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• W2016706267 abstract "In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed PabTrm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N2) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N2-monomethyl (m2G) or N2-dimethylguanosine (m22G). Interestingly, PabTrm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus (PfuTrm-G26, also known as PfuTrm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life. In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed PabTrm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N2) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N2-monomethyl (m2G) or N2-dimethylguanosine (m22G). Interestingly, PabTrm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus (PfuTrm-G26, also known as PfuTrm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life. The considerable effort of the last 5 years in sequencing entire genomes from the three domains of life (Bacteria, Archaea, and Eukaryota (1Woese C.R. Kandler O. Wheelis M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4576-4579Crossref PubMed Scopus (4515) Google Scholar)) has made possible the establishment of a list of clusters of orthologous groups (COGs) 1The abbreviations used are: COG, cluster of orthologous group; AdoMet, S-adenosylmethionine; DTT, dithiothreitol; HEPPS, N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid); MTase, methyltransferase; PSI-BLAST, position-specific iterated BLAST; TLC, two-dimensional thin layer cellulose; MOPS, 4-morpholinepropanesulfonic acid; bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. of unknown function (2Tatusov R.L. Fedorova N.D. Jackson J.D. Jacobs A.R. Kiryutin B. Koonin E.V. Krylov D.M. Mazumder R. Mekhedov S.L. Nikolskaya A.N. Rao B.S. Smirnov S. Sverdlov A.V. Vasudevan S. Wolf Y.I. Yin J.J. Natale D.A. BMC Bioinformatics. 2003; 4: 41Crossref PubMed Scopus (3415) Google Scholar, 3Tatusov R.L. Koonin E.V. Lipman D.J. Science. 1997; 278: 631-637Crossref PubMed Scopus (2758) Google Scholar, 4Tatusov R.L. Natale D.A. Garkavtsev I.V. Tatusova T.A. Shankavaram U.T. Rao B.S. Kiryutin B. Galperin M.Y. Fedorova N.D. Koonin E.V. Lipman D.J. Nucleic Acids Res. 2001; 29: 22-28Crossref PubMed Scopus (1556) Google Scholar) (available on the World Wide Web at www.ncbi.nlm.nih.gov/COG/). This continuously updated list allows functional annotation of newly sequenced genomes and genome-wide evolutionary analyses. Among these COGs, some appear to have great importance, since they are present in most, if not all, members of one or several domains of life. Distinction can be made between those conserved in all three domains (thus defining a universal subset of proteins probably present in LUCA, the last universal common ancestor of the extant life forms), those specific to a single domain, and those shared by two of the three domains of life. Several comparative studies on characterized proteins, aimed at distinguishing organisms of the three biological domains, have indicated that many, although not all, proteins involved in metabolic pathways of Archaea resemble more closely their bacterial than eukaryotic counterparts, whereas proteins involved in the organization or processing of genetic information (structures of ribosome and chromatin, translation, transcription, replication, and DNA repair) suggest a closer relationship between Archaea and Eukaryota (5Dennis P.P. Cell. 1997; 89: 1007-1010Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 6Olsen G.J. Woese C.R. Cell. 1997; 89: 991-994Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 7Makarova K.S. Koonin E.V. Genome Biol. 2003; 4: 115Crossref PubMed Scopus (75) Google Scholar, 8Terns M.P. Terns R.M. Gene. Expr. 2002; 10: 17-39PubMed Google Scholar). A series of 32 COGs of unknown function have been identified that are common to Eukaryota and Archaea but not found in Bacteria (9Matte-Tailliez O. Zivanovic Y. Forterre P. Trends Genet. 