Relevant bibliographies by topics / RRNA Methyltransferase

Academic literature on the topic 'RRNA Methyltransferase'

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Published: 6 September 2023

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Journal articles on the topic "RRNA Methyltransferase"

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Corrêa, Laís L., Marta A. Witek, Natalia Zelinskaya, Renata C. Picão, and Graeme L. Conn. "Heterologous Expression and Functional Characterization of the Exogenously Acquired Aminoglycoside Resistance Methyltransferases RmtD, RmtD2, and RmtG." Antimicrobial Agents and Chemotherapy 60, no. 1 (November 9, 2015): 699–702. http://dx.doi.org/10.1128/aac.02482-15.

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ABSTRACTThe exogenously acquired 16S rRNA methyltransferases RmtD, RmtD2, and RmtG were cloned and heterologously expressed inEscherichia coli, and the recombinant proteins were purified to near homogeneity. Each methyltransferase conferred an aminoglycoside resistance profile consistent with m7G1405 modification, and this activity was confirmed byinvitro30S methylation assays. Analyses of protein structure and interaction withS-adenosyl-l-methionine suggest that the molecular mechanisms of substrate recognition and catalysis are conserved across the 16S rRNA (m7G1405) methyltransferase family.
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Savic, Miloje, S. Sunita, Natalia Zelinskaya, Pooja M. Desai, Rachel Macmaster, Kellie Vinal, and Graeme L. Conn. "30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr." Antimicrobial Agents and Chemotherapy 59, no. 5 (March 2, 2015): 2807–16. http://dx.doi.org/10.1128/aac.00056-15.

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ABSTRACTMethylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of theSorangium cellulosumSo ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum inEscherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core β-strands 6 and 7, which forms part of theS-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m1A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition byS-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate.
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Nosrati, Meisam, Debayan Dey, Atousa Mehrani, Sarah E. Strassler, Natalia Zelinskaya, Eric D. Hoffer, Scott M. Stagg, Christine M. Dunham, and Graeme L. Conn. "Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition." Journal of Biological Chemistry 294, no. 46 (October 8, 2019): 17642–53. http://dx.doi.org/10.1074/jbc.ra119.011181.

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Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase–substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.
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Chen, Hao, Zhennan Shi, Jiaojiao Guo, Kao-jung Chang, Qianqian Chen, Cong-Hui Yao, Marcia C. Haigis, and Yang Shi. "The human mitochondrial 12S rRNA m4C methyltransferase METTL15 is required for mitochondrial function." Journal of Biological Chemistry 295, no. 25 (May 5, 2020): 8505–13. http://dx.doi.org/10.1074/jbc.ra119.012127.

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Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro. We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes.
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Rowe, Sebastian J., Ryan J. Mecaskey, Mohamed Nasef, Rachel C. Talton, Rory E. Sharkey, Joshua C. Halliday, and Jack A. Dunkle. "Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition." Journal of Biological Chemistry 295, no. 51 (October 5, 2020): 17476–85. http://dx.doi.org/10.1074/jbc.ra120.014280.

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Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m62A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity–binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.
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McGann, Patrick, Sarah Chahine, Darius Okafor, Ana C. Ong, Rosslyn Maybank, Yoon I. Kwak, Kerry Wilson, Michael Zapor, Emil Lesho, and Mary Hinkle. "Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin." Journal of Clinical Microbiology 54, no. 1 (November 4, 2015): 208–11. http://dx.doi.org/10.1128/jcm.02642-15.

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16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity inEnterobacteriaceaewith high specificity using the standard disk susceptibility test.
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van Tran, Nhan, Felix G. M. Ernst, Ben R. Hawley, Christiane Zorbas, Nathalie Ulryck, Philipp Hackert, Katherine E. Bohnsack, et al. "The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112." Nucleic Acids Research 47, no. 15 (July 22, 2019): 7719–33. http://dx.doi.org/10.1093/nar/gkz619.

