Дисертації: "TRNA Function"

1

Boonyalai, Nonlawat. "Lysyl-tRNA synthetase : structure-function studies." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429379.

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2

Kelly, Paul Michael. "Characterizing the Function of Alanyl-tRNA Synthetase Activity in Microbial Translation." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586259003806337.

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3

Dhungel, Nripesh. "tRNA Splicing Endonuclease: Novel and Essential Function Beyond tRNA Splicing and Subunit interactions." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337968400.

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4

Swinehart, William E. Jr. "A Biochemical Investigation of Saccharomyces cerevisiae Trm10 and Implications of 1-methylguanosine for tRNA Structure and Function." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429867956.

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5

Rogers, Theresa Elizabeth. "Elucidating the Function of a Pseudo-tRNA in Bacillus cereus." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1291213318.

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6

Faruggio, Dawn C. (Dawn Catherine) 1965. "Stucture-function relationships of human initiator tRNA mutants and attempted regulated expresion of tRNA genes in yeast." Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/32665.

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7

Picchioni, Daria. "Biological function of SLIMP, a mitochondrial seryl-tRNA synthetase paralog." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/283974.

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Our research group focuses on protein translation and more specifically on the mechanism of transfer RNA (tRNA) aminoacylation by a family of essential and universal enzymes called aminoacyl-tRNA synthetases (aaRSs). aaRS are essential and universal components of the genetic code and their long evolutionary history explains the growing number of functions being discovered for aaRS and for aaRS homologues, beyond their canonical role in gene translation. During the process of constructing a model for human disorders caused by mitochondrial tRNA aminoacylation deficiencies in Drosophila melanogaster in the laboratory has been identified a previously uncharacterized paralog of a seryl-tRNA synthetase (SerRS) named SLIMP. Given the conservation of SLIMP in all available insect genomes, a characterization of the phenotype upon SLIMP depletion was conducted in Drosophila melanogaster. We analyzed the specific effect of SLIMP knockdown in muscles and glia, and we observed a dramatic reduction of the lifespan in both mutants. Interestingly, SLIMP knockdown in the glia causes neurodegeneration visualized as vacuolization in the brain of mutant flies. The sequence identity that SLIMP shares with SerRS allowed to predict that this protein has an identical fold of a seryl-tRNA synthetase. It was previously shown that SLIMP does not posses tRNA aminoacylation activity, but it retains the property to bind mitochondrial tRNASer isoacceptors as a possible reflection of the evolutionary origin of the protein. We characterized the RNA-and DNA-binding property of SLIMP through in vitro and in vivo methodologies. We found that SLIMP binds in vitro all RNA sequences that are encoded in the mitochondrial genome. We suggest that the composition of these sequences (AT enrichment) and the respective folding energy (low .G) are the main determinants for SLIMP binding to RNA. We performed ribonucleoprotein immunoprecipitation assay (RIP) in cells to study SLIMP-RNA interaction in vivo and we showed that SLIMP interact with almost all mitochondrial transcripts. Pull-down experiments combined to mass spectrometry analysis revealed at least two SLIMP protein interactors: SRS2 and LON protease. We demonstrated that SLIMP and SRS2 are interdependent, as the knockdown or the overexpression of one protein reduced or increase respectively the level of the other. We suggest that SLIMP and SRS2 might form a functional complex that is maintained at a given SLIMP:SRS2 ratio. LON was the other identified SLIMP interactor. Our data shows that LON and SRS2 do not interact, but SRS2 might be a substrate of LON proteolytic activity. We also found OPA1 as a potential interactor of LON. OPA1 is a dynamin-like GTPase that is responsible for inner membrane fusion. Our results indicated that the overexpression of LON protease results in an increase of the smaller OPA1 isoforms that correlates with mitochondrial stress. Given the proposed role of SLIMP in RNA binding, we aimed to determine whether SLIMP has an effect on mitochondrial transcription. The results obtained by Northern blot screening of some mitochondrial mRNAs revealed that knockdown of SLIMP significantly reduced the steady-state levels of COX2 and COX3 mRNA, and 12S and 16S rRNAs whereas tRNA levels were found constant. Our results suggest that the defect in transcription upon SLIMP depletion, might be limited to mature mRNAs and may reveal a specific function for SLIMP in the binding and stabilization of processed mitochondrial mRNAs rather than a role in the control of their transcriptional rate or processing. We showed that knockdown of SLIMP affects also cellular growth and cell cycle progression, in fact, upon SLIMP depletion, we observed a dramatic increase of cells in G2/M phase. This result suggests that the mitochondrial role of SLIMP, or a consequence of its function, may be acting as a crosstalk between mitochondria and nuclear transcription factors that regulate cell proliferation. Collectively, the work described in this thesis has contributed to the characterization of a mitochondrial seryl-tRNA synthetase paralog that has acquired an essential function in insects despite a relatively modest divergence from a canonical SerRS structure.
El nostre grup de recerca es centra en la traducció de proteïnes i més específicament en el mecanisme d’aminoacilació dels àcids ribonucleics (ARNs) de transferència (ARNt) per una família d’enzims essencials i universals anomenats aminoacil-ARNt sintetases (aaRSs). Al laboratori s’han analitzat el paper de les aaRSs en la traducció proteica, les seves funcions no canòniques, la seva evolució, així com la seva implicació en malalties humanes. Les aaRSs són components universals i essencials del codi genètic. La seva llarga historia evolutiva explica el creixent número de funcions que s’estan descobrint, tant per a elles com per a proteïnes paràlogues, més enllà del seu paper canònic en traducció genètica. Al laboratori, durant el procés d’obtenció d’un model a Drosophila melanogaster per a l’estudi de malalties humanes degudes a deficiències en l’aminoacilació d’ARNt, es va identificar un nou gen, paràlog de la seril-ARNt sintetasa (SeRS) mitocondrial, anomenat SLIMP. La proteïna SLIMP representa un nou tipus de proteïna similar a aaRS que ha adquirit una funció essencial a insectes, tot i la relativament baixa divergència respecta a una estructura d’SeRS canònica. Tot i amb això, són necessaris estudis addicionals per a identificar el paper biològic de SLIMP. Per aconseguir aquesta fita, s’ha portat a terme el projecte descrit en aquest manuscrit, el qual consisteix en anàlisis addicionals del fenotip resultant de la depleció de SLIMP in vivo, seguits d’estudis detallats de les interaccions moleculars amb àcids nucleics i proteïnes, per acabar amb un estudi dels efectes de SLIMP en la fisiologia cel•lular. En conjunt, els nostres resultats demostren que SLIMP s’uneix específicament a ARNs mitocondrials in vivo i in vitro. SLIMP interacciona amb SerRS2 i les dues són interdependents a nivell d’estabilitat proteica. La depleció de SLIMP o de SerRS2 redueix els nivells basals d’alguns ARNm mitocondrials, però la transcripció d’ARNt és manté inalterada. Es proposa un rol en la regulació post-transcripcional o en l’estabilitat dels ARNm madurs. Hem observat també que la depleció de SLIMP indueix l’aturada del cicle cel•lular en la transició G2/M. Aquests resultats suggereixen que SLIMP, o una conseqüència de la seva funció, podria tenir un paper d’enllaç entre els mitocondris i els factors de transcripció nuclears que regulen la proliferació cel•lular.

