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PubMed2002

A human mitochondrial GTP binding protein related to tRNA modification may modulate phenotypic expression of the deafness-associated mitochondrial 12S rRNA mutation.

Human mitochondrial 12S rRNA A1555G mutation has been found to be associated with deafness. However, putative nuclear modifier gene(s) has been proposed to regulate the phenotypic expression of this mutation. In yeast cells, mutant alleles of MSS1, encoding a mitochondrial GTP-binding protein, manifest a respiratory-deficient phenotype only when coupled with mitochondrial 15S rRNA P(R)(454) mutation corresponding to human A1555G mutation. This suggests that an MSS1-like modifier gene may influence the phenotypic expression of the A1555G mutation. We report here the identification and characterization of human MSS1 homolog, GTPBP3, the first identified vertebrate gene related to mitochondrial tRNA modification. The Gtpbp3 is the mitochondrial GTPase evolutionarily conserved from bacteria to mammals. Functional conservation of this protein is supported by the observation that isolated human GTPBP3 cDNA can complement the respiratory-deficient phenotype of yeast mss1 cells carrying P(R)(454) mutation. GTPBP3 is ubiquitously expressed in various tissues as multiple transcripts, but with a markedly elevated expression in tissues of high metabolic rates. We showed that Gtpbp3 localizes in mitochondrion. These observations suggest that the human GTPBP3 is a structural and functional homolog of yeast MSS1. Thus, allelic variants in GTPBP3 could, if they exist, modulate the phenotypic manifestation of human mitochondrial A1555G mutation.

Alleles↗
PubMed2002

Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance.

The truB gene of Escherichia coli encodes the pseudouridine-55 (psi55) synthase and is responsible for modifying all tRNA molecules in the cell at the U55 position. A truB null mutant grew normally on all growth media tested, but exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype could be complemented by both the cloned truB gene and by a D48C, catalytically inactive allele of truB. The truB mutant also exhibited a defect in survival of rapid transfer from 37 to 50 degrees C. This mutant phenotype could be complemented by the cloned truB gene but not by a D48C, catalytically inactive allele of truB. The temperature sensitivity of truB mutants could be enhanced by combination with a mutation in the trmA gene, encoding an m(5)U-methyltransferase, modifying the universal U54 tRNA nucleoside, but not by mutations in trmH, encoding the enzyme catalysing the formation of Gm18. The truB mutant proteome contained altered levels of intermediates involved in biogenesis of the outer-membrane proteins OmpA and OmpX. The truB mutation also reduced the basal expression from two sigma(E) promoters, degP and rpoHP3. Three novel aspects to the phenotype of truB mutants were identified. Importantly the data support the hypothesis that TruB-effected psi55 modification of tRNA is not essential, but contributes to thermal stress tolerance in E. coli, possibly by optimizing the stability of the tRNA population at high temperatures.

Bacterial Outer Membrane Proteins↗
PubMed1997

Preliminary investigation of tRNA modification enzymes with Se in bovine liver.

We measured the amount of Se in the tRNA fractions eluted from a BD-cellulose column. Se was found in the fraction eluted early from the column as to be 3 x 10(-4) mol/mol of tRNA. This low amount suggests that there is no tRNA species that contain 1 mol of Se per 1 mol of tRNA and only few specific tRNAs contain Se-nucleotides. Next, we searched the Se modification enzymes with this tRNA fraction and found the activity in cytosol. Now the digestions of the tRNA modified with 75Se was analyzed by two dimensional TLC. tRNA(Sec) of T7 transcript was not substrate for this enzyme.

Animals↗
PubMed1983

Alteration of tRNA modification in eukaryotes: causes and consequences.

