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1.  The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA 
Nucleic Acids Research  2014;42(12):8073-8082.
Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m5U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N5,N10-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m5U54 tRNA modification. We created single and dual deletions of the trmFO homologs using a novel synthetic biology approach. Subsequent analysis of the M. capricolum RNAs by mass spectrometry shows that the TrmFO homolog encoded by Mcap0476 specifically modifies m5U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.
doi:10.1093/nar/gku518
PMCID: PMC4081110  PMID: 24939895
2.  Predicting the Minimal Translation Apparatus: Lessons from the Reductive Evolution of Mollicutes 
PLoS Genetics  2014;10(5):e1004363.
Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches.
Author Summary
In all cells, proteins are synthesized from the message encoded by mRNA using complex machineries involving many proteins and RNAs. In this process, named translation, the ribosome plays a central role. The elements involved in both ribosome biogenesis and its function are extremely conserved in all organisms from the simplest bacteria to mammalian cells. Most of the 260 known proteins involved in translation have been identified and studied in the bacteria Escherichia coli and Bacillus subtilis, two common cellular models in biology. However, comparative genomics has shown that the translation protein set can be much smaller. This is true for bacteria belonging to the class Mollicutes that are characterized by reduced genomes and hence considered as models for minimal cells. Using homology inference approach and expert analyses, we identified the translation apparatus proteins for 39 of these organisms. Although striking variations were found from one group of species to another, some Mollicutes species require half as many proteins as E. coli or B. subtilis. This analysis allowed us to determine a set of proteins necessary for translation in Mollicutes and define the translation apparatus that would be required in a cellular chassis mimicking a minimal bacterial cell.
doi:10.1371/journal.pgen.1004363
PMCID: PMC4014445  PMID: 24809820
3.  RNA-methyltransferase TrmA is a dual-specific enzyme responsible for C5-methylation of uridine in both tmRNA and tRNA 
RNA Biology  2013;10(4):572-578.
In bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5′ and 3′ ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m5UψC. In tRNAs of gram-negative bacteria, formation of m5U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m5U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m5U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m5U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m5U in RNA was performed by NMR analysis.
doi:10.4161/rna.24327
PMCID: PMC3710363  PMID: 23603891
methylation; methyltransferase; TrmA; NMR; post-transcriptional modification; t-RNA like domain (TLD); tmRNA
4.  Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile 
Nucleic Acids Research  2013;42(3):1904-1915.
Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA2Ile to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNAIle-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA2Ile is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA1Ile, in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.
doi:10.1093/nar/gkt1009
PMCID: PMC3919564  PMID: 24194599
5.  Life without tRNAArg–adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes 
Nucleic Acids Research  2013;41(13):6531-6543.
In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNAArgICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNAArgCCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNAArgCCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNAArg and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNAArgACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNAArgACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNAArgCCG and tadA, and then acquired a new tRNAArgUCG. This permitted further tRNAArgACG mutations with tRNAArgGCG or its disappearance, leaving a single tRNAArgUCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.
doi:10.1093/nar/gkt356
PMCID: PMC3711424  PMID: 23658230
6.  Decoding in Candidatus Riesia pediculicola, close to a minimal tRNA modification set? 
A comparative genomic analysis of the recently sequenced human body louse unicellular endosymbiont Candidatus Riesia pediculicola with a reduced genome (582 Kb), revealed that it is the only known organism that might have lost all post-transcriptional base and ribose modifications of the tRNA body, retaining only modifications of the anticodon-stem-loop essential for mRNA decoding. Such a minimal tRNA modification set was not observed in other insect symbionts or in parasitic unicellular bacteria, such as Mycoplasma genitalium (580 Kb), that have also evolved by considerably reducing their genomes. This could be an example of a minimal tRNA modification set required for life, a question that has been at the center of the field for many years, especially for understanding the emergence and evolution of the genetic code.
PMCID: PMC3539174  PMID: 23308034
tRNA; maturation; translation; modified nucleosides; comparative genomics
7.  MODOMICS: a database of RNA modification pathways—2013 update 
Nucleic Acids Research  2012;41(Database issue):D262-D267.