2000; 16: 533-536Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). These groups were named PACEs (for proteins from Archaea conserved in Eukaryota (see, on the World Wide Web, www-archbac.u-psud.fr/projects/pace/paceproteins.html). Interestingly, for some of these proteins, roles in transcription, translation, or replication processes have been postulated (9Matte-Tailliez O. Zivanovic Y. Forterre P. Trends Genet. 2000; 16: 533-536Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). For example, polypeptides from the PACE08 group (COG1369) have been identified as one of the subunits of ribonuclease P (RNase P), a ribonucleoprotein complex involved in 5′-end tRNA maturation (10Hartmann E. Hartmann R.K. Trends Genet. 2003; 19: 561-569Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The SSO0175 and MJ0051 archaeal proteins, as well as the Ybl057c protein from Saccharomyces cerevisiae (all from the PACE07 group), have been shown to have peptidyl-tRNA hydrolase activity and are thus involved in recycling peptidyl-tRNA molecules prematurely dissociated from the mRNA template (11Fromant M. Ferri-Fioni M.L. Plateau P. Blanquet S. Nucleic Acids Res. 2003; 31: 3227-3235Crossref PubMed Scopus (22) Google Scholar, 12Rosas-Sandoval G. Ambrogelly A. Rinehart J. Wei D. Cruz-Vera L.R. Graham D.E. Stetter K.O. Guarneros G. Soll D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16707-16712Crossref PubMed Scopus (57) Google Scholar). Among proteins of the PACE groups characterized so far, only those belonging to PACE 11 have been shown not to be related to organization or processing of cellular information. Two of us recently reported that the product of the Pyrococcus abyssi GE5 PAB0944 gene (COG1019) corresponds to a single domain polypeptide involved in the fourth step of coenzyme A biosynthesis (phosphopantetheine adenylyltransferase), whereas its human ortholog is fused to another domain, dephosphocoenzyme A kinase, which is responsible for catalyzing the last step of the coenzyme A biosynthetic pathway (13Armengaud J. Fernandez B. Chaumont V. Rollin-Genetet F. Finet S. Marchetti C. Myllykallio H. Vidaud C. Pellequer J.L. Gribaldo S. Forterre P. Gans P. J. Biol. Chem. 2003; 278: 31078-31087Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). All of these examples clearly indicate that PACE proteins have fundamental cellular functions and that several of them are obviously related to RNA metabolism. In the present study, we focus our attention on PAB1283 from the hyperthermophilic archaeon P. abyssi GE5. This protein belongs to the PACE18 group (9Matte-Tailliez O. Zivanovic Y. Forterre P. Trends Genet. 2000; 16: 533-536Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and is classified as a member of the COG1041 group (2Tatusov R.L. Fedorova N.D. Jackson J.D. Jacobs A.R. Kiryutin B. Koonin E.V. Krylov D.M. Mazumder R. Mekhedov S.L. Nikolskaya A.N. Rao B.S. Smirnov S. Sverdlov A.V. Vasudevan S. Wolf Y.I. Yin J.J. Natale D.A. BMC Bioinformatics. 2003; 4: 41Crossref PubMed Scopus (3415) Google Scholar). A search of the NCBI conserved domain data base (available on the World Wide Web at www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) (14Marchler-Bauer A. Anderson J.B. DeWeese-Scott C. Fedorova N.D. Geer L.Y. He S. Hurwitz D.I. Jackson J.D. Jacobs A.R. Lanczycki C.J. Liebert C.A. Liu C. Madej T. Marchler-Bauer A. Marchler G.H. Mazumder R. Nikolskaya A.N. Rao B.S. Panchenko A.R. Shoemaker B.A. Simonyan V. Song J.S. Thiessen P.A. Vasudevan S. Wang Y. Yamashita R.A. Yin J.J. Bryant S.H. Nucleic Acids Res. 2003; 31: 383-387Crossref PubMed Scopus (650) Google Scholar) indicates that all sequences from COG1041 encompass two domains: a putative AdoMet-dependent methyltransferase (MTase) domain (pfam01170) at the C terminus and a so-called THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain (pfam02926 (15Aravind L. Koonin E.V. Trends Biochem. Sci. 