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Abstract N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5–TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5–TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.
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White, Joshua, Zhihua Li, Richa Sardana, Janusz M. Bujnicki, Edward M. Marcotte, and Arlen W. Johnson. "Bud23 Methylates G1575 of 18S rRNA and Is Required for Efficient Nuclear Export of Pre-40S Subunits." Molecular and Cellular Biology 28, no. 10 (March 10, 2008): 3151–61. http://dx.doi.org/10.1128/mcb.01674-07.

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ABSTRACT BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5′ internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Δ mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.
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Ruszkowska, Agnieszka. "METTL16, Methyltransferase-Like Protein 16: Current Insights into Structure and Function." International Journal of Molecular Sciences 22, no. 4 (February 22, 2021): 2176. http://dx.doi.org/10.3390/ijms22042176.

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Methyltransferase-like protein 16 (METTL16) is a human RNA methyltransferase that installs m6A marks on U6 small nuclear RNA (U6 snRNA) and S-adenosylmethionine (SAM) synthetase pre-mRNA. METTL16 also controls a significant portion of m6A epitranscriptome by regulating SAM homeostasis. Multiple molecular structures of the N-terminal methyltransferase domain of METTL16, including apo forms and complexes with S-adenosylhomocysteine (SAH) or RNA, provided the structural basis of METTL16 interaction with the coenzyme and substrates, as well as indicated autoinhibitory mechanism of the enzyme activity regulation. Very recent structural and functional studies of vertebrate-conserved regions (VCRs) indicated their crucial role in the interaction with U6 snRNA. METTL16 remains an object of intense studies, as it has been associated with numerous RNA classes, including mRNA, non-coding RNA, long non-coding RNA (lncRNA), and rRNA. Moreover, the interaction between METTL16 and oncogenic lncRNA MALAT1 indicates the existence of METTL16 features specifically recognizing RNA triple helices. Overall, the number of known human m6A methyltransferases has grown from one to five during the last five years. METTL16, CAPAM, and two rRNA methyltransferases, METTL5/TRMT112 and ZCCHC4, have joined the well-known METTL3/METTL14. This work summarizes current knowledge about METTL16 in the landscape of human m6A RNA methyltransferases.
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L. Aishwarya, K. V., P. V. Geetha, M. Shanthi, and S. Uma. "Co occurrence of two 16S rRNA methyltrasferases along with NDM and OXA 48 like carbapenamases on a single plasmid in Klebsiella pneumoniae." Journal of Laboratory Physicians 11, no. 04 (October 2019): 305–11. http://dx.doi.org/10.4103/jlp.jlp_59_19.

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Abstract BACKGROUND: The carbapenemase-encoding genes, bla NDM- and bla OXA-48 - like , confer resistance to all the known beta-lactams and are encountered along with other beta-lactamase-encoding genes and/or 16S ribosomal RNA (rRNA)-methylating genes. The co-occurrence of bla NDM and bla OXA-48 - like on a single plasmid is a rare occurrence. AIM AND OBJECTIVE: The purpose of the study was to characterize the plasmids in Klebsiella pneumoniae isolates producing 16S rRNA methyltransferase along with bla NDM , bla OXA-48-like , and other resistance encoding genes. MATERIALS AND METHODS: One-hundred and seventeen K. pneumoniae clinical isolates which were resistant to aminoglycosides were collected. Polymerase chain reaction-based screening for 16S rRNA methyltransferase genes armA, rmtB, and rmtC; carbapenamase genes bla NDM , bla OXA-48-like , bla IMP, bla VIM, and bla KPC ; and other resistance genes such as bla TEM, bla SHV, bla CTX-M , and qnr (A, B, and S) determinants acc (6') Ib-cr was performed. Conjugation experiment was carried out for seven isolates that anchored bla NDM and bla OXA-48-like along with any one of the 16S rRNA methyltransferases. The plasmid-based replicon typing for different plasmid-incompatible (Inc) group was performed on the conjugatively transferable plasmids. RESULTS: Among the 16S rRNA methyltransferases, armA was more predominant. bla NDM and bla OXA-48 -like were present in 56 (47.86%) and 22 (18.80%) isolates, respectively. Out of seven isolates which were conjugatively transferable, only four had bla NDM and bla OXA-48 - like on the same plasmid and they belonged to Inc N and A/C replicon. Three isolates co-harbored 16S rRNA methyltransferases armA, rmtB, and rmtC, and out of the them, one isolate harbored two 16S rRNA methyltransferases armA and rmtB, on the single-plasmid replicon A/C. CONCLUSION: This is the first report revealing the coexistence of bla NDM and bla OXA-48 - like co-harboring two 16S rRNA methylases on a single conjugative plasmid replicon belonging to incompatibility group A/C.
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Dissertations / Theses on the topic "RRNA Methyltransferase"