MODOMICS is a database of RNA modifications that provides comprehensive information concerning the chemical structures of modified ribonucleosides, their biosynthetic pathways, RNA-modifying enzymes and location of modified residues in RNA sequences. In the current database version, accessible at http://modomics.genesilico.pl, we included new features: a census of human and yeast snoRNAs involved in RNA-guided RNA modification, a new section covering the 5′-end capping process, and a catalogue of ‘building blocks’ for chemical synthesis of a large variety of modified nucleosides. The MODOMICS collections of RNA modifications, RNA-modifying enzymes and modified RNAs have been also updated. A number of newly identified modified ribonucleosides and more than one hundred functionally and structurally characterized proteins from various organisms have been added. In the RNA sequences section, snRNAs and snoRNAs with experimentally mapped modified nucleosides have been added and the current collection of rRNA and tRNA sequences has been substantially enlarged. To facilitate literature searches, each record in MODOMICS has been cross-referenced to other databases and to selected key publications. New options for database searching and querying have been implemented, including a BLAST search of protein sequences and a PARALIGN search of the collected nucleic acid sequences.
doi:10.1093/nar/gks1007
PMCID: PMC3531130  PMID: 23118484
8.  Characterization and Structure of the Aquifex aeolicus Protein DUF752 
The Journal of Biological Chemistry  2012;287(52):43950-43960.
Background: Escherichia coli encodes a bifunctional oxidase/methyltransferase catalyzing the final steps of methylaminomethyluridine (mnm5U) formation in tRNA wobble positions.
Results: Aquifex aeolicus encodes only a monofunctional aminomethyluridine-dependent methyltransferase, lacking the oxidase domain.
Conclusion: An alternative pathway exists for mnm5U biogenesis.
Significance: Information about how an organism modifies the wobble base of its tRNA is important for understanding the emergence of the genetic code.
Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm5U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm5U34) by the MnmE-MnmG enzyme complex. The cmnm5U34 is further modified to mnm5U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm5U into 5-aminomethyluridine (nm5U34), and this reaction is immediately followed by the methylation of the free amino group into mnm5U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm5U in vitro, to form mnm5U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm5U34 in tRNAs.
doi:10.1074/jbc.M112.409300
PMCID: PMC3527978  PMID: 23091054
Crystal Structure; RNA Methyltransferase; RNA Modification; S-Adenosylmethionine (AdoMet); Transfer RNA (tRNA); Genetic Code; tRNA Anticodon; Wobble Uridine
9.  A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA 
Nucleic Acids Research  2011;39(21):9368-9375.
Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m5U modifications. However, as previously shown, the m5U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m5U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m5U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.
doi:10.1093/nar/gkr626
PMCID: PMC3241648  PMID: 21824914
10.  MODOMICS: a database of RNA modification pathways. 2008 update 
Nucleic Acids Research  2008;37(Database issue):D118-D121.
MODOMICS, a database devoted to the systems biology of RNA modification, has been subjected to substantial improvements. It provides comprehensive information on the chemical structure of modified nucleosides, pathways of their biosynthesis, sequences of RNAs containing these modifications and RNA-modifying enzymes. MODOMICS also provides cross-references to other databases and to literature. In addition to the previously available manually curated tRNA sequences from a few model organisms, we have now included additional tRNAs and rRNAs, and all RNAs with 3D structures in the Nucleic Acid Database, in which modified nucleosides are present. In total, 3460 modified bases in RNA sequences of different organisms have been annotated. New RNA-modifying enzymes have been also added. The current collection of enzymes includes mainly proteins for the model organisms Escherichia coli and Saccharomyces cerevisiae, and is currently being expanded to include proteins from other organisms, in particular Archaea and Homo sapiens. For enzymes with known structures, links are provided to the corresponding Protein Data Bank entries, while for many others homology models have been created. Many new options for database searching and querying have been included. MODOMICS can be accessed at http://genesilico.pl/modomics.
doi:10.1093/nar/gkn710
PMCID: PMC2686465  PMID: 18854352
11.  RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes 
BMC Genomics  2008;9:470.
Background
Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes.
Results
By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original predictions.
Conclusion
The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation.
doi:10.1186/1471-2164-9-470
PMCID: PMC2584109  PMID: 18844986
12.  The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity 
Nucleic Acids Research  2008;36(15):4929-4940.
