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Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes). Many of these nucleotides are important in the formation of the Michaelis complex. In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA. In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions. 相似文献
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Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly 总被引:10,自引:0,他引:10
M J Stark R J Gregory R L Gourse D L Thurlow C Zwieb R A Zimmermann A E Dahlberg 《Journal of molecular biology》1984,178(2):303-322
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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
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Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S subunits in bacterial cells. We have tested the idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of ribosomal subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S subunits was examined under the same conditions. The rate of 50S subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected sequence was found in both 50S subunits and in a subunit precursor particle sedimenting at about 30S in sucrose gradients. 23S rRNA isolated from 50S subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with (3)H-S-adenosylmethionine as a substrate. 50S subunits were not a substrate for the enzyme, but the 30S gradient region from erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation become insensitive to the inhibitory effects of the drug. 相似文献
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Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance
to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S subunits in bacterial cells. We have tested the
idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of
ribosomal subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S subunits was examined under
the same conditions. The rate of 50S subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin
binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S
rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected
sequence was found in both 50S subunits and in a subunit precursor particle sedimenting at about 30S in sucrose gradients.
23S rRNA isolated from 50S subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with 3H-S-adenosylmethionine as a substrate. 50S subunits were not a substrate for the enzyme, but the 30S gradient region from
erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests
that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by
the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation
become insensitive to the inhibitory effects of the drug.
Received: 21 June 2002 / Accepted: 21 August 2002 相似文献
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Cloacin DF12 cleavage of Escherichia coli f[3H]MettRNA-AUG-ribosome complexes affects this substrate for in vitro peptide chain termination. Codon-directed release factors' (RF) 1 and 2 release of f[3H]methionine is inhibited by cloacin. Since cloacin inhibits RF1 and -2 binding to ribosomes but not RF-directed f[3H]methionine release from f[3H]met-tRNA-AUG-ribosome complexes when reactions contain 20% ethanol, we conclude that cloacin DF 13 inhibits formation of the termination codon recognition complex. Thus, cleavage of the 3'-OH 49-nucleotide sequence of the 16 S rRNA perturbs the codon-directed binding of RF to ribosomes. 相似文献
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Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha 总被引:1,自引:0,他引:1
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Heissmeyer V Krappmann D Hatada EN Scheidereit C 《Molecular and cellular biology》2001,21(4):1024-1035
p105 (NFKB1) acts in a dual way as a cytoplasmic IkappaB molecule and as the source of the NF-kappaB p50 subunit upon processing. p105 can form various heterodimers with other NF-kappaB subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IkappaB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKKbeta phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSGPsi motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IkappaBalpha, -beta, and -epsilon. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules betaTrCP1 and betaTrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKKgamma, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing. 相似文献
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In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. 总被引:48,自引:10,他引:38
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DNA oligonucleotide complementary to sequences in the 5' third of U3 snRNA were injected into Xenopus oocyte nuclei to disrupt endogenous U3 snRNA. The effect of this treatment on rRNA processing was examined. We found that some toads have a single rRNA processing pathway, whereas in other toads, two rRNA processing pathways can coexist in a single oocyte. U3 snRNA disruption in toads with the single rRNA processing pathway caused a reduction in 20S and '32S' pre-rRNA. In addition, in toads with two rRNA processing pathways, an increase in '36S' pre-rRNA of the second pathway is observed. This is the first in vivo demonstration that U3 snRNA plays a role in rRNA processing. Cleavage site #3 is at the boundary of ITS 1 and 5.8S and links all of the affected rRNA intermediates: 20S and '32S' are the products of site #3 cleavage in the first pathway and '36S' is the substrate for cleavage at site #3 in the second pathway. We postulate that U3 snRNP folds pre-rRNA into a conformation dictating correct cleavage at processing site #3. 相似文献
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The Bacillus subtilis RNase M5 activity, responsible for the endonucleolytic maturation of 5 S rRNA, requires two proteins, alpha and beta. The beta component has been purified to homogeneity and shown to correspond to ribosomal protein BL16. The BL16 protein evidently corresponds functionally to Escherichia coli ribosomal protein EL18, as that latter protein also will complement the B. subtilis alpha protein in the RNase M5 reaction. A filter binding assay for the formation of B. subtilis 5 S rRNA-protein complexes was characterized and used to evaluate the association of BL16 protein with some RNAs. A native precursor of 5 S rRNA, containing extra sequences at both termini of the mature domain, binds the ribosomal protein no better than the mature 5 S rRNA; the precursor sequences do not facilitate that interaction. A model is considered in which the precursor segments facilitate, by refolding, the dissociation of processing products prior to the RNase M5 step. Electrostatic versus nonelectrostatic contributions to the BL16-5 S rRNA complex formation were inspected by analyzing variation in apparent association constants as a function of ionic strength. Electrostatic interactions were seen to contribute approximately 65% to the overall binding energy. 相似文献
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Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs 总被引:1,自引:0,他引:1
The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome. 相似文献
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Granato DC Gonzales FA Luz JS Cassiola F Machado-Santelli GM Oliveira CC 《The FEBS journal》2005,272(17):4450-4463
In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two-hybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing. 相似文献
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Solid-phase processing of U2 snRNA precursors 总被引:4,自引:0,他引:4
HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2. The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA. To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns. When the immobilized [3H]uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h. Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody. Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns. Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP. Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease. These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component. 相似文献
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Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. 总被引:34,自引:1,他引:33
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The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre-rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation. 相似文献
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Wenjuan Wu Sheng Liu Hannes Ruwe Delin Zhang Joanna Melonek Yajuan Zhu Xupeng Hu Sandra Gusewski Ping Yin Ian D. Small Katharine A. Howell Jirong Huang 《The Plant journal : for cell and molecular biology》2016,85(5):607-621
Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid‐localized pentatricopeptide repeat (PPR) protein with a small MutS‐related domain, is required for maturation of the 23S–4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5′ end of the 23S–4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA ‘footprint’ associated with this site in sot1 mutants. We found that more than half of the 23S–4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5′ and 3′ ends, and that the endonucleolytic cleavage product normally released from the 5′ end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5′ extremity of the 23S–4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5′ and 3′ ends. 相似文献