Wyosine derivatives are highly complex modified ribonucleic acid (RNA) bases found in archaea and eukarya. They are a modification of a genetically encoded guanosine found at position 37 of phenylalanine encoding transfer ribonucleic acid (tRNA). The second step in the biosynthesis of all wyosine derivatives, in both archaea and eukarya, is the transformation of N-methylguanosine to 4-demethylwyosine by the radical S-adenosyl-l-methionine enzyme TYW1. When these studies were initiated, the substrate of TYW1 was unknown. Four possible substrates; acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate; were tested for activity. Only incubation with pyruvate led to production of 4-demethylwyosine. As only two new carbons are incorporated into the RNA base at this step, ¹³C isotopologues were used to identify the carbons that are transferred into 4-demethylwyosine. These experiments revealed that C2 and C3 of pyruvate are incorporated into 4-demethylwyosine, with C1 lost as an unknown byproduct. Utilizing pyruvate containing deuteriums in place of protons on the C3 carbon, the regiochemistry of the addition was determined. It was found that C3 forms the methyl group of 4-demethylwyosine and C2 becomes the bridging carbon in the imidazoline ring. The site of hydrogen atom abstraction by 5'-deoxyadenosyl radical was identified as the N-methylguanosine methyl group through the use of tRNA containing a deuterated methyl group. The putative mechanism for this transformation involved the formation of an enzyme substrate Schiff base through a conserved lysine residue. Utilizing sodium cyanoborohydride a Schiff base was trapped between TYW1 and pyruvate. The mass of the trapped adduct responded as expected when different isotopologues of pyruvate were used, demonstrating that it is due to pyruvate. Moreover, the fragment of TYW1 that contained the trapped adduct contained two lysine residues, one of which was shown to be required for activity both in vivo and in vitro. It was initially proposed that TYW1 contained two iron-sulfur clusters, and then subsequently shown to have two 4Fe-4S clusters. Site directed mutagenesis, along with iron and sulfide analysis identified the cysteines; as C26, C39, and C52; coordinating the second 4Fe-4S cluster. This study identified pyruvate as the substrate of TYW1, and provided evidence for key steps in the transformation of N-methylguanosine to 4-demethylwyosine.

Neisseria gonorrhoeae (Ng) encounters different microenvironments during its life-cycle. Some of these niches have different concentrations of oxygen, which influences the rate of Ng growth; as well as iron, an element essential for Ng survival. Differential expression of several proteins allows the bacteria to adapt to the diverse conditions it comes encounters. One protein affected by environmental changes during Ng growth is Gcp, a tRNA-modification enzyme essential for protein synthesis. To study the regulation of expression of Gcp, we first analyzed the sequence of its ORF, gcp. Orthologs of this gene are found in all kingdoms of life. In silico analysis shows that among Neisseria species, gcp ranges in homology from 76% to 99%, at the nucleotide level. Reverse transcription PCR indicates that gcp is expressed as part of an operon, together with three cytochrome-associated genes cyc4, resB and resC. Rapid amplification of complementary DNA ends determined the start of transcription of cyc4 (and possibly of the cyc4-gcp operon) at 95 nucleotides from the gene start codon. Transcriptional fusions determined that the promoter region upstream of cyc4 is the strongest promoter in the operon. However, the region directly upstream of gcp also has low level of promoter activity, suggesting that the gene may be expressed from two different promoters. Semi-quantitative determination of the concentration of gcp mRNA indicates that the transcription of the gene is significantly repressed when Ng is grown under low iron or low oxygen conditions. Analysis of an fnr mutant, grown under the same conditions as its parental wild type, indicates that the FNR transcriptional regulator is involved in the repression of gcp in low iron or low oxygen conditions. Contrary to expectation, the cyc4 promoter is upregulated when Ng is grown under low oxygen or low iron conditions. However, these results cannot be compared to the original promoter strength. Determination of which was performed on bacteria grown in liquid medium. Coregulation of gcp with cytochrome genes can guarantee low levels of protein synthesis when Ng encounters adverse microenvironments and needs its energy redirected to the expression of genes that would allow it to survive.

The KsgA enzymes comprise an ancient family of methyltransferases that are intimately involved in ribosome biogenesis. Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution, and therefore were present in the last common ancestor. The first member of the family to be described, KsgA from Escherichia coli, was initially shown to be the determining factor for resistance/sensitivity to the antibiotic kasugamycin and was subsequently found to dimethylate two adenosines in 16S rRNA during maturation of the 30S subunit. Since then, numerous other members of the family have been characterized in eubacteria, eukaryotes, archaea and in eukaryotic organelles. The eukaryotic ortholog, Dim1, is essential for proper processing of the pre-rRNA, in addition to and separate from its methyltransferase function. The KsgA/Dim1 family bears sequence and structural similarity to a larger group of S-adenosyl-L-methionine dependent methyltransferases, which includes both DNA and RNA methyltransferases. In this document we report that KsgA orthologs from archaea and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life. We have characterized KsgA structurally, and discuss aspects of KsgA's activity in light of the structural data. We also propose a model for KsgA binding to the 30S subunit, based on solution probing data. This model sheds light on KsgA's unusual regulation and on the dual function of the Dim1 enzymes.