The 5-methyluridine is invariably found at position 54 in the TΨC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase (PabTrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of PabTrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 Å resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of PabTrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by PabTrmU54. These results were used to model yeast tRNAAsp in the PabTrmU54 structure to get further insights into the different RNA specificities of RumA and PabTrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to PabTrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TΨC loop conformational change has to occur for the flipping of the target uridine into the PabTrmU54 active site during catalysis.
doi:10.1093/nar/gkn437
PMCID: PMC2528175  PMID: 18653523
13.  Formation of the conserved pseudouridine at position 55 in archaeal tRNA 
Nucleic Acids Research  2006;34(15):4293-4301.
Pseudouridine (Ψ) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Ψ55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are pfuCbf5, a protein known to play a role in RNA-guided modification of rRNA, and pfuPsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. pfuPsuX is hereafter renamed pfuPus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, pfuPus10 efficiently complements an Escherichia coli strain deficient in the bacterial Ψ55 synthase TruB. These results indicate that it is probable that pfuCbf5 or pfuPus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.
doi:10.1093/nar/gkl530
PMCID: PMC1616971  PMID: 16920741
14.  THUMP from archaeal tRNA:m22G10 methyltransferase, a genuine autonomously folding domain 
Nucleic Acids Research  2006;34(9):2483-2494.
The tRNA:m22G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2,N2-dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)—containing N-terminal domain [1–152] and C-terminal catalytic domain [157–329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPα) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPα and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis). A comparative model of THUMPα structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNAAsp substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.
doi:10.1093/nar/gkl145
PMCID: PMC1459410  PMID: 16687654
15.  The RNA polymerase III-dependent family of genes in hemiascomycetes: comparative RNomics, decoding strategies, transcription and evolutionary implications 
Nucleic Acids Research  2006;34(6):1816-1835.
We present the first comprehensive analysis of RNA polymerase III (Pol III) transcribed genes in ten yeast genomes. This set includes all tRNA genes (tDNA) and genes coding for SNR6 (U6), SNR52, SCR1 and RPR1 RNA in the nine hemiascomycetes Saccharomyces cerevisiae, Saccharomyces castellii, Candida glabrata, Kluyveromyces waltii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and the archiascomycete Schizosaccharomyces pombe. We systematically analysed sequence specificities of tRNA genes, polymorphism, variability of introns, gene redundancy and gene clustering. Analysis of decoding strategies showed that yeasts close to S.cerevisiae use bacterial decoding rules to read the Leu CUN and Arg CGN codons, in contrast to all other known Eukaryotes. In D.hansenii and C.albicans, we identified a novel tDNA-Leu (AAG), reading the Leu CUU/CUC/CUA codons with an unusual G at position 32. A systematic ‘p-distance tree’ using the 60 variable positions of the tRNA molecule revealed that most tDNAs cluster into amino acid-specific sub-trees, suggesting that, within hemiascomycetes, orthologous tDNAs are more closely related than paralogs. We finally determined the bipartite A- and B-box sequences recognized by TFIIIC. These minimal sequences are nearly conserved throughout hemiascomycetes and were satisfactorily retrieved at appropriate locations in other Pol III genes.
doi:10.1093/nar/gkl085
PMCID: PMC1447645  PMID: 16600899
16.  MODOMICS: a database of RNA modification pathways 
Nucleic Acids Research  2005;34(Database issue):D145-D149.
MODOMICS is the first comprehensive database resource for systems biology of RNA modification. It integrates information about the chemical structure of modified nucleosides, their localization in RNA sequences, pathways of their biosynthesis and enzymes that carry out the respective reactions. MODOMICS also provides literature information, and links to other databases, including the available protein sequence and structure data. The current list of modifications and pathways is comprehensive, while the dataset of enzymes is limited to Escherichia coli and Saccharomyces cerevisiae and sequence alignments are presented only for tRNAs from these organisms. RNAs and enzymes from other organisms will be included in the near future. MODOMICS can be queried by the type of nucleoside (e.g. A, G, C, U, I, m1A, nm5s2U, etc.), type of RNA, position of a particular nucleoside, type of reaction (e.g. methylation, thiolation, deamination, etc.) and name or sequence of an enzyme of interest. Options for data presentation include graphs of pathways involving the query nucleoside, multiple sequence alignments of RNA sequences and tabular forms with enzyme and literature data. The contents of MODOMICS can be accessed through the World Wide Web at .
doi:10.1093/nar/gkj084
PMCID: PMC1347447  PMID: 16381833
17.  Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria—evolutionary implications 
Nucleic Acids Research  2005;33(13):3955-3964.
Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA:m5U-54 MTase). In all Eukarya and many Gram-negative Bacteria, the methyl donor for this reaction is S-adenosyl-l-methionine (S-AdoMet), while in several Gram-positive Bacteria, the source of carbon is N5, N10-methylenetetrahydrofolate (CH2H4folate). We have identified the gene for Bacillus subtilis tRNA:m5U-54 MTase. The encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m5U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate. This gene is currently annotated gid in Genome Data Banks and it is here renamed trmFO. TrmFO (Gid) orthologs have also been identified in many other bacterial genomes and comparison of their amino acid sequences reveals that they are phylogenetically distinct from either ThyA or ThyX class of thymidylate synthases, which catalyze folate-dependent formation of deoxyribothymine monophosphate, the universal DNA precursor.
doi:10.1093/nar/gki703
PMCID: PMC1178002  PMID: 16027442
18.  Trm11p and Trm112p Are both Required for the Formation of 2-Methylguanosine at Position 10 in Yeast tRNA†  
Molecular and Cellular Biology  2005;25(11):4359-4370.
N2-Monomethylguanosine-10 (m2G10) and N2,N2-dimethylguanosine-26 (m22G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m22G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m2G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m2G10 and m22G26 affects tRNA metabolism or functioning.
doi:10.1128/MCB.25.11.4359-4370.2005
PMCID: PMC1140639  PMID: 15899842
19.  A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase 
Nucleic Acids Research  2004;32(2):465-476.
The modified nucleoside 1-methyladenosine (m1A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m1A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m1A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m1I57). In yeast, the formation of m1A58 is catalysed by the essential tRNA (m1A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m1A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m1A MTases is discussed in the context of different hypotheses of the tree of life.
doi:10.1093/nar/gkh191
PMCID: PMC373318  PMID: 14739239
20.  Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures 
Nucleic Acids Research  2003;31(8):2148-2156.
N1-methyladenosine (m1A) is found at position 58 in the T-loop of many tRNAs. In yeast, the formation of this modified nucleoside is catalyzed by the essential tRNA (m1A58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p). In this report we describe the cloning, expression and characterization of a Gcd14p homolog from the hyperthermophilic bacterium Thermus thermophilus. The purified recombinant enzyme behaves as a homotetramer of ∼150 kDa by gel filtration and catalyzes the site- specific formation of m1A at position 58 of the T-loop of tRNA in the absence of any other complementary protein. S-adenosylmethionine is used as donor of the methyl group. Thus, we propose to name the bacterial enzyme TrmI and accordingly its structural gene trmI. These results provide a key evolutionary link between the functionally characterized two-component eukaryotic enzyme and the recently described crystal structure of an uncharacterized, putative homotetrameric methyltransferase Rv2118c from Mycobacterium tuberculosis. Interest ingly, inactivation of the T.thermophilus trmI gene results in a thermosensitive phenotype (growth defect at 80°C), which suggests a role of the N1-methylation of tRNA adenosine-58 in adaptation of life to extreme temperatures.
PMCID: PMC153742  PMID: 12682365
21.  Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p 
Nucleic Acids Research  2000;28(23):4604-4610.
Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S.cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.
PMCID: PMC115158  PMID: 11095668
22.  Pseudouridine Mapping in the Saccharomyces cerevisiae Spliceosomal U Small Nuclear RNAs (snRNAs) Reveals that Pseudouridine Synthase Pus1p Exhibits a Dual Substrate Specificity for U2 snRNA and tRNA 
Molecular and Cellular Biology  1999;19(3):2142-2154.
Pseudouridine (Ψ) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Ψ residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Ψ content, only the loss of the Pus1p activity was found to affect Ψ formation in spliceosomal UsnRNAs. Indeed, Ψ44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Ψ44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Ψ content, formation of Ψ residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.
PMCID: PMC84007  PMID: 10022901

Results 1-22 (22)