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8

Cacan, Ercan. "Evolutionary synthetic biology: structure/function relationships within the protein translation system." Thesis, Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45838.

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Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.

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9

Mirando, Adam Christopher. "Characterization Of A Non-Canonical Function For Threonyl-Trna Synthetase In Angiogenesis." ScholarWorks @ UVM, 2015. http://scholarworks.uvm.edu/graddis/523.

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In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

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10

Kubicek, Charles E. 1981. "Identifying targets and function of the ubiquitin related modifier Urm1 in Saccharomyces cerevisiae." Thesis, University of Oregon, 2009. http://hdl.handle.net/1794/10310.

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xi, 81 p. : ill. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.
Post-translational modification of proteins is an important cellular method of controlling various aspects of protein activity, including protein-protein interactions, half- life, and transport. An important class of post-translational modifications involves the ubiquitin family of proteins. In these modifications, a small protein, such as ubiquitin, is conjugated to a target protein through an isopeptide bond. Conjugation by a ubiquitin family member acts as a signal to regulate the activity, function, or stability of the target protein. Urm1, a ubiquitin-like protein conserved throughout all eukaryotes, was initially identified in S. cerevisiae. Loss of Urm1 leads to the disruption of a variety of cellular processes, including oxidative stress response, filamentous growth, and temperature sensitivity. This body of work comprises efforts to identify novel targets of Urm1, the mechanism by which Urm1 is attached to target proteins, and the physiological consequences of such conjugation. To gain understanding of the function and mechanism of Urm1 conjugation, the only known conjugate of Urm1, the peroxiredoxin reductase Ahp1, was examined in an effort to identify the site of modification on Ahp1 and to evaluate the physiological consequences of urmylation of Ahp1. I then completed a series of screens--a synthetic lethal screen, a two-hybrid screen, and a protein over-expression screen--to identify novel Urm1 conjugates and cellular functions dependent on Urm1. Of particular interest were genes identified in the synthetic lethal screen, namely PTC1, which encodes a protein phosphatase, and a set of genes encoding the Elongator complex, which functions in transcriptional elongation and tRNA modification. During this time period, other groups showed that thiolation of tRNAs depends on Urm1. Thus, Urm1 does not function only in protein conjugation, but also as a sulfur carrier in the thiolation of tRNA. Interestingly, I identified Elp2, a component of the Elongator complex, as a new Urm1-conjugate. Because Elp2 is also required for tRNA modification, perhaps Urm1 plays more than one role in tRNA modification. Loss of tRNA modification may disrupt many cellular functions and could explain the variety of urm1 mutant phenotypes. I have determined that all known Urm1 dependent processes are also associated with tRNA modification.
Committee in charge: Karen Guillemin, Chairperson, Biology; George Sprague, Advisor, Biology; Alice Barkan, Member, Biology; Kenneth Prehoda, Member, Chemistry; Tom Stevens, Outside Member, Chemistry

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11

Wo, Peibin. "Assessment Of A Function For Threonyl-Trna Synthetase In Angiogenesis In A Mouse Ovarian Cancer Model." ScholarWorks @ UVM, 2017. https://scholarworks.uvm.edu/graddis/839.

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Despite the high mortality rate of ovarian cancer, there are few selective biomarkers that detect its progression and none have become successful targets for therapy. A complex microenvironment that promotes angiogenesis, reduces immune responses and alters the integrity of the surrounding matrix is involved through the biology of ovarian cancer. Previous studies done by our lab and collaborators indicated that extracellular threonyl-tRNA synthetase (TARS) is a pro-angiogenic mediator of the ovarian tumor microenvironment, which is secreted in response to inflammatory signals, and actively promotes angiogenesis. In order to better understand the mechanisms underlying the angiogenic effects of TARS in ovarian cancer, it is essential to identify whether it directly affects ovarian tumor growth and invasion. Preliminary evidence indicated that TARS is secreted from ovarian cancer cells in response to TNF-α and TARS exhibits extracellular angiogenic activity. In previous studies, TARS was shown to significantly increase migration of HUVECs in a transwell assay to an extent that was similar to VEGF. The purpose of this project was to establish a role for TARS in tumor progression and its potential as a diagnostic marker using an animal model of ovarian cancer. The hypothesis tested is that TARS plays a key role in the angiogenic and invasive potential of ovarian cancer, and TARS inhibition will reduce the angiogenic effect of tumor cells which is reflected by measurement of intratumor microvessel density (MVD). The study tested the effect of BC194-mediated TARS inhibition on the development of ovarian tumors in ID8 mouse model. We found a positive correlation between TARS expression and ovarian cancer progression, and TARS inhibition with BC194 reduce the progression of ovarian cancer. These data suggest that TARS has an important role in the tumor microenvironment and that TARS inhibition should be further investigated as a therapy for ovarian and other angiogenic cancers.

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12

Longstaff, David Gordon. "Requirements and rationale for amber translation as pyrrolysine." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1196107921.

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13

Chen, Peng. "Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar Typhimurium." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknat- och Medfak), 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-328.

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Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described. A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C. cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent. During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

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14

Howard, C. Bradley Howard. "Development of gain-of-function reporters to probe trans-editing of misacylated tRNA in vivo." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469118488.

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15

Somme, Jonathan. "Structure-function relationship studies on the tRNA methyltransferases TrmJ and Trm10 belonging to the SPOUT superfamily." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209122.

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During translation, the transfer RNAs (tRNAs) play the crucial role of adaptors between the messenger RNA and the amino acids. The tRNAs are first transcribed as pre-tRNAs which are then maturated. During this maturation, several nucleosides are modified by tRNA modification enzymes. These modifications are important for the functions of the tRNAs and for their correct folding. Many of the modifications are methylations of the bases or the ribose. Four families of tRNA methyltransferases are known, among which the SPOUT superfamily. Proteins of this superfamily are characterised by a C-terminal topological knot where the methyl donor is bound. With the exception of the monomeric Trm10, all known SPOUT proteins are dimeric and have an active site composed of residues of both protomers. Interestingly, depending on the organism, the same modification can be catalysed by completely unrelated enzymes. On the other hand, homologous enzymes can have different specificities or/and activities. These differences are well illustrated for the TrmJ and Trm10 enzymes.

