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1.
Meyer B Wurm JP Kötter P Leisegang MS Schilling V Buchhaupt M Held M Bahr U Karas M Heckel A Bohnsack MT Wöhnert J Entian KD 《Nucleic acids research》2011,39(4):1526-1537
The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome. 相似文献
2.
Jan Philip Wurm Elke Duchardt Britta Meyer Belinda Z. Leal Peter Kötter Karl-Dieter Entian Jens Wöhnert 《Biomolecular NMR assignments》2009,3(2):251-254
Nep1 from Methanocaldococcus jannaschii is a 48 kDa dimeric protein belonging to the SPOUT-class of S-adenosylmethionine dependent RNA-methyltransferases and acting
as a ribosome assembly factor. Mutations in the human homolog are the cause of Bowen-Conradi syndrome. We report here 1H, 15N and 13C chemical shift assignments for the backbone of the protein in its apo state. 相似文献
3.
The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases
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Leulliot N Bohnsack MT Graille M Tollervey D Van Tilbeurgh H 《Nucleic acids research》2008,36(2):629-639
Emg1 was previously shown to be required for maturation of the 18S rRNA and biogenesis of the 40S ribosomal subunit. Here we report the determination of the crystal structure of Emg1 at 2 Å resolution in complex with the methyl donor, S-adenosyl-methionine (SAM). This structure identifies Emg1 as a novel member of the alpha/beta knot fold methyltransferase (SPOUT) superfamily. In addition to the conserved SPOUT core, Emg1 has two unique domains that form an extended surface, which we predict to be involved in binding of RNA substrates. A point mutation within a basic patch on this surface almost completely abolished RNA binding in vitro. Three point mutations designed to disrupt the interaction of Emg1 with SAM each caused>100-fold reduction in SAM binding in vitro. Expression of only Emg1 with these mutations could support growth and apparently normal ribosome biogenesis in strains genetically depleted of Emg1. We conclude that the catalytic activity of Emg1 is not essential and that the presence of the protein is both necessary and sufficient for ribosome biogenesis. 相似文献
4.
Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein 总被引:1,自引:0,他引:1
The nucleolar Saccharomyces cerevisiae protein Nep1 was previously shown to bind to a specific site of the 18S rRNA and to be involved in assembly of Rps19p into pre-40S ribosome subunits. Here we report on the identification of tma23 and nop6 mutations as recessive suppressors of a nep1(ts) mutant allele and the nep1 deletion as well. Green fluorescent protein fusions localized Tma23p and Nop6p within the nucleolus, indicating their function in ribosome biogenesis. The high lysine content of both proteins and an RNA binding motif in the Nop6p amino acid sequence suggest RNA-binding functions for both factors. Surprisingly, in contrast to Nep1p, Tma23p and Nop6p seem to be specific for fungi as no homologues could be found in higher eukaryotes. In contrast to most other ribosome biogenesis factors, Tma23p and Nop6p are nonessential in S. cerevisiae. Interestingly, the tma23 mutants showed a considerably increased resistance against the aminoglycoside G418, probably due to a structural change in the 40S ribosomal subunit, which could be the result of incorrectly folded 18S rRNA gene, missing rRNA modifications or the lack of a ribosomal protein. 相似文献
5.
In humans, the enzyme thiopurine methyltransferase (TPMT) metabolizes 6-thiopurine (6-TP) medications, commonly used for immune suppression and for the treatment of hematopoietic malignancies. Genetic polymorphisms in the TPMT protein sequence accelerate intracellular degradation of the enzyme through an ubiquitylation and proteasomal-dependent pathway. Research has led to the hypothesis that these polymorphisms destabilize the native structure of TPMT, resulting in the formation of misfolded or partially unfolded states, which are subsequently recognized for intracellular degradation. Addition of the cosubstrate, S-adenosylmethionine (SAM), prevents degradation of the TPMT polymorphs in experimental assays, presumably by stabilizing the native structure. Using a bacterial orthologue of TPMT from Pseudomonas syringae, we have used NMR spectroscopy to describe the consequences of binding sinefungin, a SAM analogue, on the structure and dynamics of the TPMT protein backbone. NMR chemical shift mapping experiments localize sinefungin to a highly conserved site in classical methyltransferases. Distal chemical shift changes involving the presumed active site cover imply indirect conformational changes induced by sinefungin, which may play a role in substrate recognition or the catalytic mechanism. Analysis of protein backbone dynamics based on NMR relaxation reveals a combination of complementary effects. Whereas the peripheral, inserted structural elements of the TPMT topology are conformationally stabilized by the presence of sinefungin, a consistent increase in backbone mobility is observed for the central, conserved structural elements. The potential implications for the structural and dynamic effects of binding sinefungin for the catalytic mechanism of the enzyme and the stabilization of the degradation-susceptible TPMT polymorphs are discussed. 相似文献
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Thomas SR Keller CA Szyk A Cannon JR Laronde-Leblanc NA 《Nucleic acids research》2011,39(6):2445-2457
Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen-Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase. 相似文献
8.
