In all the three domains of life, most RNAs undergo post transcriptional modifications both on the bases as well as the ribose sugars of the individual ribonucleotides. 2'-O-methylation of ribose sugars and isomerization of Uridines to Pseudouridines are two most predominant modifications in rRNAs and tRNAs across all domains of life. Besides 2'-O-methylation of ribose sugars, methylation of pseudouridine (Ø) at position 54 of tRNA, producing m1Ø, is a hallmark of many archaeal species but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m1Ø was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m1Ø minus phenotype of the ÄHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TØ-arm (17-mer) fragments as substrates, the sequential pathway of m1Ø54 formation in Archaea was reconstituted. The methylation reaction is AdoMet-dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TØ-loop. The presence of Ø55 allowed the efficient conversion of Ø54 to m1Ø54, whereas in the presence of C55 the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins. Another aim of this study was to investigate the mechanism of target RNA recruitment to a box C/D sRNP. From data obtained, we have made the following hypothesis- aNop5p, either alone or as a heterodimer with Fibrillarin, binds to single stranded bulges and loops of target RNA. This aNop5p bound target is then hybridized to an assembling guide sRNP complex containing the guide RNA and L7Ae or guide RNA, L7Ae and aNop5p. If the guide:target sequences are complementary to each other, they hybridize and the target nucleotide gets modified. We also think that post modification, the guide and target strands separate, the core proteins rearrange themselves on the guide RNA and then prime the guide RNA for next round of modification. Compared to the general archaeal populations, haloarchaea contain significantly fewer number of box C/D guide RNAs. In archaea, previous studies have underscored the importance of a symmetric assembly of the core proteins on the sRNA. This meant that if the core proteins were unable to bind to either the terminal box C/D or the internal box C'/D' motifs, the sRNP was not efficient to carry out the modification of the target RNA. Essentially the only two haloarchaeal box C/D sRNPs known before had a symmetric architecture. In this study we discovered the first naturally occurring asymmetric box C/D sRNP called sR-41 in Haloferax volcannii. The architecture of Haloferax volcanii sR-41 box C/D sRNP seems to be closer in conformation to eukaryal snoRNPs (eukaryal counterparts of archaeal sRNPs) in which the core proteins assemble asymmetrically on the RNA. Till date, no information regarding the catalytic mechanism of an asymmetrically arranged eukaryal box C/D snoRNPs are available, because of unavailability of any assembly systems or crystal structures. Hence, this archaeal sR-41 guide sRNP provides a unique opportunity to study mechanism of modification in an asymmetrically arranged box C/D sRNP molecule.

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

Submitted by ADAUTO LIMA CARDOSO null (adautolimacardoso@gmail.com) on 2017-08-07T20:31:46Z No. of bitstreams: 1 Tese_Final_Adauto.pdf: 1788431 bytes, checksum: 257f753cdae88b1e8797c0626e99bb5a (MD5) / Rejected by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido está sem a ficha catalográfica. A versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Corrija esta informação e realize uma nova submissão contendo o arquivo correto. Agradecemos a compreensão. on 2017-08-10T20:17:23Z (GMT) / Submitted by ADAUTO LIMA CARDOSO null (adautolimacardoso@gmail.com) on 2017-08-17T14:38:48Z No. of bitstreams: 1 Tese_Final_Adauto.pdf: 2278150 bytes, checksum: 6afc73dfaadceeb6d4aa47ea2b6cb566 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-08-23T14:20:25Z (GMT) No. of bitstreams: 1 cardoso_al_dr_bot.pdf: 2278150 bytes, checksum: 6afc73dfaadceeb6d4aa47ea2b6cb566 (MD5) / Made available in DSpace on 2017-08-23T14:20:25Z (GMT). No. of bitstreams: 1 cardoso_al_dr_bot.pdf: 2278150 bytes, checksum: 6afc73dfaadceeb6d4aa47ea2b6cb566 (MD5) Previous issue date: 2017-06-27 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Cromossomos supernumerários são polimorfismos numéricos frequentemente registrados em eucariotos, sendo que seus efeitos são pouco elucidados. Em alguns indivíduos da espécie Astatotilapia latifasciata pode-se identificar um ou dois cromossomos B, que são totalmente heterocromáticos e ricos em sequências repetitivas. Em vista de compreender sua origem, evolução e efeitos, este elemento vem sendo largamente explorado por técnicas integradas de citogenética, biologia molecular e genômica. Aqui, explorou-se o padrão de marcas epigenéticas do DNA deste cromossomo B e seus efeitos nas vias de metilação do DNA e de formação de tRFs. Usando-se imunocitogenética, ferramentas de bioinformática, quantificação global de 5mC e 5hmC e RT-qPCR, identificou-se que o cromossomo B de A. latifasciata possui padrão epigenético ativo e que não é um isocromossomo. Além disso, foram observados efeitos heterogêneos deste cromossomo na expressão de epi-miRNAs candidatos, de genes de modificações epigenéticas do DNA e de genes relacionados com a formação de tRFs. Como consequência, também foram registrados efeitos de cromossomos B nos níveis globais de 5mC e 5hmC e na formação de tRFs. Essas variações observadas parecem estar relacionadas com os mecanismos de manutenção do cromossomo B e estão em desacordo com a difundida ideia de que ele seja um elemento inerte. / Supernumerary B chromosomes are numerical polymorphisms frequently registered in eukaryotes, and their effects are not elucidated. In some individuals of the species Astatotilapia latifasciata one or two B isochromosomes can be identified, which are totally heterochromatic and enriched by repetitive sequences. In order to understand its origin, evolution and effects, this element has been widely explored by integrated techniques of cytogenetics, molecular biology and genomics. Here, the pattern of epigenetic marks of the DNA of this B chromosome and its effects on the pathways of DNA methylation and formation of tRFs. Using immunocytogenetics, bioinformatics tools, global quantification of 5mC and 5hmC and RT-qPCR, it was identified that the B chromosome of A. latifasciata has active epigenetic pattern and it is not an isochromosome. In addition, heterogeneous effects of this chromosome were observed in the expression of candidate epi-miRNAs, genes of epigenetic modifications of DNA and genes related to the formation of tRFs. As a consequence, effects of B chromosomes were also registered at the global levels of 5mC and 5hmC and in the formation of tRFs. These variations appear to be related to the maintenance mechanisms of chromosome B and are in opposition with the widespread idea that they are inert elements. / FAPESP: 2012/21546-9 / FAPESP: 2016/07743-7