In the first part of this work we have identified the TrmJ enzyme of Sulfolobus acidocaldarius (the model organism of hyperthermophilic Crenarchaeota) which 2’-O-methylates the nucleoside at position 32 of tRNAs. This protein belongs to the SPOUT superfamily and is homologous to TrmJ of the bacterium Escherichia coli. A comparative study shows that the two enzymes have different specificities for the nature of the nucleoside at position 32 as well as for their tRNA substrates. To try to understand these shifts of specificity at a molecular level we solved the crystal structure of the SPOUT domains of the two TrmJ proteins.

In the second part of this work, we have determined the crystal structure of the Trm10 protein of S. acidocaldarius. This is the first structure of a 1-methyladenosine (m1A) specific Trm10 and also the first structure of a full length Trm10 protein. The Trm10 protein of S. acidocaldarius is distantly related to its yeast homologues which are 1-methylguanosine (m1G) specific. To understand the difference of activity between the Trm10 enzymes, we compared the yeast and the S. acidocaldarius Trm10 structures. Remarkably several Trm10 proteins (such as Trm10 of Thermococcus kodakaraensis) are even able to form both m1A and m1G. To understand the capacity of the T. kodakaraensis protein to methylate A and G, a mutational study was initiated./Lors de la traduction, les ARN de transfert (ARNt) jouent le rôle crucial d’adaptateurs entre l’ARN messager et les acides aminés. Les ARNt sont transcrits sous forme de pré-ARNt qui doivent être maturés. Lors de cette maturation, plusieurs nucléosides sont modifiés. Un grand nombre de ces modifications sont des méthylations des bases ou du ribose. Quatre familles d’ARNt méthyltransferases sont actuellement connues, dont la superfamille des SPOUT. Les membres de cette superfamille sont caractérisés par un nœud dans la chaîne polypeptidique du côté C-terminal. C’est au niveau de ce nœud que se lie la S-adénosylméthionine qui est le donneur de groupement méthyle. A l’exception de Trm10 qui est monomérique, toutes les protéines SPOUT connues sont dimériques et leur site actif est formé de résidus provenant des deux protomères. Selon l’espèce, une même modification peut être formée à la même position dans la molécule d’ARNt par des enzymes qui appartiennent à des familles différentes. A l’opposé, des enzymes homologues peuvent présenter des spécificités ou des activités différentes.

Au cours de ce travail, nous avons identifié l’enzyme TrmJ de Sulfolobus acidocaldarius (l’organisme modèle des Crénarchées hyperthermophiles) qui méthyle le ribose du nucléoside en position 32 des ARNt. Cette protéine est un homologue de l’enzyme TrmJ de la bactérie Escherichia coli. L’étude comparative que nous avons réalisée a révélé que ces deux enzymes présentent une différence de spécificité pour la nature du nucléoside en position 32 ainsi que pour les ARNt substrats. Afin de comprendre ces différences de spécificité au niveau moléculaire, les structures des domaines SPOUT des deux TrmJ ont été déterminées et comparées.

En parallèle, nous avons résolu la structure cristalline de la protéine Trm10 de S. acidocaldarius. C’est la première structure disponible d’un enzyme Trm10 formant de la 1-méthyladénosine (m1A). C’est aussi la première structure complète d’une protéine Trm10. Les enzymes homologues des levures Saccharomyces cerevisiae et Schizosaccharomyces pombe qui n’ont que peu d’identité de séquence avec l’enzyme de S. acidocaldarius, forment de la 1-méthylguanosine (m1G). Dans le but de comprendre comment ces enzymes homologues peuvent présenter des activités différentes, leurs structures ont été comparées. De manière surprenante, certains homologues de Trm10 (comme l’enzyme de l’Euryarchée Thermococcus kodakaraensis) sont capables de former du m1A et du m1G. Afin de mieux comprendre comment ces protéines sont capables de méthyler deux types de bases, nous avons initié l’étude de l’enzyme Trm10 de T. kodakaraensis par mutagenèse dirigée.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

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16

Wallweber, Gerald J. "Characterization of Neurospora mitochondrial group I introns and the function of tyrosyl-tRNA synthetase in RNA splicing /." The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487942739806006.

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17

Fill, Mary-Margaret Anne. "Establishment of a tRNA over-expression system in Trypanosoma brucei to study the role of post-transcriptional modifications on function." Connect to resource, 2007. http://hdl.handle.net/1811/28390.

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Thesis (Honors)--Ohio State University, 2007.
Title from first page of PDF file. Document formatted into pages: contains x, 25 p.; also includes graphics. Includes bibliographical references (p. 24-25). Available online via Ohio State University's Knowledge Bank.

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18

Sanford, Brianne. "Role of Coupled Dynamics and a Strictly Conserved Lysine Residue in the Function of Bacterial Prolyl-tRNA Synthetase and Substrate Binding by a Related trans-Editing Enzyme ProXp-ala." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397645941.

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19

Tyagi, Kshitiz. "A systems biology approach unravels the biological function of two tRNA wobble base modifications in fine-tuning translation and the response to environmental stress." Thesis, University of Dundee, 2014. https://discovery.dundee.ac.uk/en/studentTheses/32e734c5-198d-4c8a-9f56-7d5d19d1ef3d.

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tRNA molecules undergo extensive post-transcriptional modifications to produce several variations of the four canonical nucleotides. Despite the huge number of nucleotide modifications that have been identified in tRNAs, to date their biological roles and regulations continue to be poorly understood. The uridines at the wobble position of the eukaryotic cytoplasmic tRNAs tK^UUU, tQ^UUG and tE^UUC are methoxycarbonylmethylated and thiolated to form mcm⁵s²U₃₄ by the ELP- and URM1-pathways, respectively. Several in vitro experiments have implicated these modifications in modulating wobbling capacity. Moreover, mutations in the ELP- and URM1-pathway genes have been associated with physiological defects in several organisms, including complex neurological disorders in humans. In this thesis we used a systems biology approach to study the in vivo functional relevance of mcm⁵s²U₃₄. A sensitive, robust and quantitative proteomics workflow was developed and applied to investigate differential proteome composition in budding yeast mutants deficient in U₃₄ modifications. We find that, in vivo and under normal conditions, mcm⁵s²U₃₄ fine tunes proteome composition by ensuring efficient translation of mRNAs biased for AAA, CAA and GAA codons. Importantly, our results connect these tRNA modifications with various cellular stress response pathways. Follow up analyses of yeast cells subjected to environmental stresses were conducted and led to the discovery that the biosynthesis of mcm⁵s²U₃₄ is dynamically regulated in response to growth conditions in a URM1-pathway dependent fashion. We propose that this regulation allows the cells to adjust their translational capacity during unfavourable growth conditions and contributes to the management of the environmental stress response. Overall, this thesis presents the first extensive investigation of the functional relevance of tRNA nucleotide modifications and reports one of the few known cases wherein cells regulate the levels of modified nucleotides to fine tune their metabolism in response to environmental cues. We expect that dynamic modulation of RNA modifications will prove to be a more general regulatory mechanism of cellular processes. The experimental and analytical approaches presented in this dissertation will provide a general framework for future studies in this field.