2001; 26: 215-217Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar)) of about 110 amino acid residues at the N terminus. The reactions catalyzed by MTases are very diverse and can transfer the AdoMet methyl group onto either nitrogen, oxygen, or carbon atoms in DNA, RNA, proteins, lipids, or small molecules such as catechol (reviewed in Ref. 16Fauman E.B. Blumenthal R.M. Cheng X. Cheng X. Blumenthal R.M. S-Adenosylmethionine-dependent Methyltransferases: Structures and Functions. World Scientific Publishing, Singapore1999: 1-38Crossref Google Scholar). The THUMP domain has been identified in the ThiI enzyme catalyzing the formation of 4-thiouridine in bacterial tRNAs as well as in several other putative RNA modification enzymes, such as MTases and pseudouridine synthases (see Ref. 15Aravind L. Koonin E.V. Trends Biochem. Sci. 2001; 26: 215-217Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and references therein). For this reason, it is expected to be an ancient RNA-binding domain. Therefore, we hypothesized that COG1041 proteins are as yet uncharacterized RNA MTases for which the target nucleoside within RNA molecules has still to be discovered. Most stable cellular RNAs (tRNAs, rRNAs, and small nuclear (small nucleolar)RNAs) contain many post-transcriptionally modified nucleosides. To date, at least 96 different chemically distinct modified nucleosides have been identified in the many naturally occurring RNA analyzed (see, on the World Wide Web, medstat.med.utah.edu/RNAmods/). Among them, ribose and/or base methylations are among the most frequently encountered (17Rozenski J. Crain P.F. McCloskey J.A. Nucleic Acids Res. 1999; 27: 196-197Crossref PubMed Scopus (350) Google Scholar) (for a review, see Ref. 18Hopper A.K. Phizicky E.M. Genes Dev. 2003; 17: 162-180Crossref PubMed Scopus (255) Google Scholar). They have been shown to play a role in stabilization, structural folding, protection from endonucleases, and molecular recognition of the functional RNAs by numerous proteins as well as in RNA-RNA interactions (for a review, see many chapters in Ref. 19Grosjean H. Benne R. Grosjean H. Benne R. Modification and Editing of RNA. American Society for Microbiology Press, Washington, D. C.1998Crossref Google Scholar). To verify that proteins belonging to COG1041 are AdoMet-dependent RNA MTases, we purified to homogeneity and characterized a recombinant form of the PAB1283 gene product from the hyperthermophilic archaeon P. abyssi. We identified in vitro both the reaction catalyzed by the purified enzyme and the target nucleotide within its RNA substrates. Our results demonstrate for the first time that a THUMP-containing MTase is associated with a tRNA modification function and that the PAB1283 gene product corresponds to a site-specific tRNA: m22G dimethyltransferase acting on the N2-exocyclic amine group of guanosine at position 10 in tRNA. Construction of an N-terminal His6-tagged PAB1283-overexpressing Plasmid—A construct was designed in order to get overexpression of a slightly modified version of P. abyssi PAB1283 open reading frame. The Met-ATG located at 1592098 (numbering refers to whole P. abyssi genomic sequence), and not the upstream Leu-TTG codon that genome annotators mentioned in the data base, was taken as the most probable initiator codon. This was the choice because TTG codons are rarely used as initiators in Archaea and also because the resulting N-terminal PAB1283 sequence aligns better with those of the other archaeal homologs. Thirteen amino acid residues (MRGSHHHHHHGMAS) were introduced with the N-terminal His6 tag. Two synthetic oligonucleotide primers were designed in order to amplify the PAB1283 gene from P. abyssi total DNA: oAD34 (5′-catatggctagcATGTTCTACGTTGAAATCCTAGGTTTG-3′) that contains two engineered restriction sites (NdeI and NheI) and oAD35 (5′-ggatcctcaTCACCTCGCC TCCATTATGTAGAAG-3′) that contains an engineered BamHI site just