To evaluate the role of the modified nucleosides in tRNA function, especially their involvement in regulatory mechanisms of development, differentiation, or neoplastic transformation we use the following organisms: eubacteria, the slime mold D. discoideum, the topminnow Xiphophorus, and mice. Ribosylthymine, a common modified nucleoside at position 54 in tRNAs of prokaryotes and the major class of eukaryotic elongator tRNAs, is involved in the binding to the ribosomal A-site and is important for the proper functioning of tRNA during translation. Alterations in the extent of this modification occur early in the development of D. discoideum. The fully methylated species are found on polysomes, actively synthesizing protein. The partially methylated tRNAs accumulate in the nuclei, and might be involved in regulatory mechanisms at the transcriptional level. The Q base, a modified deazaguanine derivative, is present at position 34, the first position of the anticodon of tRNAAsn, tRNATyr, and tRNAHis. Alterations in the extent of this modification occur in corresponding tRNAs during the first minutes after the onset of development in D. discoideum and before final differentiation into spores, indicating that Q is important for developmental processes. Changes in the modification of G34 to Q34 in specific tRNAs of the melanophoric system of the topminnow Xiphophorus further support the view that Q is necessary in differentiation. In plasmacytomas and in Ehrlich ascites tumor cells of mice, the amount of unmodified G34 in corresponding tRNAs is correlated to the growth rate, density, or age of the tumor cells.

Animals↗
PubMed1985

Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation.

Previous work indicates that aminoacyl-tRNA synthetases make a transient covalent adduct with cognate tRNAs, through Michael addition of an enzyme nucleophile to the carbon-6 position of uridine 8. We report the selective reduction of the 5,6 double bond of 4-thiouridine at position 8 in Escherichia coli tyrosine tRNA, so as to prevent formation of the presumed covalent enzyme-nucleic acid adduct. The completely reduced tRNA molecules are inactivated for aminoacylation. With partial reduction, a mixed pool of active and inactive molecules is created and the degree of inactivation exactly matches the extent of 4-thiouridine reduction. The active molecules recovered from this mixed pool are specifically unaltered at position 8. The results are consistent with the view that the covalent enzyme-RNA adduct is an obligatory intermediate for aminoacylation of this tRNA.

Borates↗
PubMed1983

Tumor-specific tRNA modifications in mouse plasmacytomas and other tumors.

RPC-5 chromatography has been used to analyze the aa-tRNA populations found in normal organs and in various tumors in experimental animals. The most extensively studied animal systems have been mineral-oil-induced mouse plasmacytomas and carcinogen-induced rat hepatomas. Certain aa-tRNA species appear to be tumor-specific, e.g., rat hepatoma phenylalanyl-tRNA1 and plasmacytoma asparaginyl-tRNAs2-4. In addition, one of the tumor-specific peaks of asparaginyl-tRNA can be found in normal livers of animals bearing plasmacytomas at a distant site. Many other significant quantitative and qualitative differences among histologically similar plasmacytomas and between normal tissues and plasmacytomas were observed in the chromatographic patterns of isoaccepting aa-tRNAs for 11 of 20 amino acids. Some of the qualitative differences in chromatographic patterns could be correlated with the tumorous nature of the tissue using computer analysis. The program utilized cluster analysis to compare the RPC-5 patterns of aa-tRNAs from 11 plasmacytomas and two normal tissues for each of the 20 amino acids. The variations in these chromatographic profiles are though to be caused by varying degrees of incomplete synthesis of some of the normally modified nucleosides in tRNAs.

Amino Acid Sequence↗
PubMed2005

The Cm56 tRNA modification in archaea is catalyzed either by a specific 2'-O-methylase, or a C/D sRNP.

We identified the first archaeal tRNA ribose 2'-O-methylase, aTrm56, belonging to the Cluster of Orthologous Groups (COG) 1303 that contains archaeal genes only. The corresponding protein exhibits a SPOUT S-adenosylmethionine (AdoMet)-dependent methyltransferase domain found in bacterial and yeast G18 tRNA 2'-O-methylases (SpoU, Trm3). We cloned the Pyrococcus abyssi PAB1040 gene belonging to this COG, expressed and purified the corresponding protein, and showed that in vitro, it specifically catalyzes the AdoMet-dependent 2'-O-ribose methylation of C at position 56 in tRNA transcripts. This tRNA methylation is present only in archaea, and the gene for this enzyme is present in all the archaeal genomes sequenced up to now, except in the crenarchaeon Pyrobaculum aerophilum. In this archaea, the C56 2'-O-methylation is provided by a C/D sRNP. Our work is the first demonstration that, within the same kingdom, two different mechanisms are used to modify the same nucleoside in tRNAs.