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20

Muchenditsi, Abigael M. "Effects of Metal Ions and Loop Stability on the Structure and Function of the T Box Antiterminator RNA and its complex with Model tRNA." Ohio University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1251219465.

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21

Prévost, Gilles. "Essais de clonage du gene de l'arginyl-arnt synthetase de saccharomyces cerevisiae : determination des domaines fonctionnels de l'aspartyl-arnt synthetase de saccharomyces cerevisiae." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13015.

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Plusieurs techniques de clonage n'ont pas permis l'obtention du gene de l'arg-arnt synthetase de levure. Par contre, l'isolement du gene aspartyl-trna synthetase de levure a permis d'aborder l'etude des relations structure-fonction de cette enzyme. Delimitation de certaines zones sensibles de la molecule: a) l'insertion de deux ou quatre acides amines via des insertions d'oligonucleotides dans le gene montre que la premiere moitie n- terminale de l'enzyme est responsable de la premiere etape catalytique (activation de l'acide aspartique). B) deletions a partir de l'extremite c terminale, mise en evidence du role dans le positionnement fonctionnel de l'arnt**(asp). L'aspartyl-trna synthetase a ete surproduite dans la levure et dans escherichia coli ou l'enzyme conserve son activite. Les dix derniers residus de l'extremite c terminale sont impliques aussi bien dans le positionnement fonctionnel de l'arnt**(asp) que dans l'etape d'activation

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22

Wiencek, Patrick. "Secondary Functions And Novel Inhibitors Of Aminoacyl-Trna Synthetases." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/941.

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The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the aminoacylation of proteins. Additionally, we provide evidence of the inhibitory abilities of the compound EHTS-0 on both E. coli and human threonyl-tRNA synthetases. Further, we also show that an EHTS-0 analog, EHTS-1, also significantly inhibits E. coli threonyl-tRNA synthetase but not the human enzyme. These data could be useful in determining the potential for EHTS-0 and EHTS-1 as possibly anti-angiogenic drugs.

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23

Karim, Loukmane. "Organisation sous-mitochondriale de l'aspartyl-ARNt synthétase humaine et implication dans le syndrome LBSL." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ073/document.

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Les travaux présentés dans cette thèse ont eu pour objectif de contribuer à la compréhension du lien entre l’aspartyl-ARNt synthétase mitochondriale (AspRSmt) humaine et le syndrome LBSL, en étudiant les propriétés de cette enzyme au niveau cellulaire. Les objectifs étaient : 1) d’explorer l’organisation de l’AspRSmt dans la mitochondrie (Chapitre 1), 2) d’identifier la forme mature de l’AspRSmt après son import, ainsi que la localisation sous-mitochondriale de cette enzyme (Chapitre 2), 3) d’évaluer l’impact de quelques mutations, impliquées dans le syndrome LBSL, sur les propriétés de l’AspRSmt (Chapitre 3). Nous avons démontré que l’AspRSmt existe sous différentes formes de produits de maturation, et qu’elle est retrouvée, au moins, dans deux complexes, suggérant potentiellement différents partenaires et/ou fonctions pour cette enzyme. Nous avons établi la localisation sous-mitochondriale de l’AspRSmt, et démontré que cette dernière est doublement localisée avec une fraction soluble et une fraction périphérique interagissant avec la membrane. Nous avons également découvert que, sous certaines conditions de stress, l’AspRSmt est relarguée de la mitochondrie et pourrait avoir un lien avec le processus d’apoptose. En outre, nous avons évalué l’impact de quelques mutations, impliquées dans le syndrome LBSL, et trouvé qu’elles n’ont pas d’effet significatif sur les propriétés de l’AspRSmt. L’ensemble des résultats souligne, d’une part, les lacunes restant à combler concernant les propriétés de l’AspRSmt dans la compréhension du lien mutations/pathologie (LBSL), et d’autre part, suggère fortement l’existence d’une éventuelle fonction non canonique (alternative) de l’AspRSmt
The aim of the PhD project was to contribute to the understanding of the link between mutations in the human mitochondrial aspartyl-tRNA synthetase (mt-AspRS) and LBSL disease, by studying the properties of this enzyme at the cellular level. Our objectives were: 1) to explore the organization of mt-AspRS in mitochondria (Chapter 1), 2) to identify the mature form of mt-AspRS after its import, and to characterize its submitochondrial localization (Chapter 2), 3) to assess, in cellulo, the impact of some LBSL-causing mutations on some properties of mt-AspRS (Chapter 3). We showed that mt-AspRS is processed into different mature forms, and that mt-AspRS belongs to two complexes likely suggesting different partners and/or functions. We demonstrated that mt-AspRS is dually localized with soluble and peripherally membrane-associated fractions. We also demonstrated that, under stress conditions, mt-AspRS is released outside mitochondria with a possible link to the apoptosis. Furthermore, we assessed the impact of some LBSL-causing mutation on some cellular properties of mt-AspRS, and showed that most mutations do not have a significant impact. This underscores the need for more studies about mt-AspRS properties, and strongly suggests a potential non-canonical (alternative) function of the enzyme

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24

Xu, Hao. "Functional aspects of modified nucleosides in tRNA." Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-109491.

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Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m1A58 of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U34 modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m1A58. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity. First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may be essential for reading frame maintenance was investigated. The results show that mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys. Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact. Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

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25

Long, Yicheng. "Characterization of the diverse functions of a family of 3'-5' reverse polymerases." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437563497.

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26

Mechulam, Yves. "Controles de l'expression de l'operon phest-hima d'escherichia coli : attenuation et repression transcriptionnelle par hima et le systeme sos." Paris 7, 1987. http://www.theses.fr/1987PA077134.