after the stop codon. Nucleotides in lowercase type were not present in the original coding sequence. PCR performed with Pwo polymerase (Roche Applied Science) gave a 1.0-kb homogeneous product that was cloned into pCRScript-cam (Stratagene), resulting in plasmid pSBTN-AB89. The 994-bp insert from pSBTN-AB89 was removed by digestion with NheI and BamHI and ligated with T4-DNA ligase with plasmid pSBTNAB23, 2J. Armengaud and V. Chaumont, unpublished data. a derivative of pCR T7/NT-topo (Invitrogen) containing a T7 promoter and His6 tag, previously digested with compatible endonucleases (NheI and BglII). The resulting plasmid was sequenced in order to ascertain the integrity of the nucleotide sequence and was named pSBTN-AC18. Purification of Recombinant PAB1283 Protein—Overexpression of the PAB1283 construct was achieved with the Escherichia coli Rosetta(DE3)pLysS strain (Novagen) transformed with pSBTN-AC18. Large scale liquid cultures were set up at 30 °C and induced with 1 mm isopropyl-γ-d-thiogalactopyranoside as described earlier (13Armengaud J. Fernandez B. Chaumont V. Rollin-Genetet F. Finet S. Marchetti C. Myllykallio H. Vidaud C. Pellequer J.L. Gribaldo S. Forterre P. Gans P. J. Biol. Chem. 2003; 278: 31078-31087Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Cells (63 g of wet material) were resuspended in 300 ml of cold 50 mm Tris/HCl buffer (pH 8.3 at 20 °C) and disrupted by means of a BasicZ cell disrupter (Constant Systems Ltd.) operated at 900 bars. The cold cell extract was then centrifuged at 20,000 × g for 20 min at 10 °C to remove cellular debris and aggregated proteins. PAB1283 was purified from 45 ml of this cell extract (corresponding to7gofwet cells). The sample was subjected to a 20-min heat treatment at 65 °C and centrifuged at 20,000 × g for 15 min at 10 °C. The resulting 40-ml clear supernatant was diluted with an equal volume of 50 mm Tris/HCl buffer (pH 8.3) containing 100 mm KCl and 50 mm imidazole (buffer A). The sample was applied at room temperature onto a 5-ml HiTrap chelating HP column (Amersham Biosciences) at a flow rate of 0.6 ml/min. After a 5-column volume wash with buffer A at a flow rate of 1 ml/min, the N-terminal His6-tagged PAB1283 protein was eluted over a 15-ml linear gradient comprising 50-300 mm imidazole. The major peak, which eluted from the column at about 150 mm imidazole, was desalted by gel filtration at a flow rate of 1.5 ml/min on an Amersham Biosciences XK26/40 column containing 175 ml of packed G25SF gel (Amersham Biosciences) previously equilibrated with 50 mm Tris/HCl buffer (pH 8.3) containing 50 mm NaCl. The protein was further purified by chromatography either on MonoQ or hydroxyapatite columns. In the first case, samples were injected at a 0.5 ml/min flow rate onto a 1-ml MonoQ HR5/5 column (Amersham Biosciences) previously equilibrated with buffer A, and the bound proteins were eluted over a 40-ml linear gradient comprising 50-550 mm NaCl. The proteins, which eluted between 100 and 150 mm NaCl, were then dialyzed against 50 mm Tris/HCl buffer (pH 8.3) containing 20 mm NaCl and stored at -80 °C. Alternatively, samples were first dialyzed against 100 mm NaH2PO4/Na2HPO4 buffer at pH 7.2 containing 300 mm NaCl and 2 mm DTT (buffer H1) and then injected at a 0.5 ml/min flow rate onto an HR10/10 column (Amersham Biosciences) packed with 7 ml of hydroxyapatite Bio-gel HT (Bio-Rad) and previously equilibrated with buffer H1. Proteins were resolved with a 3-column volume linear gradient from 100 to 500 mm NaH2PO4/Na2HPO4 and then dialyzed against 50 mm Tris/HCl buffer (pH 8.3) containing 20 mm NaCl. Protein concentrations were determined by UV spectrophotometry at 280 nm using the molar absorption coefficient of 29,300 m-1 cm-1 obtained from calculation of the amino acid composition of the recombinant protein (20Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5060) Google Scholar) (available on the World Wide Web