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Query: UNIPROT:P06889 (Mol)
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RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.
Mol Cell Biol 1995 Aug
PMID:Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene. 762 11

Strains of Escherichia coli have been produced which express very high levels of the tRNA(1Leu) isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA(1Leu) gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA(1Leu). As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA(1Leu). We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA(1Leu) was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m1G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA(1Leu) operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA(1Leu) levels. This level of tRNA(1Leu) titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.
Mol Microbiol 1993 Jan
PMID:Effects of tRNA(1Leu) overproduction in Escherichia coli. 768 Apr 10

The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons. A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity. In this study, the mechanism of this trmD3-induced frameshift involving mutant tRNA(Pro) and tRNA(Leu) species has been investigated. Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain. Hybrid beta-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing. The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNA(Pro) deficient in m1G is the frameshifting agent. Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3-dependent +1 frameshifting. Of the four potential sites for tRNA(Leu) (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant. Thus, species of tRNA(Leu) may also act as +1 frameshift suppressors. No -1 frameshifting activity was found with the trmD3 mutant.
J Mol Biol 1993 Aug 05
PMID:Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation. 768 13

The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.
Mol Microbiol 1994 Dec
PMID:Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable. 771 56

Sphingomyelin and product of its enzymatic hydrolysis, sphingosine, influence on the degree of DNA methylation by EcoRII cytosine DNA-methyltransferase. Sphingomyelin activates (up to 30%), whereas sphingosine inhibits this in vitro DNA methylation. A single intraperitoneal injection of the synthetic antioxidant, butylated hydroxytoluene (BHT, 30 mg/kg body weight) results in DNA hypermethylation as well as increase in sphingomyelin content in rat liver nuclei and RNA-polymerase activity. Thus, phospholipids may control DNA methylation in cell, and by this way they seem to modulate replication and transcription.
Biochem Mol Biol Int 1995 Jan
PMID:Effect of sphingomyelin and antioxidants on the in vitro and in vivo DNA methylation. 773 43

Heterogenous nuclear ribonucleoproteins (hnRNPs) bind pre-mRNAs and facilitate their processing into mRNAs. Many of the hnRNPs undergo extensive posttranslational modifications including methylation on arginine residues. hnRNPs contain about 65% of the total NG,NG-dimethylarginine found in the cell nucleus. The role of this modification is not known. Here we identify the hnRNPs that are methylated in HeLa cells and demonstrate that most of the pre-mRNA-binding proteins receive this modification. Using recombinant human hnRNP A1 as a substrate, we have partially purified and characterized a protein-arginine N-methyltransferase specific for hnRNPs from HeLa cells. This methyltransferase can methylate the same subset of hnRNPs in vitro as are methylated in vivo. Furthermore, it can also methylate other RNA-binding proteins that contain the RGG motif RNA-binding domain. This activity is evolutionarily conserved from lower eukaryotes to mammals, suggesting that methylation has a significant role in the function of RNA-binding proteins.
Mol Cell Biol 1995 May
PMID:In vivo and in vitro arginine methylation of RNA-binding proteins. 773 61

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activity that synthesizes phosphatidylcholine (PtdCho) via formation of methylated intermediates (phosphatidyl-N-monomethylethanolamine, PtdEtnMe and phosphatidyl-N,N-dimethylethanolamine, PtdEtnMe2) was comparatively studied in rat heart sarcolemmal (SL), sarcoplasmic reticular (SR) and mitochondrial fractions during Ca2+ paradox. Perfusion (5 min) with Ca(2+)-free medium followed by reperfusion (5 min) with Ca(2+)-containing medium produced a marked rise in resting tension without any recovery of contractile force. Methyltransferase catalytic sites I, II and III which synthesize PtdEtnMe, PtdEtnMe2 and PtdCho, respectively, were assayed by measuring the [3H] methyl group incorporation from 0.055, 10 and 150 microM S-adenosyl-L-[3H-methyl] methionine into membrane PtdEtn molecules. Five minutes of perfusion with Ca(2+)-free medium did not affect either SL or SR N-methyltransferase systems. Ca(2+)-readmission for 1 to 5 min induced a selective, time-dependent depression of SL site II and SR site I methyltransferase activities. Individual N-methylated phospholipids specifically formed at the two sites reflected these changes. The above abnormalities were differently influenced by the duration (1-5 min) of Ca(2+)-free perfusion and were characterized by different kinetic alterations. The mitochondrial methylation system was not affected under Ca2+ paradox. The results suggest that reduced synthesis of SL N-methylated phospholipids may contribute to the contractile dysfunction observed in Ca2+ paradox.
J Mol Cell Cardiol 1995 Jan
PMID:Abnormal synthesis of N-methylated phospholipids during calcium paradox of the heart. 776 Mar 78