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L'operon phest d'e. Coli code pour deux sous unite de la phenylalanyl-arnt synthetase. La transcription de cet operon est modulee par un mecanisme d'attenuation dependant du taux d'aminoacylation de l'arnt phe. La determination de la sequence nucleotidique des regions distales de l'operon a permis de mettre en evidence le gene hima transcrit dans la meme direction que phet, et demarrant seulement 7 nucleotides en aval de celui-ci. Hima est sous controle sos et autoregule. L'autoregulation de hima intervient via une modulation de l'activite des deux promoteurs: le promoteur principal, dont depend la transcription phest et le promoteur secondaire, interne phet. L'induction sos de l'expression de hima intervient au niveau des deux promoteurs. Ces controles impliquent un lien entre l'appareil de traduction et la reponse sos. Le controle hima par attenuation permet de coupler la synthese de son produit a l'etat fonctionnel de l'appareil de traduction

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27

Tran, Thi thanh tam. "Comparative and functional genome analysis of Acidithiobacillus bacteria." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4060.

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Les bactéries acidophiles du genre Acidithiobacillus joue un rôle important dans les activités industrielles de récupération des métaux au sein des sites miniers. Dans cette thèse, la séquence du génome de la bactérie psychro-tolerante Acidithiobacillus ferrivorans CF27 a été re-séquencée. L’analyse comparative du génome de CF27 et des autres bactéries du genre Acidithiobacillus a permis de montrer: (i) une synthénie conservée entre 2 clusters de tRNAs trouvés dans les génomes de At. ferrivorans CF27 et At. ferrooxidans ATCC 23270, et qui ont contribué à la redondance génique des tRNAs chez ces 2 organismes. Notre analyse in silico à grande échelle de ces clusters de tRNAs au sein des génomes procaryotes a montré que les clusters de tRNAs sont présents dans très peu de phyla bactériens; (ii) la présence d’une importante proportion de gènes spécifiques chez CF27 et SS3, ce qui indique la très grande variabilité du contenu génique dans les génomes d’Acidithiobacillus et ainsi la nature unique de chaque groupe d’espèces. L’expression de ces gènes spécifiques a été confirmée chez CF27 cultivés en présence de Fer et soufre; et (iii) une composition taxonomique chimérique des génomes de la classe des Acidithiobacillia, confirmant ainsi que ce groupe appartient à une classe taxonomique particulière. Ces résultats apporte de nouvelles connaissances sur l’adaptation de CF27 à son environnement, ainsi que la nature chimérique des génomes de la classe taxonomique Acidithiobacillia. J’ai participé au projet ‘Thioredoxine réductase (TR)’ dont l’objectif est de définir la fonction biochimique, la structure moléculaire, ainsi que l’histoire évolutive de TRi, une réductase atypique
The acidophilic Acidithiobacillus bacteria play an important role in industrial biomining operations for metal recovery. In this thesis, the genome sequence of a psychrotolerant Acidithiobacillus ferrivorans CF27 were first refined. The comparative genome analysis between CF27 and the closely related Acidithiobacillus genomes revealed: (i) a syntenic conservation of two tRNA array units which are only present in At. ferrivorans CF27 and At. ferrooxidans ATCC 23270 genomes and mainly contribute to the tRNA gene redundancy in both organisms. Moreover, our large-scale genome survey of the tRNA array units in prokaryotic organisms showed that tRNA arrays appear in few phyla; (ii) a high proportion of species-specific genes in CF27 and SS3 strains indicated the high variability of gene content in Acidithiobacillus genomes and therefore the unique nature of each group of species. Given that mRNA expression of some CF27 specific genes were confirmed in Fe(II)-grown cells and sulfur attached cells in CF27, these results highlighted the functional importance of specific genes for CF27 lifestyle; and (iii) the mosaic taxonomic composition of members of the Acidithiobacillia class, and thus confirmed that this group belongs to a particular taxonomic class, distinct to other proteobacterial groups. Taken together, our results provide insights into At. ferrivorans lifestyle as well as the chimeric genome nature of the Acidithiobacillus organisms. In addition, I also participated to the ‘Thioredoxin reductase’ project which aims to define the biochemical function, molecular structure and evolution of TRi, an atypical thioredoxin reductase

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28

Esberg, Anders. "Functional aspects of wobble uridine modifications in yeast tRNA." Doctoral thesis, Umeå : Department of Molecular Biology, Umeå Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1093.

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29

Davis, Matthew W. (Matthew Warren) 1966. "Functional analysis of a class II aminoacyl-tRNA synthetase." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/26867.

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30

Wu, Shiying. "Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis." Doctoral thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-159312.

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RNase P is a ubiquitous ribonuclease responsible for removing the 5’ leader of tRNA precursor. Bacterial RNase P contains one RNA (RPR) and one protein (RPP) subunit. However, the number of protein variants depends on the origin. The RNA subunit is the catalytic subunit that in vitro cleaves its substrate with and without the protein subunit. Therefore RNase P is a ribozyme. However, the protein subunit is indispensable in vivo. The objective of this thesis was to understand the mechanism of and substrate interaction in RPR-mediated cleavage, in particular the contributions of the two domains of RPR and the roles of the base at the -1 residue in the substrate. As model systems I have used bacterial (Eco) and archaeal (Pfu) RPRs. The TSL (T-stem-loop) region of a tRNA precursor and the TBS (TSL-binding site) in the RPR S-domain interact upon RPR-substrate complex conformation. A productive TSL/TBS-interaction affects events at the cleavage site by influencing the positioning of chemical groups and/ or Mg2+ such that efficient and correct cleavage occurs consistent with an induced fit mechanism. With respect to events at the cleavage site, my data show that the identity of the residue immediately upstream the 5’ of the cleavage site (at -1) plays a significant role for efficient and accurate cleavage although its presence is not essential. My data also show that the RPR C-domain can cleave without the S-domain. However, the presence of the S-domain increases the efficiency of cleavage but lowers the accuracy. The structure of the S-domain of Pfu RPR differs from that of Eco RPR and my data suggest that the Pfu S-domain does not affect the accuracy in the same way as for Eco RPR. It also appears that the proteins that bind to the Pfu S-domain play a role in formation of a productive TSL/TBS-interaction. It is therefore possible that the proteins of Pfu RNase P have evolved to take over the role of the S-domain with respect to the interaction with the TSL-region of the substrate.

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So, Byung Ran. "Characterization of the Cys-tRNA^Pro Editing Mechanism and Functional Interactions of Bacterial YbaK Protein." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1268257970.

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32

Walker, Sarah Elizabeth. "Functional Studies of Transfer RNA Interactions in the Ribosome." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1217605676.

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33

Takita, Teisuke. "The Structure and Functions of the Lysyl-tRNA Synthetase of Bacillus stearothermophilus." Kyoto University, 1996. http://hdl.handle.net/2433/78070.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第6479号
農博第892号
新制||農||720(附属図書館)
学位論文||H8||N2923(農学部図書室)
UT51-96-F358
京都大学大学院農学研究科食品工学専攻
(主査)教授外村辨一郎, 教授左右田健次, 教授佐々木隆造
学位規則第4条第1項該当

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34

Wang, Tingzhang. "Make inferences about bacterial gene functions with the concept of neighborhood in silico." Thesis, Evry-Val d'Essonne, 2010. http://www.theses.fr/2010EVRY0036/document.