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Zarubica, Tamara. "SPECIFICITY DETERMINANTS OF ArmA, A RIBOSOMAL RNA METHYLTRANSFERASE THAT CONFERS ANTIBIOTIC RESISTANCE." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2273.

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Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the 30S ribosomal subunit, has been traced to the arm/rmt family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosylmethionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA in the 30S ribosomal subunit. Neither 16S rRNA alone nor intact 70S ribosome is an efficient substrate for armA methyltransferase. To more fully characterize this family of enzymes, xiii we have investigated the substrate requirements of ArmA. We determined the Mg2+ dependence of ArmA activity and found that the enzyme could recognize both translationally active and translationally inactive forms of 30S subunits. To identify the site of interaction between ArmA and the 30S subunit, we used hydroxyl radical cleavage of 16S rRNA mediated by ferrous iron chelated to several sites on the ArmA molecule that were mutated to cysteine. This data suggests that significant conformational changes in 30S structure are involved in binding of ArmA. We hypothesized that a precursor intermediate in the biogenesis of the 30S subunit might be the optimal substrate for ArmA enzymes in vivo. To test this, we prepared 30S particles partially depleted of proteins by treatment with increasing concentrations of LiCl and assayed them for ArmA methylation. Even low concentrations of LiCl alter the 30S particles and greatly diminish their susceptibility to methylation. Additionally, a previously identified pre-30S particle isolated from an E. coli culture was assayed for its ability to support methylation by ArmA and found to be inferior to intact 30S particles as a methylation substrate. Thus, testing of immature particles prepared from in vitro and in vivo sources suggest that ArmA works very late in the 30S biogenesis pathway. Initiation factor 3 (IF3), a factor that only binds fully mature 30S particles, does not inhibit the ArmA methylation, while kasugamycin methyltransferase (KsgA) abolishes ArmA activity by sharing the same binding site with ArmA. From aforementioned experiments, we conclude that ArmA is most active toward 30S ribosomal subunits that are at or very near full maturation.
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Kshetri, Man B. "N-TERMINAL DOMAIN OF rRNA METHYLTRANSFERASE ENZYME RsmC IS IMPORTANT FOR ITS BINDING TO RNA AND RNA CHAPERON ACTIVITY." Kent State University Honors College / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1621007414429417.

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Treede, Irina. "Aufklärung der Funktion von zwei rRNA-Methyltransferasen und Untersuchungen zur Eurekanat-Biosynthese in Streptomyces viridochromogenes Tü57." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973266686.

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Punekar, Avinash S. "Ribosomal RNA Modification Enzymes : Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-212394.

