14 items with the tag “magnesium

  • Ribosome Paleontology
    NAI 2009 Georgia Institute of Technology Annual Report

    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
  • High Level Theory - the Role of Mg2+ in Ribosome Assembly
    NAI 2009 Georgia Institute of Technology Annual Report

    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
  • Experimental Model System - an Ancestral Magnesium-RNA-Peptide Complex
    NAI 2009 Georgia Institute of Technology Annual Report

    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
  • Ribosome Paleontology
    NAI 2010 Georgia Institute of Technology Annual Report

    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
  • High Level Theory - the Role of Mg2+ in Ribosome Assembly
    NAI 2010 Georgia Institute of Technology Annual Report

    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
    NAI 2010 Georgia Institute of Technology Annual Report

    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
  • Project 5C: Fluid-Mineral Fractionation of Mg Isotopes and Tracing the Origin of Sulfate Minerals
    NAI 2010 University of Wisconsin Annual Report

    We are developing an experimental program to characterize the Mg isotope fractionation between fluids and minerals in order to use the Mg isotope system to characterize the paleoenvironmental conditions of ancient terrestrial rocks and samples from Mars. Our initial work has focused on Mg isotope fractionation between aqueous Mg and epsomite. Magnesium sulfate is present on the surface of Mars, where, for example, up to 36 wt. % sulfate has been found in some outcrops on the Martian surface, of which Mg-sulfate is the most abundant (Clark et al., 2005). Sulfates are a major water reservoir for the Martian surface and thus it is inferred that there was a period of aqueous alteration on Mars (e.g., Wang et al., 2008). Knowledge of the controls on Mg isotope fractionation in the system fluid and Mg sulfate will allow us to ultimately characterize the evaporation rates and Mg fluxes that occurred during one of the wettest periods in Mars History.

    ROADMAP OBJECTIVES: 2.1 4.1 7.1 7.2
  • Ribosome Paleontology
    NAI 2011 Georgia Institute of Technology Annual Report

    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 time 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 4.2
  • High Level Theory - the Role of Mg2+ in Ribosome Assembly
    NAI 2011 Georgia Institute of Technology Annual Report

    Magnesium plays a special role in RNA function and folding. Although water is magnesium’s most common first-shell ligand, magnesium has significant affinity for the oxyanions of RNA phosphates. Here we provide a quantum mechanical (QM) description of first shell RNA-magnesium and DNA-magnesium interactions, demonstrating unique features that appear to be required for folding of large RNAs. Our work focuses on multidentate chelation of magnesium by RNA and DNA, where multiple phosphate oxyanions enter the first coordination shell of magnesium. The results suggest that magnesium, compared to calcium and sodium, has enhanced ability to form bidentate chelation complexes with RNA. Sodium complexes, in particular, are unstable and spontaneously open. A magnesium cation is closer to the oxyanions of RNA than the other cations, and is stabilized not only by electrostatic interaction with the oxyanions but also by charge transfer and polarization interactions. Those interactions are quite substantial at close distances. The quantum effects are less pronounced for calcium due to its larger size, and for sodium due to its smaller charge. Additionally, we find that magnesium complexes with RNA are more stable than those with DNA. The nature of the additional stability is twofold: it is due to a slightly greater energetic penalty of ring closure to form chelation complexes for DNA, and elevated electrostatic interactions between the RNA and cations. In sum it can be seen that even at high concentration, sodium and calcium cannot replicate the structures or energetics of RNA-magnesium complexes.

    ROADMAP OBJECTIVES: 3.2 4.2
  • 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
  • Ironing Out the RNA World
    NAI 2011 Georgia Institute of Technology Annual Report

    Life originated during the early Archean, which was characterized by a non-oxidative atmosphere and abundant soluble Fe2+. Current theories on the origin of life focus on RNA-based genetic and metabolic systems. Here we show, by theory and experiment, that critical roles of Mg2+ in extant RNA folding and function can be better served by Fe2+ in the absence of oxygen. The results of our high-level quantum mechanical calculations show that the geometry of coordination of Fe2+ by RNA phosphates is similar to that of Mg2+. The conformation of Tetrahymena Group I intron P4-P6 domain is conserved between complexes of Fe2+ and Mg2+. Additionally, a ribozyme obtained previously by in vitro selection in the presence of Mg2+ and a natural ribozyme both have significantly greater catalytic competence in the presence of Fe2+ than in Mg2+. The combined biochemical and paleogeological data are consistent with an RNA-Fe2+ world that could have supported an array of RNA structures and catalytic functions far more diverse that of an RNA-Mg2+ world.

    ROADMAP OBJECTIVES: 4.1 4.2
  • 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
  • Ironing Out the RNA World
    NAI 2012 Georgia Institute of Technology Annual Report

    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