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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 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 commenced the synthesis of a series of oxazolidinone-peptide conjugates (Zyvotides) that places nascent peptide at a different window to the exit tunnel. We will characterize the interaction of these zyvotides with the ribosome exit tunnel using the tools we have developed during our investigation of the peptolides.

  ROADMAP OBJECTIVES: 3.2

• Extremophile Ribosomes

  Diapausing embryos (resting eggs) from brachionid rotifers are able to withstand desiccation and thermal stress. Resting eggs can remain viable for decades, and develop normally once placed in a permissive environment that allows for hatching, growth and development. The exact mechanisms of resistance are not known, although several molecules have been suggested to confer protection during desiccation and thermal stress. In this study, we have identified by mass spectrometry two thermostable proteins, LEA (late embryogenesis abundant) and VTG (vitellogenin-like), found exclusively in the resting eggs of Brachionus manjavacas. This is the first observation that LEA proteins may play a role in thermostability and the first report of a VTG-like protein in the phylum Rotifera. These proteins exhibited increased expression in rotifer resting eggs when compared to amictic females. Our data suggest the existence of alternate pathways of desiccation and thermal resistance in brachionid rotifers.

  ROADMAP OBJECTIVES: 3.2 4.2 5.3

• 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 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

• Deconstruction of the Ribosome

  In the ribosome, RNA and protein are fully interdependent, part of the highly complex system of translation. We demonstrate here that isolated Domain III of 23S rRNA from Thermus thermophilus retains function after being split essentially in half. Chemical footprinting shows that a core of Domain III (DIII^core), obtained replacement of helices 54-59 with a simple stem-loop, folds to a near-native state in the presence of Mg²⁺ ions. Both DIII and DIII^core form specific complexes with ribosomal protein L23 in vivo, as indicated with a yeast three-hybrid experiment. L23 has a globular domain on the LSU surface and an extension (L23peptide) that penetrates into the ribosomal large subunit. In the assembled LSU, L23peptide (amino acids 58-79) traverses the surface of DIII^core. In solution, DIII^core forms a stable 1:1 complex with L23peptide, as observed with spectroscopic assay. The experiments described here are intended to recapitulate steps in early ribosomal evolution. We have previously proposed that some of the extensions of ribosomal proteins are molecular fossils that predate the globular protein domains in evolution. In our favored model of ribosomal origins, small independently-folding RNA elements associated with short peptides. Such complexes assembled to form a primitive peptidyl transferase center. The PTC evolved into the modern LSU, in a series of cooptions that left unaltered the basic structure and function of the PTC. This model predicts a continuous size distribution of folding and assembly elements within the LSU. We anticipate autonomy and specificity of folding and interaction of small, mid-sized and large rRNA and protein components.

  ROADMAP OBJECTIVES: 3.2 4.1 4.2

• Ironing Out the RNA World

  In RNA World models of evolution, RNA was once the primary biopolymer of genetics and catalysis (1). Ancient RNA-based life would have inhabited an earth with abundant soluble iron and no free oxygen (2,3). Anoxic life persisted for around 1.0-1.5 billion years before photosynthesis began producing substantial free oxygen. The ‘great oxidation’ led to Fe²⁺/O₂ mediated cellular damage (4) and depletion of soluble iron from the biosphere (5). We hypothesize that Fe2²⁺ was an RNA cofactor when iron was benign and abundant and that Fe²⁺ was replaced by Mg²⁺ during the great oxidation. The RNA-Fe²⁺ to RNA-Mg²⁺ hypothesis is in close analogy with known metal substitutions in some metalloproteins (6-11). An ancestral ribonucleotide reductase (RNR), for example, spawned di-iron, di-manganese, and iron-manganese RNRs (12). Our hypothesis is supported by observations (13) 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⁺. Here, we demonstrate 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 (14), 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.

  ROADMAP OBJECTIVES: 4.1 4.2

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

  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

• Life on the Edge: Astrobiology Summer Learning Program

  For the fourth year in a row, the Georgia Tech Team ran a one-week, non-residential Summer Learning Program (SLP), called Life on the Edge: Astrobiology. This year 20 high school students from the Atlanta metro area participated in the SLP. The GT Team recruited a high school Biology teacher and a high school Chemistry teacher teacher into the Georgia Intern Fellowship for Teachers program (GIFT, https://www.ceismc.gatech.edu/gift). The teachers this year were from the DeKalb County School System, one of the most diverse systems in the state of Georgia serving over 100,000 students; 75% are African American, 8% Hispanic, and 66% qualify for free and reduced lunch. In an effort to promote and encourage entry into science teaching careers, the GT Team paired the teachers with two Georgia Tech undergraduate students (biology majors), through the NSF sponsored “Tech to Teaching” Program (http://nsf-i3.org/projects/view/tech_to_teaching/). The teachers and undergraduate students collaborated over the summer with GT team members to prepare and develop, conduct, document, scrutinize and analyze the SLP. They updated and improved the previous years experiments, and ensured and documented that the program is aligned with the Georgia Performance Standards in Biology. Using materials from our previous camps, the teachers and the undergraduate students prepared student and teacher guides for the 2012 SLP. They worked with GT team members, in their laboratories, to prepare the SLP experiments. The teachers and undergraduate students, actually ran the camp, with participation from other GT team members.

  ROADMAP OBJECTIVES: None Selected

• 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.

  ROADMAP OBJECTIVES: 3.2 4.2