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1.
The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere with methylation at C1962 in 23 S rRNA. Purified recombinant YebU protein retains its specificity for C1407 in vitro, and methylates 30 S subunits (but not naked 16 S rRNA or 70 S ribosomes) isolated from yebU knockout strains. Nucleotide C1407 is located at a functionally active region of the 30 S subunit interface close to the P site, and YebU-directed methylation of this nucleotide seems to be conserved in bacteria. The yebU knockout strains display slower growth and reduced fitness in competition with wild-type cells. We suggest that a more appropriate designation for yebU would be the rRNA small subunit methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification. 相似文献
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Sonja ?ubrilo Fedora Babi? Stephen Douthwaite Gordana Maravi? Vlahovi?ek 《RNA (New York, N.Y.)》2009,15(8):1492-1497
Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m7G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m4Cm1402 and m5C1407. Modification at m5C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m4Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell''s own indigenous methyltransferases can play an important role in determining resistance levels. 相似文献
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Steven T. Gregory Hasan Demirci Riccardo Belardinelli Tanakarn Monshupanee Claudio Gualerzi Albert E. Dahlberg Gerwald Jogl 《RNA (New York, N.Y.)》2009,15(9):1693-1704
The RsmG methyltransferase is responsible for N7 methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 Å resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG. 相似文献
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Galimand M Schmitt E Panvert M Desmolaize B Douthwaite S Mechulam Y Courvalin P 《RNA (New York, N.Y.)》2011,17(2):251-262
Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6′)-Ii that encodes a 6′-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m5C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m5C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m5C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes'' respective specificities for nucleotides C1404 and C1407. 相似文献
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Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibiotic-resistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function. 相似文献
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Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE
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The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly. 相似文献
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Summary The nucleotide sequences of 5S rRNAs from three protozoa,Bresslaua vorax, Euplotes woodruffi andChlamydomonas sp. have been determined and aligned together with the sequences of 12 protozoa species including unicellular green algae already reported by the authors and others. Using this alignment, a phylogenic tree of the 15 species of protozoa has been constructed. The tree suggests that the ancestor for protozoa evolved at an early time of eukaryotic evolution giving two major groups of organisms. One group, which shares a common ancestor with vascular plants, contains a unicellular green flagellate (Chlamydomonas) and unicellular green algae. The other group, which shares a common ancestor with the multicellular animals, includes various flagellated protozoa (includingEuglena), ciliated protozoa and slime molds. Most of these protozoa appear to have separated from one another at a fairly early period of eukaryotic evolution. 相似文献
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针对细菌rRNA研发抑制细菌增殖的新型抗菌素是抗生素研究领域的新课题。细菌rRNA与基因mRNA一样自然形成折叠卷曲高级结构,其结构上可以结合反义核酸的位点即靶点,靶点的阐明是设计有效反义核酸、核酶(Ribozyme)和脱氧核酶(DNAzyme)的关键。MAST方法固定16S rRNA,将其与寡核苷酸文库杂交筛选出靶点,获得了大肠杆菌16S rRNA的6个反义核酸结合靶点,并鉴定5个靶点有效,其中1个为高效。5个靶点的反义核酸能在通透性大肠杆菌菌株培养中不同程度地抑制其生长,针对高效靶点的核酶在转化大肠杆菌中表达而抑制其生长。 相似文献
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PCR-generated artefact from 16S rRNA gene-specific primers 总被引:2,自引:0,他引:2
Artefacts consisting of concatenated oligonucleotide primer sequences were generated during sub-optimally performing polymerase chain reaction amplification of bacterial 16S rRNA genes using a commonly employed primer pair. These artefacts were observed during amplification for terminal restriction fragment length polymorphism analyses of complex microbial communities, and after amplification from DNA from a microbial culture. Similar repetitive motifs were found in gene sequences deposited in GenBank. The artefact can be avoided by using different primers for the amplification reaction. 相似文献
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Purta E Kaminska KH Kasprzak JM Bujnicki JM Douthwaite S 《RNA (New York, N.Y.)》2008,14(10):2234-2244
Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Ψ) sites in Escherichia coli rRNA, and further modification is found only at Ψ1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of the E. coli methyltransferase that specifically catalyzes methyl group addition to form m3Ψ1915. Analyses of E. coli rRNAs using MALDI mass spectrometry showed that inactivation of the ybeA gene leads to loss of methylation at nucleotide Ψ1915. Methylation is restored by complementing the knockout strain with a plasmid-encoded copy of ybeA. Homologs of the ybeA gene, and thus presumably the ensuing methylation at nucleotide m3Ψ1915, are present in most bacterial lineages but are essentially absent in the Archaea and Eukaryota. Loss of ybeA function in E. coli causes a slight slowing of the growth rate. Phylogenetically, ybeA and its homologs are grouped with other putative S-adenosylmethionine-dependent, SPOUT methyltransferase genes in the Cluster of Orthologous Genes COG1576; ybeA is the first member to be functionally characterized. The YbeA methyltransferase is active as a homodimer and docks comfortably into the ribosomal A site without encroaching into the P site. YbeA makes extensive interface contacts with both the 30S and 50S subunits to align its active site cofactor adjacent to nucleotide Ψ1915. Methylation by YbeA (redesignated RlmH for rRNA large subunit methyltransferase H) possibly functions as a stamp of approval signifying that the 50S subunit has engaged in translational initiation. 相似文献
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Joseph Pitula William T Ruyechan Noreen Williams 《Biochemical and biophysical research communications》2002,290(1):569-576
We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei which we have termed p34 and p37. The predicted primary structures of the two proteins are highly homologous with one major difference, an 18-amino-acid insert in the N-terminal region of p37. These two proteins have been localized to the nucleus based on immunofluorescence microscopy. To gain insight into their function, we have utilized UV crosslinking, coimmunoprecipitation, and sucrose density gradients to identify T. brucei RNA species that associate with p34 and p37. These experiments have demonstrated a specific interaction of both p34 and p37 with the 5S ribosomal RNA and indicate that other RNA species are unlikely to be specifically bound. This suggests a role for p34 and p37 in the import and/or assembly pathway of T. brucei 5S rRNA in ribosome biogenesis. 相似文献
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Virginia S. Lioy Sylvie Goussard Vincent Guerineau Eun-Jeong Yoon Patrice Courvalin Marc Galimand Catherine Grillot-Courvalin 《RNA (New York, N.Y.)》2014,20(3):382-391
In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution—ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness. 相似文献
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Mycoplasma gallisepticum 16S rRNA genes 总被引:1,自引:0,他引:1
Andrei Skamrov Marie Goldman Jana Klasova Robert Beabealashvilli 《FEMS microbiology letters》1995,128(3):321-325
Abstract The genome of Mycoplasma gallisepticum A5969 contains a truncated pseudogene for 16S rRNA in addition to a single unsplit rRNA-operon and a second discontinuous set of rRNA genes. Other M. gallisepticum strains tested do not posses the truncated gene. This gene is almost identical to full-size isolated 16S rRNA gene starting from at least 500 nucleotides upstream of the coding sequence and ending at the 977th nucleotide within the structural part of 16S rRNA. 相似文献
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4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome-bound nascent peptides to the endoplasmic reticulum. To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA. Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only. These data are consistent with a dual role for 4.5S RNA, one involving co-translational protein secretion by a 4.5S-Ffh complex, the other involving free 4.5S RNA. 相似文献