Ribosomal RNAs (rRNA) undergo several post-transcriptional modifications inside the cell. These modifications can be (1) RNA- independent (enzyme only) and (2) guide RNA-mediated. In the latter mechanism, a group of small, metabolically stable, non-coding RNAs, present as ribonucleoprotein (RNP) particles, modify ribosomal RNAs inside the cell. One of the highly abundant rRNA modifications is pseudouridine (Y) formation. In Archaea and Eukarya, pseudouridine synthases, with the help of small RNAs, form pseudouridines at functionally important regions in rRNA. Cbf5, the pseudouridine synthase, three other core proteins, and a box H/ACA RNA form the ribonucleoprotein complex in sRNP-mediated rRNA pseudouridylation. Certain Ys in rRNAs are evolutionarily conserved from Bacteria to human. Among those, two Ys are present in helix 69 of rRNA and one in helix 90. We successfully deleted Cbf5 in Haloferax volcanii, a haloarchaeon, and showed that the deleted strain was viable. It was the first report where Cbf5 deletion was achieved, because deletion or mutation of cbf5 or of its homologs is lethal in eukaryotes. We also found that the cbf5 deleted strain was unable to produce the three highly conserved Ys in rRNA of H. volcanii (position 1940, 1942 in helix 69, and 2605 in helix 90), whereas the tRNA Ys were intact. To identify the specific structural features of Cbf5 involved in rRNA Ψ formation, we used a cbf5 deleted strain which was complemented with a plasmid borne copy of the gene. Using the crystal structure of Pyrococcous furiosus Cbf5 as template, we created a homology model of H. volcanii Cbf5 (HvCbf5) and identified several residues and motifs/domains of HvCbf5 that might be important to the protein's enzymatic activity. By using an in vivo mutational approach, we confirmed some previously predicted and certain unidentified residues/motifs/domains that serve as positive determinants of rRNA Ys1940, 1942, and 2605 formation inside the cell. A box H/ACA RNA, sR-h45, was bioinformatically predicted before. We confirmed its presence as a double hairpin RNA inside the cell whose level goes down in the absence of Cbf5. We identified that sR-h45 is the guide RNA for sRNP-mediated Ys at the three above mentioned rRNA positions in H. volcanii. Each hairpin of this RNA can independently modify the substrate, both in vivo and in vitro. To characterize the structure of sR-h45, we have used a sR-h45 deleted strain where the function of sR-h45 was complemented with a plasmid-borne copy of the gene. By a combination of in vivo and in vitro mutagenic approaches, we determined specific nucleotides/structures of this RNA, involved in binding to the core proteins and also to the substrate RNA. We also identified that one hairpin of sR-h45 can modify two successive positions (1940 and 1942) in rRNA.

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京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24756号 / 生博第497号 / 新制||生||66(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 北島 智也, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

La traduction représente un processus central au sein de la cellule, elle assure le transfert de l’information génétique de l’ARNm vers les protéines. De nombreux acteurs y sont impliqués directement ou indirectement et parmi eux, chez les eucaryotes, la petite protéine Trm112. Celle-ci participe à la modification de plusieurs acteurs directs en interagissant et en activant quatre MTases. Le facteur de terminaison eRF1 est méthylé par le complexe Mtq2-Trm112, l’ARNr 18S par Bud23-Trm112 et certains ARNt par les complexes Trm9-Trm112 et Trm11-Trm112. Au cours de ce travail, les structures cristallographiques de Trm9-Trm112 et de Bud23-Trm112 de levure ont été résolues. L’étude comparative structurale de ces complexes et de la structure connue de Mtq2-Trm112, a permis de mettre en évidence que dans un même organisme, les séquences des trois protéines ont évolué de manière à conserver l’interaction avec Trm112. Même si les quatre partenaires présentent moins de 20% d’identité de séquence, les résidus clés pour l’interaction avec la petite protéine activatrice sont conservés ou partagent des caractéristiques identiques. En plus de l’analyse structurale, le complexe Trm9-Trm112 a fait l’objet d’une étude fonctionnelle chez S. cerevisiae ce qui a permis de cartographier le site actif de l’enzyme et de proposer un modèle de mécanisme d’action. Enfin, les premières études in vivo réalisées chez Haloferax volcanii suggèrent que cette plateforme serait également présente chez certains organismes procaryotes. / Protein synthesis is a central process in the cell; it ensures the transfer of genetic information from mRNA in to protein. A lot of actors are involved directly or indirectly in translation. In Eukaryotes, Trm112, a small protein, interacts with and activates four methyltransferases modifying direct actors of translation. The termination factor eRF1 is methylated by the Mtq2-Trm112 complex, the 18S rRNA by Bud23-Trm112 and some tRNA by the Trm9-Trm112 and Trm11-Trm112 complexes. During this work, the crystal structures of Trm9-Trm112 and Bud23-Trm112 complexes from yeast were solved. The comparative analysis of these two new structures with Mtq2-Trm112 structure highlights the structural plasticity allowing Trm112 to interact through a very similar mode with its partners although those share less than 20% sequence identity. In the same organism, the key residues for the interaction with Trm112 are conserved or share similar characteristics. In addition to the structural analysis, the function of the Trm9-Trm112 complex was studied in S. cerevisiae. This analysis allowed to map the active site of the enzyme and to propose a model of its mechanism of action. Finally, the first data obtained in vivo, with the Archaea Haloferax volcanii suggest that the Trm112 platform might also be present in some prokaryotic organisms.