at www.expasy.org/tools/protparam.html). Estimation of Quaternary Structure of Purified Recombinant Enzyme by Gel Filtration—The native molecular mass of PAB1283 protein was first estimated by gel filtration chromatography on a Superdex 200 gel packed into a HR10/30 column (Amersham Biosciences) with a final bed volume of 24 ml. The column was equilibrated at room temperature at a flow rate of 0.5 ml/min with 50 mm Tris/HCl buffer, pH 8.3, containing 50 mm NaCl and eluted with the same buffer. A second, more accurate estimation was made on a Superdex75 gel packed into a HR10/30 column (Amersham Biosciences) with a final bed volume of 24 ml. This column was equilibrated at room temperature at a flow rate of 0.75 ml/min with 25 mm sodium phosphate buffer, pH 7.2, containing 5 mm MgCl2 and 2 mm DTT. Samples containing the purified recombinant PAB1283 protein (2.3 and 3.7 nmol, respectively) were chromatographed on Superdex200 and Superdex75 columns. Complex formation between PAB1283 protein and various samples of tRNAs (bulk yeast tRNAs, bulk E. coli tRNAs, or purified yeast tRNAAsp) were evaluated by comparing the elution profiles of tRNAs and protein alone with those of tRNA-protein complexes on a Superdex75 gel filtration column. Transfer RNAs from both yeast and E. coli were obtained from Sigma, whereas purified yeast tRNAAsp and purified 74-nucleotide RNA that includes part of helix 22 and helices 34 and 35 from E. coli 23 S rRNA (described in Ref. 21Lebars I. Yoshizawa S. Stenholm A.R. Guittet E. Douthwaite S. Fourmy D. EMBO J. 2003; 22: 183-192Crossref PubMed Scopus (29) Google Scholar) were generous gifts of Dr. Anne Theobald-Ditriech (CNRS-IBMC, Strasbourg, France) and Dr. Dominique Fourmy (CNRSICSN, Gif-sur-Yvette, France), respectively. Before use, the RNAs were precipitated with ethanol and resuspended in 10 mm Tris/HCl buffer, pH 7.4, containing 10 mm MgCl2 and 150 mm NaCl. Differential Scanning Calorimetry—The transition temperature, Tm, of purified PAB1283 was determined with a high sensitivity differential scanning microcalorimeter, VP-DSC (MicroCal). The reference buffer was 30 mm HEPPS/NaOH, pH 8.0, containing 50 mm NaCl and 5 mm MgCl2. Prior to the calorimetric analysis, the sample was dialyzed at room temperature against this reference buffer. Recombinant His6-tagged PAB1283 protein was analyzed at a protein concentration of 5.3 μm. Calorimetric scans against the reference buffer were carried out in 0.5 ml of fixed-in-place cells. A self-contained pressurizing system (0-45 p.s.i.) allowed the scanning of solutions above their boiling point. Scans were recorded between 30 and 110 °C with a heating scan rate of 1.0 °C/min. tRNA Methylation Assays—To determine the tRNA (G10) MTase activity of PAB1283, various 32P-radiolabeled tRNA transcripts were used as substrates. They were obtained by in vitro transcription with T7 polymerase (Promega) of linearized plasmids harboring appropriate synthetic tRNA genes using α-32P-labeled GTP, UTP, or CTP (Amersham Biosciences). Both preparation and purification of resulting transcripts on urea gels have been described elsewhere (22Grosjean H. Droogmans L. Giege R. Uhlenbeck O.C. Biochim. Biophys. Acta. 1990; 1050: 267-273Crossref PubMed Scopus (41) Google Scholar). For testing enzyme activity, an aliquot (a few nmol) of purified recombinant PAB1283 protein in 2.5 μl of 25 mm sodium phosphate buffer (pH 7.2) containing 1 mm MgCl2, 10% glycerol, and 1 mg/ml serum albumin was added to 22.5 μl of the standard reaction mixture consisting in 25 mm sodium phosphate buffer (pH 7.2), 5 mm MgCl2, 2 mm DTT, 40 μg/ml polyuridylic acid (Sigma), 80 μm AdoMet (Sigma), with enough 32P-radiolabeled tRNA to obtain maximum incorporation of the methyl group from AdoMet to the tRNA substrate after 1 h of incubation at 50 °C. AdoMet stock solutions were made at 20 mm in hydrochloric acid, pH 2.0, and stored at -70 °C. Prior to the addition to