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1.
The Escherichia coli RrmJ gene product has recently been shown to be the 23S rRNA:U2552 specific 2'-O-ribose methyltransferase (MTase) (RrmJ). Its structure has been solved and refined to 1.5 A resolution, demonstrating conservation of the three-dimensional fold and key catalytic side chains with the vaccinia virus VP39 protein, which functions as an mRNA 5'm(7)G-cap-N-specific 2'-O-ribose MTase. Using the amino acid sequence of RrmJ as an initial probe in an iterative search of sequence databases, we identified a homologous domain in the sequence of the L protein of non-segmented, negative-sense, single-stranded RNA viruses. The plausibility of the prediction was confirmed by homology modeling and checking whether important residues at substrate/ligand-binding sites were conserved. The predicted structural compatibility and the conservation of the active site between the novel putative MTase domain and genuine 2'-O-ribose MTases, together with the available results of biochemical studies, strongly suggest that this domain is a 5'm(7)G-cap-N-specific 2'-O-ribose MTase (i.e. the cap 1 MTase). Evolutionary relationships between these proteins are also discussed. 相似文献
2.
The heat shock protein RrmJ (FtsJ), highly conserved from eubacteria to eukarya, is responsible for the 2'-O-ribose methylation of the universally conserved base U2552 in the A-loop of the 23 S rRNA. Absence of this methylation, which occurs late in the maturation process of the ribosome, appears to cause the destabilization and premature dissociation of the 50 S ribosomal subunit. To understand the mechanism of 2'-O-ribose methyltransfer reactions, we characterized the enzymatic parameters of RrmJ and conducted site-specific mutagenesis of RrmJ. A structure based sequence alignment with VP39, a structurally related 2'-O-methyltransferase from vaccinia virus, guided our mutagenesis studies. We analyzed the function of our RrmJ mutants in vivo and characterized the methyltransfer reaction of the purified proteins in vitro. The active site of RrmJ appears to be formed by a catalytic triad consisting of two lysine residues, Lys-38 and Lys-164, and the negatively charged residue Asp-124. Another highly conserved residue, Glu-199, that is present in the active site of RrmJ and VP39 appears to play only a minor role in the methyltransfer reaction in vivo. Based on these results, a reaction mechanism for the methyltransfer activity of RrmJ is proposed. 相似文献
3.
Recent analyses identified a putative catalytic tetrad K-D-K-E common to several families of site-specific methyltransferases (MTases) that modify 2'-hydroxyl groups of ribose in mRNA, rRNA and tRNA (designated the RrmJ class after one of the structurally characterized members; 1eiz in Protein Data Bank) [Genome Biol. 2(9) (2001) 38]. Subsequently, three residues of the tetrad (K-D-K) were shown to be essential for catalysis in RrmJ [J. Biol. Chem. 277 (2002) 41978]. Here, we report identification of a similar conserved tetrad (K-D-K-H) in the family of snoRNA-guided ribose 2'-O-MTases related to fibrillarin (represented by the Mj0697 protein structure; 1fbn in PDB). The corresponding functional groups of putative catalytic tetrads of RrmJ and Mj0697 may be superimposed in space. However, one of the invariant residues (K(164) in RrmJ and K(179) in Mj0697) is observed in two distinct locations in the primary sequence, suggesting an interesting case of 'migration' of the conserved side chain within the framework of the active site. RrmJ and Mj0697 sequences were used as starting points to carry out comprehensive sequence database searches, resulting in identification of a similar conserved tetrad (and hence, prediction of a ribose 2'-O-specificity) in several families of putative MTases, including TlyA hemolysins, novel proteins from Trypanosoma, and large multidomain proteins from Flaviviriruses, Nidoviruses, and Alphaviruses. The results of our analysis of phylogenetic relationships in the RrmJ/fibrillarin superfamily provide insight into the evolution of site-specific and snoRNA-guided ribose 2'-O-MTases from a common ancestor. 相似文献
4.
The 23S rRNA methyltransferase RrmJ (FtsJ) is responsible for the 2'-O methylation of the universally conserved U2552 in the A loop of 23S rRNA. This 23S rRNA modification appears to be critical for ribosome stability, because the absence of functional RrmJ causes the cellular accumulation of the individual ribosomal subunits at the expense of the functional 70S ribosomes. To gain insight into the mechanism of substrate recognition for RrmJ, we performed extensive site-directed mutagenesis of the residues conserved in RrmJ and characterized the mutant proteins both in vivo and in vitro. We identified a positively charged, highly conserved ridge in RrmJ that appears to play a significant role in 23S rRNA binding and methylation. We provide a structural model of how the A loop of the 23S rRNA binds to RrmJ. Based on these modeling studies and the structure of the 50S ribosome, we propose a two-step model where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2'-O position of the ribose. 相似文献
5.