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Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli. RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V. RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity. These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.
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Sharma, Sunny [Verfasser], Karl-Dieter [Akademischer Betreuer] Entain, Jens [Akademischer Betreuer] Wöhnert, Heinz Dieter [Akademischer Betreuer] Osiewacz, and Micheala [Akademischer Betreuer] Müller-McNicoll. "Identification of novel base methyltransferases of the 25S rRNA in Saccharomyces cerevisiae / Sunny Sharma. Gutachter: Karl-Dieter Entain ; Jens Wöhnert ; Heinz Dieter Osiewacz ; Micheala Müller-McNicoll." Frankfurt am Main : Univ.-Bibliothek Frankfurt am Main, 2014. http://d-nb.info/1062604032/34.

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Schillewaert, Stéphanie. "Etude de la maturation et de l'assemblage du ribosome eucaryote: caractérisation fonctionnelle de nouveaux facteurs trans-." Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209826.

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La synthèse du ribosome est un processus compliqué, très hiérarchisé et essentiel à toutes les cellules vivantes. La complexité de ce processus tient notamment au fait que les différentes étapes de la biogenèse du ribosome eucaryote sont temporellement et spatialement organisées dans des compartiments cellulaires différents (le nucléole, le nucléoplasme et le cytoplasme). Il est toutefois connu que le pré-ARNr 35S (le précurseur de trois des quatre ARNr, les ARNr 18S, 5.8S et 25S) est pris en charge dès sa synthèse par des facteurs impliqués dans sa maturation. Ainsi, la formation d’un ribosome requiert l’association, sur le transcrit naissant, des facteurs de synthèse, au nombre de 400. Ces facteurs essentiels interagissent transitoirement avec l’ARNr et ne font pas partie des particules ribosomiques matures impliquées dans la traduction. Leur rôle est d’assister le remodelage constant du pré-ribosome et le processus d’assemblage des sous-unités.

Parmi ces facteurs de synthèse, nous avons caractérisé en détail, chez la levure et chez l’homme, la protéine Las1 impliquée dans la maturation des deux extrémités de l’ITS2, séquence qui sépare les ARNr 5.8S et 25S/28S. Chez la levure, en absence de la protéine Las1, les analyses de profils de polysomes révèlent un déficit de sous-unité 60S et l’apparition d’« halfmères ». Les techniques de purification d’affinité et de gradient de sédimentation nous indiquent que Las1 est associée aux pré-ribosomes 60S et qu’elle interagit avec de nombreux facteurs de synthèse de la petite, de la grande sous-unité ou des deux. De plus, Las1 copurifie avec des pré-ribosomes qui contiennent aussi les exoribonucléases 5’-3’ Rat1/Rai1 et Xrn1. Rai1 coordonne la maturation aux deux extrémités de l’ARNr 5.8S. Nous suggérons que Las1 appartient à un macrocomplexe connectant spatialement des sites de clivages éloignés sur la séquence primaire du pré-ARNr qui seraient rapprochés suite au reploiement de l’ITS2.

Un autre aspect de ce travail de thèse consiste en l’étude de l’assemblage des particules ribonucléoprotéiques et plus spécifiquement du pré-ribosome et des sous-unités ribosomiques eucaryotes. Nous avons utilisé la technique d’immunoprécipitation de chromatine (Ch-IP) pour caractériser l’assemblage d’une structure appelée le « SSU processome ». Celui-ci correspond à un pré-ribosome en formation ainsi que l’assemblage des protéines ribosomiques sur l’ARNr naissant.

Enfin, nous avons étudié le rôle d’une plateforme d’activation de méthyltransférases d’ARN et de protéines, la protéine Trm112 dans la ribogenèse. Nous avons montré que chez la levure, Trm112 est impliquée dans la synthèse du ribosome et dans la progression de la mitose. En absence de cette protéine, les pré-ARNr sont dégradés par un mécanisme de surveillance. Trm112 copurifie avec plusieurs facteurs de synthèse du ribosome dont la méthyltransférase Bud23, impliquée dans la modification post-transcriptionnelle de l’ARNr18S. Trm112 est requise pour cette méthylation et nous postulons que la protéine Bud23 est incapable de se lier aux pré-ribosomes en l’absence de Trm112.