the reaction mixture, the solutions of tRNA substrates contained in siliconized Eppendorf tubes were renatured by a 3-min incubation in a water bath at 75 °C followed by gradual cooling to room temperature. After incubation, the reaction was stopped by adding 200 μl of cold 0.3 m sodium acetate (pH 5.3) immediately followed by the addition of an equal volume of phenol/chloroform (24:1). Denatured proteins were then removed by centrifugation at 13,000 × g for 3 min at room temperature, and nucleic acids present in the upper phase were precipitated with ethanol, collected by centrifugation, washed once with 70% ethanol, dried, and finally completely digested into 5′-monophosphate nucleosides by overnight incubation at 37 °C with an excess (0.4 μg) of nuclease P1 (Roche Applied Science) in 10 μl of 50 mm ammonium acetate/acetic acid buffer at pH 5.3. Alternatively, complete digestion of dried RNA samples into 3′-phosphate nucleosides was performed by overnight incubation at 37 °C with 10 μl of 50 mm ammonium acetate/acetic acid buffer at pH 4.6 containing a home-made mixture of RNase T1 and RNase T2 (23Grosjean H. Keith G. Droogmans L. Methods Mol. Biol. 2004; 265: 357-392PubMed Google Scholar). The resulting hydrolysates were then analyzed by two-dimensional thin layer chromatography on 10 × 10-cm cellulose plates as described elsewhere together with the necessary reference maps (23Grosjean H. Keith G. Droogmans L. Methods Mol. Biol. 2004; 265: 357-392PubMed Google Scholar). Localization of radioactive spots on the thin layer plates and evaluation of their relative radioactivity were performed after exposure of the plates to a PhosphorImager screen followed by scanning with a STORM instrument (Amersham Biosciences). Using this approach, the optimal experimental conditions described above for testing tRNA methylation were obtained by varying the nature of the buffering system, the nature and concentration of monovalent salt and Mg2+ ions, and the protein and tRNA stabilizers such as bovine serum albumin and polyuridilic acid. Maximum activity of PAB1283 protein was obtained in 25 mm sodium phosphate buffer (pH 7.2) containing 5 mm MgCl2, 40 μg/ml polyuridylic acid, and 100 μg/ml of bovine serum albumin. For testing the incorporation of the tritiated methyl group into tRNA, the same reaction mixture as described above was used, except that unlabeled bulk E. coli tRNA (Sigma) and methyl-3H-labeled AdoMet (15 Ci/mmol; Amersham Biosciences) were used as substrate and methyl donor, respectively. Evaluation of tritium incorporation into tRNA was measured after trichloroacetic acid precipitation of the nucleic acid and filtration on a Millipore membrane. COG1041 Proteins Are Almost Ubiquitous in Archaea and Eukaryota—A search of the nonredundant protein data base from NCBI using the PSI-BLAST tool (24Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59929) Google Scholar) and PAB1283 sequence as query revealed a set of 20 full-length closely related proteins (hits reported in the first iteration with E values below 10-14 and sequence identity above 30%), all from Archaea. Among more distantly related proteins, eukaryotic sequences were detected in the second iteration, such as AAQ10284 from Homo sapiens (gi33329797) and Yol124c from S. cerevisiae (gi6324448). Reciprocal data base searches yielded 41 presumed orthologs from Eukaryota and Archaea grouped in COG1041. No close homologs (E value below 10-12 in the second iteration) with full-length sequence matching to PAB1283 were found among Bacteria, except closely related proteins from Bacillus anthracis A2012 and Bacillus cereus ATCC14579. However, reciprocal BLAST searches revealed no further close bacterial homologs of these proteins, suggesting that COG1041 members are not generic to Bacteria and might have been introduced to this domain by horizontal gene transfer. Remarkably, another related sequence, Vng2242c, was detected in