Elzbieta Purta Michelle O'Connor Janusz M. Bujnicki Stephen Douthwaite 《Molecular microbiology》2009,72(5):1147-1158
The rRNAs of Escherichia coli contain four 2'- O- methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, the E. coli rRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small complementary RNAs. We show here that the ygdE gene encodes the methyltransferase that catalyses 2'- O- methylation at nucleotide C2498 in the peptidyl transferase loop of E. coli 23S rRNA. Analyses of rRNAs using MALDI mass spectrometry showed that inactivation of the ygdE gene leads to loss of methylation at nucleotide C2498. The loss of ygdE function causes a slight reduction in bacterial fitness. Methylation at C2498 was restored by complementing the knock-out strain with a recombinant copy of ygdE . The recombinant YgdE methyltransferase modifies C2498 in naked 23S rRNA, but not in assembled 50S subunits or ribosomes. Nucleotide C2498 is situated within a highly conserved and heavily modified rRNA sequence, and YgdE's activity is influenced by other modification enzymes that target this region. Phylogenetically, YgdE is placed in the cluster of orthologous groups COG2933 together with S -adenosylmethionine-dependent, Rossmann-fold methyltransferases such as the archaeal and eukaryotic RNA-guided fibrillarins. The ygdE gene has been redesignated rlmM for r RNA l arge subunit m ethyltransferase M . 相似文献
6.
7.
核糖体是所有细胞中负责蛋白质合成的分子机器。它自身在细胞内的组装成熟过程受到严密调控,需要诸多组装因子的参与。RrmJ是原核生物中一类保守的甲基转移酶,能够甲基化修饰核糖体上肽基转移酶中心(peptidyl transferase center, PTC)内A环的U2552位点。敲除rrmJ基因的大肠杆菌表现出显著的生长缺陷及50S亚基组装前体的累积,因而RrmJ在50S亚基组装中具有重要作用。本研究对细菌生长实验与核糖体图谱分析表明,回补表达RrmJ的质粒对于ΔrrmJ菌株生长缺陷有显著改善,50S前体累积现象也得到有效缓解。通过共沉淀实验证明,RrmJ与ΔrrmJ菌株中提取的50S前体结合能力显著强于缺失型或野生型菌株中纯化的成熟50S;当加入S-腺苷甲硫氨酸时,该酶与50S前体结合能力显著下降。冷冻电镜三维重构数据进一步阐明,缺失型菌株50S前体主要停滞在组装晚期两个PTC区域成熟程度不同的特定时段。综合上述结果表明,U2552位点的修饰发生在50S亚基组装晚期特定阶段,这一事件不仅会加速A环的RNA螺旋折叠,另有可能促进附近PTC区域结构成熟。 相似文献
8.
Pintard L Lecointe F Bujnicki JM Bonnerot C Grosjean H Lapeyre B 《The EMBO journal》2002,21(7):1811-1820
The genome of Saccharomyces cerevisiae encodes three close homologues of the Escherichia coli 2'-O-rRNA methyltransferase FtsJ/RrmJ, designated Trm7p, Spb1p and Mrm2p. We present evidence that Trm7p methylates the 2'-O-ribose of nucleotides at positions 32 and 34 of the tRNA anticodon loop, both in vivo and in vitro. In a trm7Delta strain, which is viable but grows slowly, translation is impaired, thus indicating that these tRNA modifications could be important for translation efficiency. We discuss the emergence of a family of three 2'-O-RNA methyltransferases in Eukaryota and one in Prokaryota from a common ancestor. We propose that each eukaryotic enzyme is located in a different cell compartment, in which it would methylate a different RNA that can adopt a very similar secondary structure. 相似文献
9.
Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity
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During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented. 相似文献
10.
Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase
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The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other E. coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E. coli genes that were capable of rescuing the rrmJ mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA. 相似文献
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12.
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
13.
Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not. 总被引:12,自引:5,他引:7
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P C Yelick R Balhorn P A Johnson M Corzett J A Mazrimas K C Kleene N B Hecht 《Molecular and cellular biology》1987,7(6):2173-2179
The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa. 相似文献
14.
Mitochondria of the yeast Saccharomyces cerevisiae assemble their ribosomes from ribosomal proteins, encoded by the nuclear genome (with one exception), and rRNAs of 15S and 21S, encoded by the mitochondrial genome. Unlike cytoplasmic rRNA, which is highly modified, mitochondrial rRNA contains only three modified nucleotides: a pseudouridine (Psi(2918)) and two 2'-O-methylated riboses (Gm(2270) and Um(2791)) located at the peptidyl transferase centre of 21S rRNA. We demonstrate here that the yeast nuclear genome encodes a mitochondrial protein, named Mrm2, which is required for methylating U(2791) of 21S rRNA, both in vivo and in vitro. Deletion of the MRM2 gene causes thermosensitive respiration and leads to rapid loss of mitochondrial DNA. We propose that Mrm2p belongs to a new class of three eukaryotic RNA-modifying enzymes and is the orthologue of FtsJ/RrmJ, which methylates a nucleotide of the peptidyl transferase centre of Escherichia coli 23S rRNA that is homologous to U(2791) of 21S rRNA. Our data suggest that this universally conserved modified nucleotide plays an important function in vivo, possibly by inducing conformational rearrangement of the peptidyl transferase centre. 相似文献
15.