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Avec l'accroissement du nombre de génomes séquencés, l'organisation de ces données brutes et des données dérivées, l'extraction de l'information et des connaissances associées défie l'imagination. La notion de voisinage a été d'abord été introduite pour l'organisation des données dans des bases de données relationnelles. Pour extraire des informations pertinentes à partir de données massives, différents types de voisinages ont été étudiés ici. Tout d'abord, avec l'analysedes correspondances (CA) et en utilisant le regroupement supervisé ("model clustering" MBC), la proximité mutuelle des éléments formant deux entités biologiques centrales, les gènes (codant les protéines) et les acides aminés a été analysée. Nous montrons par exemple que les protéines de Psychromonas ingrahamii, bactérie psychrophile extrêmes, sont regroupées en six classes, et qu'il y a une forte opposition entre le comportement de l'asparagine (N) et des acides aminés sensibles à l'oxygène, ce que nous expliquons en terms de résistance au froid. Ensuite, nous avons analysé la répartition entre les îlots génomiques (GI) et le squelette du génome de base à partir d'une nouvelle méthode combinant composition en bases et en gènes, caractéristiques GI et de briser les synténies. L'application de cette approche à E. coli et B. subtilis a révélé que cette nouvelle méthode permet d'extraire certaines régions significative, non publiées auparavant.Enfin, pour illustrer un voisinage fin, la régulation de l'expression d'un gène et son évolution, nous avons étudié la relation entre les régions en amont du gène et la zone codante du gène thrS de façon approfondie. Nous avons constaté que ces deux régions associées à un gène, se sont comportés différemment dans l'histoire évolutive. Certaines des régions en amont porteuses de la fonction non-essentielle de régulation (qui contrôle l'expression de gène) ont évoluédifféremment de la région codante
With more and more genomes being sequenced, the organization of those raw data and the derived data, the extraction of information and knowledge from these data has become a challenge. A key concept in this field is that of the neighborhood, especially with respect to the organization of data in relational databases. To extract information from bulk data, different kinds of neighborhoods were studied and each show interesting results in current study. .Firstly, through the Correspondence Analysis (CA) and later Model Based Clustering (MBC), two kinds of neighbors i.e. the genes (proteins) and amino acids were analyzed respectively, and it was found that proteins from Psychromonas ingrahamii are clustered into six classes, and there is strong opposition between asparagine (N) and the oxygen-sensitive amino acids. Secondly, the relationship between genomic islands and core genome (i.e. two closely linked neighbors withlarge range on the chromosome) was studied by a new method combining composition, GI features and synteny break. On applying to E. coli and B. subtilis it was revealed that this new method can extract some meaningful regions not published before. Thirdly, the relationship between upstream and coding regions of thrS gene (i.e. a case for two closely linked neighbors with small range on the chromosome) was studied extensively. It was found that these two regions associated to one gene, behaved differently in the evolutionary history.. Some of the upstream regions bearing non-essential function (i.e. regulation of gene expression) evolved more slowly than the coding region

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35

Bou, Nader Charles. "Structural and Functional characterization of flavoenzymes involved in posttranscriptional modification of tRNA." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066205/document.

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La modification posttranscriptionnelle des acides ribonucléiques (ARNs) est une étape de maturation conservée dans tous les domaines du vivant. Mes travaux de thèse ont porté sur la caractérisation fonctionnelle et structurale de flavoenzymes impliquées dans la modification des ARN de transfert (ARNt) : les dihydrouridines synthases (Dus) dictant la formation de dihydrouridine via la flavine mononucléotide (FMN) et TrmFO responsable de la méthylation en C5 de l'uridine 54 via la flavine adénosine dinucléotide (FAD) ainsi que le methylènetétrahydrofolate. Afin d'élucider le mécanisme de TrmFO, nous avons élaboré une apoenzyme grâce à une simple mutation qui est efficacement reconstituée in vitro. Nous avons chimiquement synthétisé l'intermédiaire catalytique qui consiste en un FAD-iminium comportant un methylène sur le N5 de l'isoalloxazine. Cette espèce synthétique a été caractérisée par spectrométrie de masse et absorption UV-visible. La reconstitution de TrmFO avec cette molécule restore l'activité in vitro sur un ARNt transcrit prouvant le rôle du FAD comme agent méthylant via une méthylation réductrice. Dus2 réduit spécifiquement U20 et est constituée d'un Dus domaine néanmoins, chez les mammifères un double-stranded RNA-binding domaine (dsRBD) est présent. Afin de comprendre la fonction de cette organisation modulaire, nous avons montré que seule l'enzyme sauvage est active contrairement aux domaines isolés. Nous avons résolu les structures cristallographiques des deux domaines suggérant une redistribution des charges positives en surface. Ce dsRBD dicte la reconnaissance de l'ARNt en se fixant à la tige acceptrice/Tpsi. Ceci est régulé par une extension N-terminal, mis en évidence par des mutations, des titrations RMN ainsi qu’une structure cristallographique en complexe avec un ARN de 22 nucléotides. Ce travail illustre l’acquisition d’un dsRBD au cours de l’évolution dont la fonction est étendu à la reconnaissance des ARNts
Posttranscriptional modification of ribonucleic acids (RNAs) is a crucial maturation step conserved in all domains of life. During my thesis, I have brought structural and functional insights on flavoenzymes involved in transfer RNA (tRNA) modifications: dihydrouridine synthase (Dus) responsible for dihydrouridine formation using flavin mononucleotide (FMN) and TrmFO responsible for C5 methylation of uridine position 54 relying on flavin adenosine dinucleotide (FAD) and methylenetetrahydrofolate. To elucidate the chemical mechanism of TrmFO we designed an apoprotein via a single mutation that could be reconstituted in vitro with FAD. Furthermore, we chemically synthesized the postulated intermediate active species consisting of a flavin iminium harboring a methylene moiety on the isoalloxazine N5 that was further characterized by mass spectrometry and UV-visible spectroscopy. Reconstitution of TrmFO with this molecule restored in vitro activity on a tRNA transcript proving that TrmFO uses FAD as a methylating agent via a reductive methylation.Dus2 reduces U20 and is comprised of a canonical Dus domain however, mammals have an additional double-stranded RNA-binding domain (dsRBD). To bring functional insight for this modular organization, we showed that only full length human Dus2 was active while its isolated domains were not. tRNA recognition is driven by the dsRBD via binding the acceptor and TΨ stem of tRNA with higher affinity then dsRNA as evidenced by NMR. We further solved the X-ray structures for both domains showing redistribution of surface positive charges justifying the involvement of this dsRBD for tRNA recognition in mammalian Dus2. This was attributed to a peculiar N-terminal extension proven by mutational analysis and an X-ray structure of dsRBD in complex with 22-nucleotide dsRNA. Altogether our work illustrates how during evolution, Dus2 enzymes acquired an engineered dsRBD for efficient tRNA binding via a ruler mechanism

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36

Rao, Bhalchandra Shantikumar. "Diverse Biological Functions For 3'-5' Nucleotide Addition Reactions: tRNA Repair to tRNAHis Identity." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397425994.

