structure, function and origins.

  ROADMAP OBJECTIVES: 3.2 4.2