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

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Hang, Pei Chun. "Investigations into Streptomyces azureus Thiostrepton-resistance rRNA Methyltransferase and its Cognate Antibiotic." Thesis, 2008. http://hdl.handle.net/10012/4143.

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Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.
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Kovalic, David Karel. "Methylation of minimalist 23S rRNA sequences in vitro by ErmSF (TlrA) N-methyltransferase." 1997. http://catalog.hathitrust.org/api/volumes/oclc/40100371.html.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1997.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 168-191).
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Treede, Irina [Verfasser]. "Aufklärung der Funktion von zwei rRNA-Methyltransferasen und Untersuchungen zur Eurekanat-Biosynthese in Streptomyces viridochromogenes Tü57 / vorgelegt von Irina Treede." 2004. http://d-nb.info/973266686/34.

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Bhattacharyya, Souvik. "Fidelity Of Translation Initiation In E. coli : Roles Of The Transcription-recycling Factor RapA, 23S rRNA Modifications, And Evolutionary Origin Of Initiator tRNA." Thesis, 2016. http://etd.iisc.ernet.in/handle/2005/2574.

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Abstract:
CSIR
Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.
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Book chapters on the topic "RRNA Methyltransferase"

1

Schomburg, Dietmar, and Dörte Stephan. "rRNA (adenine-N6-)-methyltransferase." In Enzyme Handbook 11, 211–14. Berlin, Heidelberg: Springer Berlin Heidelberg, 1996. http://dx.doi.org/10.1007/978-3-642-61030-1_46.

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Schomburg, Dietmar, and Dörte Stephan. "rRNA (guanine-N1-)-methyltransferase." In Enzyme Handbook 11, 223–25. Berlin, Heidelberg: Springer Berlin Heidelberg, 1996. http://dx.doi.org/10.1007/978-3-642-61030-1_49.

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Schomburg, Dietmar, and Dörte Stephan. "rRNA (guanine-N2-)-methyltransferase." In Enzyme Handbook 11, 227–29. Berlin, Heidelberg: Springer Berlin Heidelberg, 1996. http://dx.doi.org/10.1007/978-3-642-61030-1_50.

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4

Schomburg, Dietmar, and Dörte Stephan. "rRNA (adenosine-2′-O-)-methyltransferase." In Enzyme Handbook 11, 283–85. Berlin, Heidelberg: Springer Berlin Heidelberg, 1996. http://dx.doi.org/10.1007/978-3-642-61030-1_63.

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5

Schomburg, Dietmar, and Ida Schomburg. "16S rRNA (guanine1207-N2)-methyltransferase 2.1.1.172." In Class 2–3.2 Transferases, Hydrolases, 41–44. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-36240-8_10.

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Schomburg, Dietmar, and Ida Schomburg. "23S rRNA (guanine2445-N2)-methyltransferase 2.1.1.173." In Class 2–3.2 Transferases, Hydrolases, 45–46. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-36240-8_11.

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Schomburg, Dietmar, and Ida Schomburg. "23S rRNA (guanine1835-N2)-methyltransferase 2.1.1.174." In Class 2–3.2 Transferases, Hydrolases, 47–48. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-36240-8_12.

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Schomburg, Dietmar, and Ida Schomburg. "16S rRNA (cytosine967-C5)-methyltransferase 2.1.1.176." In Class 2–3.2 Transferases, Hydrolases, 53–55. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-36240-8_14.

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Schomburg, Dietmar, and Ida Schomburg. "23S rRNA (pseudouridine1915-N3)-methyltransferase 2.1.1.177." In Class 2–3.2 Transferases, Hydrolases, 56–57. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-36240-8_15.

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Schomburg, Dietmar, and Ida Schomburg. "16S rRNA (cytosine1407-C5)-methyltransferase 2.1.1.178." In Class 2–3.2 Transferases, Hydrolases, 58–60. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-36240-8_16.

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