A new locus, designated pilK, located immediately adjacent to the previously described Pseudomonas aeruginosa pilG-J gene cluster, has been identified. Sequence analysis of a 1.3 kb region revealed the presence of a single open reading frame of 291 amino acid residues (M(r) 33,338) that contained significant homology to the chemotactic methyltransferase proteins of Escherichia coli, Bacillus subtilis and the gliding bacterium Myxococcus xanthus. The 60 bp pilJ-pilK intergenic region was devoid of promoter consensus sequences, suggesting that pilJ and pilK are contained within the same transcriptional unit. The intergenic region did contain, however, a large, highly GC-rich, inverted repeat that prevented PilK production in expression studies. To investigate the regulatory role of these sequences, pilK-lacZ gene fusions, as well as derivatives containing sequence alterations in the potential stem-loop region, were constructed and analysed in E. coli and P. aeruginosa. Modification of the inverted repeat region in pilK-lacZ protein fusion constructs resulted in as much as a 24-fold increase in beta-galactosidase activity, whereas similar modifications in pilK-lacZ transcriptional fusions had only a marginal effect on beta-galactosidase levels. These results indicated that PilK production may be largely regulated at the level of translation. In stark contrast to pilG-J mutants, which are dramatically impaired in pilus production and/or function, a PAO1 pilK deletion mutant was indistinguishable from the wild type. In addition, complementation studies suggested that the PilK and E. coli CheR proteins are not functionally interchangeable.
Mol Microbiol 1995 Feb
PMID:The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated. 778 42

Changes in the pattern of DNA methylation have been a consistent finding in cancer cells. The mostly descriptive nature of these studies and the fact that both hypo- and hypermethylation have been observed at various loci have made it difficult to assess whether these changes are causally involved in the transformation process or whether they reflect the altered physiology of rapidly dividing cancer cells. It is clear, however, that DNA methylation plays an important role in the generation of mutations in human tumors. The high incidence of C-to-T transitions found in the p53 tumor-suppressor gene is attributed to the spontaneous deamination of 5-methylcytosine residues. The multiple observations linking DNA methylation to cancer can be resolved in a model proposing that the high rate of mutation at CpG dinucleotides is due in part to methyltransferase-facilitated deamination. Support for a role of DNA methyltransferase as a mutator enzyme is provided by work with a prokaryotic DNA methyltransferase under S-adenosyl-methionine methyl-donor limiting conditions. Methyl-donor limiting conditions might arise in early stages of tumor development, leading to high rates of methyltransferase-mediated CpG mutagenesis, as seen in human tumors. Such a mechanism is consistent with the frequently reported methionine auxotrophy of cancer cells and with the tumorigenic effects of methyl-deficient diets. Methyl deficiency in tumor cells is also consistent with the commonly observed global hypomethylation of tumor cell DNA, despite normal or even high levels of DNA methyltransferase expression.
Hum Mol Genet 1994
PMID:DNA methylation and cancer. 784 43

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) N-methyltransferase (protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco. With limited internal amino acid sequence information obtained from HPLC-purified peptic polypeptides from Rubisco LSMT, a full-length cDNA clone was isolated utilizing polymerase chain reaction-based technology and conventional bacteriophage library screening. The 1802 bp cDNA of Rubisco LSMT encodes a 489 amino acid polypeptide with a predicted molecular mass of ca. 55 kDa. A derived N-terminal amino acid sequence with features common to chloroplast transit peptides was identified. The deduced sequence of Rubisco LSMT did not exhibit regions of significant homology with other protein methyltransferases. Southern blot analysis of pea genomic DNA indicated a low gene copy number of Rubisco LSMT in pea. Northern analysis revealed a single mRNA species of about 1.8 kb encoding for Rubisco LSMT which was predominately located in leaf tissue. Illumination of etiolated pea seedlings showed that the accumulation of Rubisco LSMT mRNA is light-dependent. Maximum accumulation of Rubisco LSMT transcripts occurred during the initial phase of light-induced leaf development which preceded the maximum accumulation of rbcS and rbcL mRNA. Transcript levels of Rubisco LSMT in mature light-grown tissue were similar to transcript levels in etiolated tissues indicating that the light-dependent accumulation of Rubisco LSMT mRNA is transient. This is the first reported DNA and amino acid sequence for a protein methylase III enzyme.
Plant Mol Biol 1995 Jan
PMID:Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase. 788 16


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