This thesis addresses several aspects of mitochondrial iron sulfur (Fe-S) cluster biogenesis and mitochondrial tRNA import and modifications in Trypanosoma brucei. Using RNA interference it uncovers essential role of Fe-S cluster assembly in tRNA(s) thiolation in both the cytosol and the mitochondrion of T. brucei. Further, this thesis describes the role of modifications in tRNA editing and in mitochondrial import of tRNAs. Finally, it provides evidence that in contrast to protein import, mitochondrial membrane potential is dispensable for import of tRNAs into the mitochondrion of T. brucei.

abstract: The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated. / Dissertation/Thesis / Ph.D. Chemistry 2013

tRNA genes have the fundamental role of translating the genetic code during protein synthesis. Beyond solely a passive decoding role, the tRNA pool exerts selection pressures on the codon usage of organisms and the viruses that infect them because processing codons read by rare tRNAs can be slow or even erroneous. To better understand the interactions of codons and anticodons in eukaryotic species, we first investigated whether tRNAs packaged into HIV-1 particles may relate to the poor codon usage of HIV-1 genes. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding poorly adapted codons are overrepresented in HIV-1 virions. Because the affinity of Gag-Pol for all tRNAs is non-specific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Moreover, differences that we found in the codon usage between early and late genes suggest alterations in the tRNA pool are induced late in viral infection. Next, we tested whether a reduced tRNA anticodon pattern, which was called into question by predicted tRNA datasets, is maintained across eukaryotes. tRNA prediction methods are prone to falsely identifying tRNA-derived repetitive sequences as functional tRNA genes. Thus, we proposed and tested a novel approach to identify falsely predicted tRNA genes using phylogenetics. Phylogenetic analysis removed nearly all the genes deviating from the anticodon pattern, therefore the anticodon pattern is reaffirmed across eukaryotes.

No description available.

A 30-mer oligonucleotide probe encoding the "A box" and anticodon loop regions of a human glycine tRNA gene was used to isolate a 581bp DNA fragment from a bovine genomic DNA library. Although the cross-hybridizing segment of DNA was found not to encode any tRNA gene or pseudogene, a region with homology to the "C-element" of the "BOV-tA" type Alulike artiodactyl retroposons was identified. This cross-hybridization was determined to be the result of conserved RNA polymerase III promoter elements in the probe portion of the tRNA gene and these repetitive elements. A microsatellite repeat (TC) was also found associated with this element. Future screening for bovine tRNA genes will require the use of a) longer probes and higher stringency hybridization conditions or b) the simultaneous screening with probes from the 5' and 3' ends of the gene which avoid the conserved Pol III promoter boxes.

No description available.

No description available.