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37

Gustavsson, Marie. "Studies of Intracellular Transport and Anticancer Drug Action by Functional Genomics in Yeast." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9408.

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This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

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38

FLORENTZ, EGELE CATHERINE. "L'extremite 3'oh aminoacyable du rna du virus de la mosaique jaune du navet : relations entre structure et fonctions." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13181.

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Analyse structurale (structure secondaire et tertiaire) d'un fragment contenant la sequence "trna like" (extremite 3' oh possedant plusieurs caracteristiques d'un arn de transfert). Modelisation de la structure sur ecran graphique. Analyse des zones de contact entre ce fragment d'arn et la valyl-trna synthetase, a l'aide de differentes sondes structurales. Discussion sur le role de l'extremite "trna like" dans le cycle de developpement du virus (regulation de la traduction de l'information genetique portee par l'arn viral)

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39

Simoneau, Steve. "Functional interactions between the p75 neurotrophin receptor and TrkA." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0029/MQ64452.pdf.

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40

Simoneau, Steve. "Functional interactions between the p75 neurotrophin receptor and TrkA." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=30747.

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The p75NTR neurotrophin receptor has previously been shown to increase the responsiveness of TrkA to NGF, the preferred ligand of TrkA. We performed structure/function studies to determine the domains of p75 NTR involved in this functional interaction. Co-expression of p75NTR in 293T cells enhances NGF-mediated TrkA autophosphorylation while the intracellular domain of p75NTR is not required for this function. Indirect evidence has previously suggested that p75 NTR may also play a role in reducing non-preferred ligand activation of TrkA. We tested this directly and found that p75NTR reduces both basal and NT-3/NT-4-mediated TrkA autophosphorylation. In addition, we show that the intracellular domain of p75NTR can by itself reduce basal TrkA activity, suggesting the involvement of intracellular signaling. These data imply that the p75NTR receptor can functionally interact with the TrkA receptor through two distinct domains. We hypothesize that these interactions function in combination to help the TrkA receptor discriminate between preferred and non-preferred ligands, therefore increasing the specificity of activation of the TrkA receptor.

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41

Chu, Hui-Yi. "Genome-wide Investigation of Cellular Functions for tRNA Nucleus-Cytoplasm Trafficking in the Yeast Saccharomyces cerevisiae." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1343397048.

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42

Hou, Zhiwei. "Fluorescence in blue light (FLU): Functional analysis of its structural domains for light and dark-dependent control of ALA synthesis." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/20951.

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Fluorescence in blue light (FLU) ist ein negativer Feedbackregulator der Chlorophyllbiosynthese, welcher an der Dunkelrepression der 5-Aminolävulinsäure (ALA)-Synthese beteiligt ist. FLU ist Teil eines Komplexes, der die Enzyme umfasst, welche an der Katalyse der finalen Schritte der Chlorophyllbiosynthese beteiligt sind. Drei funktionelle Domänen wurden für das Arabidopsis FLU Protein postuliert: eine Tetratricopeptid-Wiederholungsdomäne (TPR) befindet sich am C-Terminus; eine Transmembrandomäne (TM) ist am N-Terminus lokalisiert; eine Coiled-coil-Domäne (linker) liegt dazwischen. Die TPR-Domäne von FLU Domäne interagiert mit dem C-terminalen Ende der Glutamyl-tRNA Reduktase (GluTR), dem geschwindigkeitsbestimmenden Enzym der ALA-Synthese. Diese Arbeit zur Erweiterung des Wissen über die Funktion von FLU im Licht sowie über die Rolle der funktionellen Domänen von FLU bei der Inaktivierung der ALA-Synthese bei.
Fluorescence in blue light (FLU), a negative feedback regulator of chlorophyll biosynthesis, is involved in dark repression of 5-aminolevulinic acid (ALA) synthesis. FLU is part of a complex comprising the enzymes catalyzing the final steps of chlorophyll synthesis. Three functional domains were proposed in the Arabidopsis FLU protein: a tetratricopeptide repeat (TPR) domain is at the C-terminus; a transmembrane domain (TM) is at the N-terminus; a coiled-coil domain (linker) is in between. The TPR(FLU) domain interacts with the C-terminal end of glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme of ALA synthesis. This thesis contributes to the extended knowledge about the function of FLU in light as well as the role of the structural domains of FLU in the inactivation of ALA synthesis.

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43

Cho, I.-Ming. "Characterizing the Role of Ribosomal Protein L7Ae in Archaeal RNase P Catalysis and Exploring the Use of Archaeal RNase P as a Functional Genomics Tool." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1290555279.

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44

LUNGHI, GIULIA. "GM1 OLIGOSACCHARIDE MODULATION OF CALCIUM SIGNALLING IN NEURONAL FUNCTIONS." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/792078.