the archaeon Halobacterium sp. NRC-1 but matched only the C-terminal part of all of the other COG1041 sequences. After analysis of the corresponding nucleic acid sequence (NC_002607), we found that a 322-amino acid polypeptide exhibiting significant similarity to all COG1041 proteins along the whole sequence would be encoded by the reverse strand from a GTG codon at 1,669,809 (and not from the ATG reported by genome annotators at 1,669,326). Except for the early divergent eukaryon Encephalitozoon cuniculi that has an exceptionally small genome, all of the sequenced Archaea and Eukaryota were found to have at least one PAB1283 homolog. Fig. 1 shows the results of an NCBI conserved domain search with PAB1283 as the query (14Marchler-Bauer A. Anderson J.B. DeWeese-Scott C. Fedorova N.D. Geer L.Y. He S. Hurwitz D.I. Jackson J.D. Jacobs A.R. Lanczycki C.J. Liebert C.A. Liu C. Madej T. Marchler-Bauer A. Marchler G.H. Mazumder R. Nikolskaya A.N. Rao B.S. Panchenko A.R. Shoemaker B.A. Simonyan V. Song J.S. Thiessen P.A. Vasudevan S. Wang Y. Yamashita R.A. Yin J.J. Bryant S.H. Nucleic Acids Res. 2003; 31: 383-387Crossref PubMed Scopus (650) Google Scholar). All of the sequences from COG1041 encompass two conserved domains: a THUMP domain (15Aravind L. Koonin E.V. Trends Biochem. Sci. 2001; 26: 215-217Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) in the N terminus (pfam 02926) and an MTase domain in the C terminus (pfam 01170). Fig. 1 also presents a sequence alignment of the COG1041 C-terminal regions from a selection of 10 representative organisms, revealing a pattern of conserved motifs characteristic of an AdoMet-dependent MTase. Both the AdoMet-binding and the catalytic residues are located within a single domain with an α/β Rossmann-like fold. Assuming that the THUMP domain is primarily involved in substrate binding (15Aravind L. Koonin E.V. Trends Biochem. Sci. 2001; 26: 215-217Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), the linear arrangement of structural elements seen in the COG1041 proteins makes them members of the ζ class of Rossmann fold MTases (16Fauman E.B. Blumenthal R.M. Cheng X. Cheng X. Blumenthal R.M. S-Adenosylmethionine-dependent Methyltransferases: Structures and Functions. World Scientific Publishing, Singapore1999: 1-38Crossref Google Scholar, 25Cheng X. Curr. Opin. Struct. Biol. 1995; 5: 4-10Crossref PubMed Scopus (105) Google Scholar). On the basis of comparisons with other MTases (26Bujnicki J.M. Rychlewski L. BMC Bioinformatics. 2002; 3: 10Crossref PubMed Scopus (26) Google Scholar, 27Bujnicki J.M. Leach R.A. Debski J. Rychlewski L. J. Mol. Microbiol. Biotechnol. 2002; 4: 405-415PubMed Google Scholar), three Asp residues in PAB1283 are probably crucial to cofactor binding (Fig. 1). Asp185, Asp208, and Asp235 are predicted to coordinate the methionine moiety via a water molecule (28Bugl H. Fauman E.B. Staker B.L. Zheng F. Kushner S.R. Saper M.A. Bardwell J.C. Jakob U. Mol. Cell. 2000; 6: 349-360Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), the 2′- and 3′-hydroxyl groups of the ribose moiety, and the N6 and/or N3 atoms of the adenine moiety, respectively. The predicted substrate-binding pocket comprises motifs IV, VI, VIII, and X. The highly conserved consensus pentapeptide sequence DPPYG (including Asp254) from the β4 region (highlighted in red in Fig. 1) comprises a typical motif IV signature, when compared with the consensus (D/N/S)PP(F/Y/W/H) pattern identified in a subset of MTases that methylate exocycli" @default.
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• W2016706267 creator A5003280938 @default.
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• W2016706267 title "N2-Methylation of Guanosine at Position 10 in tRNA Is Catalyzed by a THUMP Domain-containing, S-Adenosylmethionine-dependent Methyltransferase, Conserved in Archaea and Eukaryota" @default.
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