S-adenosylhomocysteine inhibition of three purified tRNA methyltransferases from rat liver. 总被引:3,自引:1,他引:2
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Three tRNA methyltransferases from rat liver have been fractionated and purified greater than 100-fold. These enzymes have been examined for their sensitivity to inhibition by S-adenosylhomocysteine (SAH). The methyltransferase which forms m2-guanine in the region between the dihydrouridine loop and the acceptor stem of tRNA (m2-guanine methyltransferase I) is least sensitive to SAH inhibition, with a Ki of 8 muM. The enzyme responsible for forming m2-guanine between the dihydrouridine and anticodon loops (m2-guanine methyltransferase II) has a Ki of 0.3 muM, while m1-adenine methyltransferase shows intermediate sensitivity to SAH (Ki = 2.4 muM). All three methyltransferases have similar Km's for the S-adenosylmethionine substrate (1.5-2.0 muM). These results are consistent with the hypothesis that activity of individual tRNA methyltransferases may be controlled by enzyme systems which alter cellular SAH levels. 相似文献
9.
White J Li Z Sardana R Bujnicki JM Marcotte EM Johnson AW 《Molecular and cellular biology》2008,28(10):3151-3161
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 bud23Delta 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. 相似文献
10.
Rachel Macmaster Natalia Zelinskaya Miloje Savic C. Robert Rankin Graeme L. Conn 《Nucleic acids research》2010,38(21):7791-7799
X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus
tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m7G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides. 相似文献
11.
RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding. 相似文献
12.
The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps. 相似文献
13.
Boehringer D O'Farrell HC Rife JP Ban N 《The Journal of biological chemistry》2012,287(13):10453-10459
The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. 相似文献
14.
Bang WY Chen J Jeong IS Kim SW Kim CW Jung HS Lee KH Kweon HS Yoko I Shiina T Bahk JD 《The Plant journal : for cell and molecular biology》2012,71(1):122-134
The Spo0B-associated GTP-binding protein (Obg) GTPase, essential for bacterial viability, is also conserved in eukaryotes, but its primary role in eukaryotes remains unknown. Here, our functional characterization of Arabidopsis and rice obgc mutants strongly underlines the evolutionarily conserved role of eukaryotic Obgs in organellar ribosome biogenesis. The mutants exhibited a chlorotic phenotype, caused by retarded chloroplast development. A plastid DNA macroarray revealed a plastid-encoded RNA polymerase (PEP) deficiency in an obgc mutant, caused by incompleteness of the PEP complex, as its western blot exhibited reduced levels of RpoA protein, a component of PEP. Plastid rRNA profiling indicated that plastid rRNA processing is defective in obgc mutants, probably resulting in impaired ribosome biogenesis and, in turn, in reduced levels of RpoA protein. RNA co-immunoprecipitation revealed that ObgC specifically co-precipitates with 23S rRNA in vivo. These findings indicate that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development. Furthermore, complementation analysis can provide new insights into the functional modes of three ObgC domains, including the Obg fold, G domain and OCT. 相似文献
15.
Michela Bollati Eloise Mastrangelo Xavier de Lamballerie Martino Bolognesi 《Biochemical and biophysical research communications》2009,382(1):200-32595
Presently known flaviviruses belong to three major evolutionary branches: tick-borne viruses, mosquito-borne viruses and viruses with no known vector. Here we present the crystal structure of the Yokose virus methyltransferase at 1.7 Å resolution, the first structure of a methyltransferase of a Flavivirus with no known vector. Structural comparison of three methyltransferases representative of each of the Flavivirus branches shows that fold and structures are closely conserved, most differences being related to surface loops flexibility. Analysis of the conserved residues throughout all the sequenced flaviviral methyltransferases reveals that, besides the central cleft hosting the substrate and cofactor binding sites, a second, almost continuous, patch is conserved and points away from active site towards the back of the protein. The high level of structural conservation in this region could be functional for the methyltransferase/RNA interaction and stabilization of the ensuing complex. 相似文献
16.
Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m1A1408) methyltransferase (‘CacKam’). The apo CacKam structure closely resembles other m1A1408 methyltransferases within its conserved SAM-binding fold but the region linking core β strands 6 and 7 (the ‘β6/7 linker’) has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the β6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover. 相似文献
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19.
In yeast, guide snoRNAs have been assigned to 51 of the 55 rRNA ribose methylation sites. LSU-Um2918 is one of the four remaining positions. This residue is highly conserved and located in the peptidyl transferase center of the ribosome. The equivalent position on the E. coli 23S rRNA is methylated by FtsJ/RrmJ which has three yeast homologs: Spb1, involved in biogenesis of LSU; Trm7, a tRNA methyltransferase; and Mrm2, a mitochondrial 21S rRNA methyltransferase. We demonstrate that a point mutation in the Ado-Met binding site of Spb1p affects cell growth but does not abolish methylation of U2918. When this mutation is combined with disruption of snR52 (a snoRNA C/D), cell growth is severely impaired and U2918 is no longer methylated. In vitro, Spb1p is able to methylate U2918 on 60S subunits. Our results reveal the importance of this methylation for which two mechanisms coexist: a site-specific methyltransferase (Spb1p) and a snoRNA-dependent mechanism. 相似文献
20.
Foster PG Nunes CR Greene P Moustakas D Stroud RM 《Structure (London, England : 1993)》2003,11(12):1609-1620
The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m(5)C methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate. 相似文献