C B Kurtz E O'Toole S M Christensen J H Weis 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(9):3581-3591
16.
K Fukuhara T Tsuji K Toi T Takao Y Shimonishi 《The Journal of biological chemistry》1985,260(19):10487-10494
Proteolytic digests of interleukin-2 from a human leukemic T-cell line produced by Escherichia coli carrying a recombinant DNA were analyzed by fast atom bombardment mass spectrometry. The mass values of intense signals observed in the mass spectrum were consistent with peptides predicted from the nucleotide sequence of cDNA for human interleukin-2, an indication that the protein with the predicted amino acid sequence was produced by E. coli. BrCN and proteolytic digests of interleukin-2 obtained from cultured cells were also examined by fast atom bombardment mass spectrometry. The observed mass values were identical with those from interleukin-2 from E. coli except for that of the NH2-terminal sequence, in which the Thr residue at position 3 was bound to a sugar moiety. The mass spectra of the digests of the two interleukin-2 preparations and synthetic peptides with sequences from 117 to 128 and 121 to 128 predicted from the nucleotide sequence of cDNA for a human interleukin-2 indicated that Cys residues at positions 58 and 105 are linked by a disulfide bond and that the Cys residue at position 125 is free. 相似文献
17.
The sequence of the 3' 4508 nucleotides (nt) of the genomic RNA of the Therien strain of rubella virus (RV) was determined for cDNA clones. The sequence contains a 3189-nt open reading frame (ORF) which codes for the structural proteins C, E2 and E1. C is predicted to have a length of 300 amino acids (aa). The N-terminal half of the C protein is highly basic and hydrophilic in nature, and is putatively the region of the protein which interacts with the virion RNA. At the C terminus of the C protein is a stretch of 20 hydrophobic aa which also serves as the signal sequence for E2, indicating that the cleavage of C from the polyprotein precursor may be catalyzed by signalase in the lumen of the endoplasmic reticulum. E2 is 282 aa in length and contains four potential N-linked glycosylation sites and a putative transmembrane domain near its C terminus. The sequence of E1 has been previously described [Frey et al., Virology 154 (1986) 228-232]. No homology could be detected between the amino acid sequence of the RV structural proteins and the amino acid sequence of the alphavirus structural proteins. From the position of a region of 30 nt in the RV genomic sequence which exhibited significant homology with the sequence in the alphavirus genome at which subgenomic RNA synthesis is initiated, the RV subgenomic RNA is predicted to be 3346 nt in length and the nontranslated region from the 5' end of the subgenomic RNA to the structural protein ORF is predicted to be 98 nt. In a different translation frame beginning at the 5' end of the RV nt sequence reported here is a 1407 nt ORF which is the C terminal region of the nonstructural protein ORF. This ORF overlaps the structural protein ORF by 149 nt. A low level of homology could be detected between the predicted amino acid sequence of the C-terminus of the RV nonstructural protein ORF and the replicase proteins of several positive RNA viruses of animals and plants, including nsp4 of the alphaviruses, the protein encoded by the C-terminal region of the alphavirus nonstructural ORF. However, the overall homology between RV and the alphaviruses in this region of the genome was only 18%, indicating that these two genera of the Togavirus family are only distantly related. Intriguingly, there is a 2844-nt ORF present in the negative polarity orientation of the RV sequence which could encode a 928-aa polyprotein. 相似文献
18.
Caldas T Binet E Bouloc P Costa A Desgres J Richarme G 《The Journal of biological chemistry》2000,275(22):16414-16419
Ribosomal RNAs undergo several nucleotide modifications including methylation. We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA. The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S(150) supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ-deficient strain. In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ. FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle. We identified the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ. 相似文献
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20.
The amino acid sequence of a Bacillus subtilis phosphoprotein that matches an orfY-tsr coding sequence 总被引:1,自引:0,他引:1
Clayton Mitchell Paul W. Morris Leanna Lum George Spiegelman James C. Vary 《Molecular microbiology》1992,6(10):1345-1349
Bacillus subtilis contains a 30 kDa protein which was phosphorylated during late vegetative growth and sporulation. The sequence for the N-terminal 16 amino acids was found to be identical to the predicted sequence for the N-terminus of a small open reading frame, orfY, but diverged from the predicted sequence thereafter. The orfY region was resequenced and contained one less adenine residue than previously reported, resulting in an open reading frame from within orfY through the entire coding region for tsr which follows orfY. The predicted orfY-tsr amino acid sequence showed 24% identity to Escherichia coli fructose-1,6-bisphosphate aldolase. Two mutants in the tsr region had 2-5% of wild-type aldolase and the nucleotide sequences showed missense mutations. These results indicate that orfY-tsr encodes aldolase and should be renamed fba1. 相似文献