Browsing by Subject "Ribosomes"
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Item Assembly of gold nanoparticles by ribosomal molecular machines(2005) Pavel, Ioana Simona; Browning, Karen S.The goal of this work is to develop novel method for organizing gold nanoparticles by linking them to chemically reactive side chains amino acids (e.g., lysine and cysteine) and then incorporating these modified amino acids into a polypeptide through in vitro translation of a synthetic mRNA template. The use of this system not only provides a method for the supramolecular assembly of new bio-inorganic heterostructures, using ribosomes as natural molecular machines and an artificial coding template, but also provides a new tool for probing fundamental biological processes (e.g., translation). Transfer RNA (tRNA) is transcribed in vitro from a plasmid-template containing the tRNA gene under control of the T7 promoter. A novel coupled transcription– aminoacylation (CTA) system which prepares large amounts of pure aminoacyl-tRNA in a single reaction mixture was developed. Radiolabeled [32P]-UTP and [14C] or [32S]- amino acid are used to monitor the coupled transcription- aminoacylation reactions. The ability to further modify the tRNA with a biotinylated probe is also demonstrated. The tRNA synthesized by CTA, or further modified with biotin is shown to retain its functionality in in vitro translation, allowing the synthesis and detection of biotin-labeled protein. Following the charging of the tRNA, the side chain of the amino acid is covalently linked to a gold nanoparticle, i.e. Monomaleido or Mono-sulfo-Nhydroxysuccinimide Nanogolds, either directly or through a heterobifunctional linker, which may vary in length and/or chemical properties, depending on the system used. The result is a novel gold nanoparticle-conjugated tRNA. Each of the steps is verified by mass spectroscopy (ESI and/or MALDI). The attachment of the gold nanoparticle to modified aminoacyl-tRNA is confirmed using High angle annular dark field images (HTEM) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The incorporation of the quantum dot-conjugated tRNA into a polypeptide is measured using an in vitro E. coli S30 cell-free coupled transcription-translation system, and verified by electrophoresis, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), High angle annular dark field (HAADF) images and electron loss spectroscopy .Item Characterization of an Orphan Riboswitch: Identification of a Metal-Sensing Regulatory RNA(2011-09-30T18:58:50Z) Wakeman, Catherine Ann; Winkler, Wade C.Riboswitches are RNA-based genetic control elements found in untranslated regions of the mRNA transcript that they regulate. These RNA motifs are highly structured and bind metabolites to elicit control of gene expression. Typically, the metabolite sensed by these RNAs is a component of the metabolic pathway in which the regulated gene product resides. The focus of this project has been the identification of the ligand for a riboswitch that was discovered using bioinformatics-based search methods. This riboswitch was designated the ykoK RNA element due to its location in the 5' UTR of the B. subtilis ykoK (mgtE) gene, which appears to be a magnesium transporter. Therefore, the possibility that this RNA senses magnesium levels was explored. The data revealed that the RNA element imparts magnesium-responsive regulation to the ykoK gene. These data also indicated which portions of the RNA are essential for genetic regulation. The results of a battery of biochemical tests demonstrated that magnesium triggers a concerted conformational change in the RNA such that it adopts a compacted tertiary structure. Resolution of the three-dimensional structure of the RNA in the magnesium bound state revealed the basis of this metal-induced tertiary conformation and how this relates to genetic control. Intriguingly, this structure revealed the presence of six magnesium ions, making this the first example of multiple ligands binding to a single riboswitch aptamer. When individual metal-binding sites were eliminated using phosphorothioate substitutions, it became evident that all six of these magnesium-binding sites and up to three additional metal-binding sites are required for function of this RNA. Therefore, these data demonstrate that the ykoK RNA element, now designated the M-box RNA, directly senses intracellular magnesium levels for the purposes of genetic control. These findings should have broad implications given that this RNA element is wide spread among Gram-positive bacteria and appears to regulate many additional gene categories such as ABC transporters, cell division proteins, and proteins of unknown function. The exploration of the connection between magnesium concentration and the expression levels of these proteins might provide insights into previously undefined functional roles. [Keywords: regulatory RNA; metal homeostasis; RNA structure; magnesium; riboswitch]Item From developing protein-protein interaction strategies to identifying gene functions: case studies for transcription factor complexes and ribosome biogenesis genes(2007-12) Li, Zhihua, doctor of cell and molecular biology; Marcotte, Edward M.Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.Item Functional characterization of the recycling mechanism for the 60S nuclear export adapter Nmd3p in yeast(2004) Hedges, John Benjamin; Johnson, Arlen W.Ribosomes are essential macromolecular machines that translate, through a messenger RNA intermediate, the information encoded in the DNA sequence of all cells into proteins. Because of their fundamental role in cell survival, an enormous amount of cellular resources must be dedicated to ribosome synthesis. Eukaryotic cells must contend with transport of materials between two compartments, the nucleus and cytoplasm. During ribosome biogenesis, these cells assemble ribosomal subunits at a specific subnuclear structure called the nucleolus and only release them into the nucleoplasm upon completion of initial assembly. The subunits must then traverse the nucleoplasm before reaching the nuclear pore complex (NPC) where they are exported to the cytoplasm to act in translation. Processing of the small (40S) subunit through this pathway occurs relatively quickly. However, processing of the large subunit (60S) involves a greater number of maturation steps. One of the last of these steps being export of the large subunit through the NPC, mediated through the export adapter protein Nmd3. The large ribosomal subunit protein Rpl10 is required for 60S export and subunit joining in yeast. It is believed that the role of Rpl10p in export is to provide the 60S binding site for Nmd3p in the nucleus. Through examination of rpl10 mutant effects on the 60S export pathway, I’ve instead found that the role of Rpl10p is indirect. This work shows that disruption of either Rpl10p or the Rpl10p 60S loading factor, Sqt1p, leads to a block in export due to entrapment of Nmd3p on 60S subunits in the cytoplasm. For rpl10 mutants these effects are suppressed by specific alleles of NMD3 that restore recycling to the nucleus. To gain a better understanding of the export function of Nmd3p, this work also examines the NES and 60S binding domains of Nmd3p and, in light of the Rpl10p results shown here, establishes an assay to identify other 60S components required for this binding. From these findings, I propose the model that Rpl10p is required for the release of Nmd3p from subunits in the cytoplasm to support further rounds of 60S export and to provide a final “quality control” step in 60S maturation prior to 40S joining.Item Nmd3p, the nuclear export adapter for the 60S ribosomal subunit: characterization of its recycling mechanism and novel interaction with the nuclear pore complex in yeast(2005) West, Matthew Blaine; Johnson, Arlen W.Item The ribosome biogenesis factor Arx1p: characterization of its recycling mechanism and its role in ribosome export(2007) Hung, Nai-Jung, 1976-; Johnson, Arlen W.Translation is an essential and fundamental process that coverts genetic codes into functional polypeptides by an apparatus called ribosome. In eukaryotic cells, ribosomes are composed of two subunits: the large (60S) subunit and small (40S) subunits. In Saccharomyces cerevisiae, ribosome biogenesis is complex and requires the involvement of over ~170 trans-acting factors. As a growing number of factors were identified related to this essential metabolic pathway, our lab has contributed to functional characterization of the late 60S subunit biogenesis pathway that centers on Nmd3p. This work particularly focuses on characterizing of the nuclear shuttling trans-acting factor Arx1p found in the Nmd3p-60S subunit particle. A working model that describes how Rei1p, another cytosolic trans-acting factor, recycles Arx1p is presented. This work also shows a similar mode of Arx1p recycling by the Hsp40 J-protein, Jjj1p. Furthermore, I have investigated functional interplay between Arx1p and Rpl25p, a 60S ribosomal protein at the polypeptide exit tunnel. These findings further reveal the involvement of Arx1p at the polypeptide exit tunnel in mediating association of other factors with 60S subunits. Beyond its function at the polypeptide exit tunnel, this work also focuses on a function for Arx1p in the export of 60S subunits. In yeast and higher eukaryotes, 60S subunit export depends on the export adaptor Nmd3p via Crm1-dependent pathway. I show that ARX1 interacts with the NES of Nmd3p and nucleoporins. From these results, I propose that Arx1p acts as another export receptor to facilitate 60S subunit export.Item Structural studies of ribosomal RNA based on cross-analysis of comparative models and three-dimensional crystal structures(2003) Lee, Jung Chull; Whitman, Christian P.; Gutell, RobinBasepair mapping of the rRNAs in the high-resolution crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits not only proved the authenticity of covariation analysis in predicting RNA secondary structures with high accuracy, but also provided a great wealth of information on RNA structure, ranging from diverse basepair conformations to structure motifs. Based on cross-analysis of comparative structure models and crystal structures of rRNAs, a systematic and unambiguous classification for basepair conformations is established, and the structural features and biological implications for a wide variety of RNA sequence and structure motifs, including the AA.AG@helix.ends motif, the sticky motif consisting of AGUA/GAA, GUA/GAA, and GGA/GAA motifs, and the lonepair triloop motif, are discussed.Item Studies in asteraceae : nuclear ribosomal DNA and macaronesian endemics(2001-08) Goertzen, Leslie Richard; Jansen, Robert K., 1954-; Levin, Donald A.The phylogenetic and biogeographical history of the Asteriscus alliance in Macaronesia was investigated with DNA sequence data from the nuclear and chloroplast genomes. Relationships among taxa were fully resolved by combining data from these sources. The alliance is divided into two groups, the monophyletic genus Pallenis, and a larger clade containing Asteriscus and the monotypic Ighermia. The direction of island colonization events is difficult to infer because of the wide distributions assumed for ancestral taxa. ITS sequences from related Inuleae and other species of Asteraceae were used to derive a secondary structure model by comparative analyses. Tribal relationships reconstructed from the resulting alignment show considerable congruence with previous molecular and morphological studies and demonstrate the retention of deep phylogenetic signal in ITS sequence data. Incongruence between Argyranthemum nuclear and chloroplast data was assessed as part of an investigation into hybridization in this Macaronesian endemic genus. ETS sequence data are incongruent with chloroplast restriction site data but the differences are more likely due to insufficient phylogenetic signal than different evolutionary histories.