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It has been already demonstrated that the oligosaccharide chain (OligoGM1) of the ganglioside GM1, β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal-(1-4)-β-Glc-(1-1)-Ceramide, promotes neurodifferentiation in the Neuro2a murine neuroblastoma cells, used as a model, by directly interacting with the NGF specific receptor TrkA, leading to the activation of ERK1/2 downstream pathway. In this context, my PhD work aimed to investigate which other biochemical pathways, in addition to TrkA-MAPK cascade activation, are prompted by OligoGM1, with an emphasis on Ca2+ modulating factors. A proteomic analysis (nLC-ESi-MS-MS) performed on Neuro2a cells treated with 50 µM OligoGM1 for 24 hours led to the identification and quantification of 324 proteins exclusively expressed by OligoGM1-treated cells. Interestingly, some of these proteins are involved in the regulation of Ca2+ homeostasis and in Ca2+-dependent differentiative pathways. In order to evaluate if OligoGM1 administration was able to modulate Ca2+ flow, we performed calcium-imaging experiments on Neuro2a cells using the Ca2+-sensitive Fluo-4 probe. Starting from 5 minutes upon OligoGM1 administration to undifferentiated Neuro2a, a significant increase in Ca2+ influx occurs. At the same time an increased activation of TrkA membrane receptor was observed and, importantly, the addition of a specific TrkA inhibitor abolished the OligoGM1 mediated increase of the cytosolic Ca2+, suggesting that the opening of the cell Ca2+ channels following OligoGM1 administration depends on the activation of TrkA receptor. To unveil which cellular pathway activated by OligoGM1 could lead to the increase of intracellular Ca2+, time-course immunoblotting analyses were performed. The data revealed that following TrkA activation, OligoGM1 induced the activation of phospholipase PLCγ1 which converts phosphatidylinositol 4,5-bisphosphate (PIP2) to diacyglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), the second messengers that propagate cellular signalling via Ca2+ mobilization. Moreover, we observed a hyperphosphorylation of the DAG substrate, protein kinase C (PKC), which is a priming event that enables its catalytic activation in response to lipid second messengers, and we found its enrichment in lipid rafts, events that consolidate its activation. When calcium-imaging experiments where performed in the presence of xestospongin C, a potent inhibitor of IP3 receptors on endoplasmic reticulum, a reduction of about 50% of Ca2+ influx was observed, suggesting that the Ca2+ flows moved by the OligoGM1 come not only from intracellular storages, but probably also from the extracellular environment. Accordingly, in the presence of both extracellular (EGTA) and intracellular (BAPTA-AM) Ca2+ chelators the neuritogenic effect induced by OligoGM1 was abolished. The work described in this thesis confirms that the effects of GM1 ganglioside on neuronal differentiation are mediated by its oligosaccharide portion. In particular, here I highlight that the oligosaccharide, initiating a signalling cascade on the cell surface, is responsible alone for the balancing of the intracellular Ca2+ levels that underlie neurite sprouting, which have historically been attributed to the whole GM1 ganglioside and its role as lipid inserted into the plasma membrane. In this way, these data give additional information on the molecular characterization of the mechanisms by which GM1 exerts its neuronal functions.

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45

Cherniack, Andrew David. "Involvement of mitochondrial tyrosyl tRNA synthetase in splicing : identification of an N-terminal domain that functions in splicing /." The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu148775817823818.

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46

Mo, Fan. "Functional role of the conserved amino acids Cysteine 81, Arginine 279, Glycine 280 and Arginine 283 in elongation factor Tu from Escherichia coli." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, c2011, 2011. http://hdl.handle.net/10133/3107.

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During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu.
x, 85 leaves : ill. (some col.) ; 29 cm

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47

Mueller, Markus. "In vivo function of NGF/TrkA signaling in the cholinergic neurons of the murine basal forebrain." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-43939.

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48

Gurha, Priyatansh. "Functional characterization of archaeal pseudouridine synthases responsible for formation of conserved pseudouridines in tRNAs /." Available to subscribers only, 2008. http://proquest.umi.com/pqdweb?did=1650501181&sid=14&Fmt=2&clientId=1509&RQT=309&VName=PQD.

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Thesis (Ph.D.)--Southern Illinois University Carbondale, 2008.
"Department of Molecular Biology, Microbiology and Biochemistry." Includes bibliographical references (p. 101-120). Also available online.

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49

Condon, Peter J. "Novel, Functional Interactions Between TrkA Kinase and p75 Neurotrophin Receptor in Neuroblastoma Cells: A Dissertation." eScholarship@UMMS, 2003. http://escholarship.umassmed.edu/gsbs_diss/148.

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To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we employed several lines of investigation including biophysical, biochemical and cellular assays. A high-affinity nerve growth factor (NGF) receptor is thought to be a complex of two receptors, p75 and the receptor tyrosine kinase, TrkA. The existence of a gp75-TrkA complex was demonstrated by a copatching technique. p75 on the surface of intact cells is patched with an anti-p75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with an anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression system, which allows high level expression of wild-type and mutated NGF receptors. TrkA and p75 copatch in both the absence and presence of NGF. This association is specific, since p75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-β and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with p75. A chimeric receptor with TrkA transmembrane and intracellular domains shows partial copatching with p75. Deletion of the intracellular domain of p75 decreases but does not eliminate copatching. A point mutation that inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with p75. Hence, although interactions between the p75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role. To study what signal transduction mechanisms were activated by the two receptors to bring about differentiation and survival, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR) and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation, but enhanced the EGF-induced response, leading to differentiation of almost all of the cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhances apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF appears to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.

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Sun, Yishan. "Novel functions of drosophila TRPA channels pain and pyx in gravity sensing and the DEG/ENaC channel ppk11 in metabolic homeostasis." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/893.

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My thesis research comprises two projects looking into physiological functions of Drosophila ion channels: first, contribution of several T ransient R eceptor P otential (TRP) channels to gravity sensing; second, regulation of metabolic homeostasis by a D egenerin/ E pithelial Na + C hannel (DEG/ENaC). Many animal species sense gravity for spatial orientation. In humans recurrent vertigo and dizziness are often attributable to impairment of gravity sensing in the vestibular organs. However, the molecular bases for gravity sensing and its disruption in vestibular disease remain uncertain. Here I studied gravity sensing in the model organism Drosophila melanogaster, with a combination of genetic, behavioral and electrophysiological methods. My results show that gravity sensing requires Johnston’s organ, a mechanosensory structure located in the antenna that also mediates hearing. Johnston’s organ neurons fire action potentials in a phasic manner in response to body rotations in the gravitational field. Furthermore, gravity sensing and hearing require different TRP channels with distinct anatomical localizations, implying separate neural mechanisms underlying gravity sensing and hearing. These findings set the stage for understanding how TRP channels contribute to the sensory transduction of gravity. Drosophila melanogaster has over 20 genes belonging to the DEG/ENaC family, which are collectively referred to as pickpockets (ppks) . Genetic analyses have implicated ppk genes in salt taste, tracheal liquid clearance, pheromone detection, and developmental timing. These results, together with the conserved presence of DEG/ENaC genes through evolution, suggest that further studies on fly ppk genes may help gain insights to a number of physiological processes. Here I report that the ppk11 gene regulates metabolic homeostasis. A ppk11 enhancer/promoter fragment labels the fat body, the lipid storage organ of Drosophila. ppk11 mutants are lean — they store less triacylglyceride (TAG), possess smaller lipid droplets and are sensitive to starvation compared to wild–type flies. ppk11 mutants also show signs of enhanced insulin sensitivity — they store more glycogen and maintain a lower level of circulating carbohydrate (trehalose). Moreover, the mutants have extended life span, suggesting ppk11–dependent activities of the fat body have systematic and long–term effects on the fly body. Understanding the cellular function of ppk11 may offer new insights into mechanisms that regulate metabolic homeostasis.

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