Amino Acid Sequence↗
PubMed2006

Amount changes of tRNA modification enzymes in Thermus thermophilus HB8 cells according to culture temperatures.

Thermus thermophilus HB8 is an extreme thermophilic eubacrium, which grows at 50-80 degrees C. Transfer RNA molecules in T. thermophilus HB8 contains modified nucleosides such as Gm18, m(7)G46, m(5)s(2)U54, Psi55, and m(1)A58. Recently, all responsible genes for these modifications have been identified. To clarify the relationship of the amounts of the RNA modification enzymes (proteins) and culture conditions, we investigated the activities and quantities of the proteins. In this meeting, we report the amount changes of tRNA (Gm18) methyltransferase [TrmH] in T. thermophilus HB8 cells cultured at 52, 67, 75, and 79 degrees C. 60 mug of total proteins in the crude extract from cells cultured at 67 degrees C contained about 10-30 ng of the TrmH protein. Thus, the content of the TrmH protein was estimated to be around 1/5000 of the total proteins. Through the log phase, the content of the TrmH protein was not changed obviously. However, in the stationary phase, the content of the TrmH protein was slowly reduced. The TrmH contents in the cells cultured at 75 or 79 degrees C were similar to that at 67 degrees C. However, the content of the TrmH protein in the extract from 52 degrees C cultured cells slightly decreased as compared with that in the 67 degrees C cultured cells.

Bacterial Proteins↗
PubMed1990

Role of tRNA modification in translational fidelity.

In transfer RNA many different modified nucleosides are found, especially in the anticodon region. In this region, pseudouridine (psi) is found in positions 38, 39 or 40 in a subset of tRNA species, 2-methylthio-6-hydroxyisopentenyladenosine (ms2io6A) is found in position 37 in tRNAs that read codons starting with U and 1-methylguanosine (m1G) is found in position 37 in tRNAs reading codons of the UCCNG type. We have used the mutants hisT, miaA and miaB and trmD, which are deficient in the biosynthesis of psi, ms2io6A, and m1G, respectively, to study the functional aspects of the respective modified nucleosides. We have shown: (1) Presence of psi improved the cellular growth rate, the polypeptide step-time, and the efficiency of an amber suppressor, but did not appreciably sense the codon context. (2) Presence of ms2io6A improved the cellular growth rate, the polypeptide step-time and the efficiency of several amber suppressor tRNAs. It also had a profound effect on the codon context sensitivity of the tRNA. (3) Presence of m1G improved the cellular growth rate and the polypeptide steptime and also prevented the tRNA from shifting the reading frame. Thus, these three modified nucleosides present in the anticodon region have apparently different functions.

Anticodon↗
PubMed2006

Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli.

In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an alpha2beta2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent.

Antibodies, Bacterial↗
PubMed2000

Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression.

The Saccharomyces cerevisiae Mod5 protein catalyzes isopentenylation of A to i(6)A on tRNAs in the nucleus, cytosol, and mitochondria. The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis. Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i(6)A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression. We devised a colony color/growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression. We identified SAL6, TEF4, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery. We also identified ARC1, whose product interacts with aminoacyl synthetases. Interestingly, we identified ERG20. Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i(6)A on tRNAs. Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate. Identification of ERG20 reveals that yeast have a limited pool of dimethylallyl pyrophosphate. It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.

Alkyl and Aryl Transferases↗
PubMed1993

tRNA modification activity is necessary for Tet(M)-mediated tetracycline resistance.

Tet(M) protein interacts with the protein biosynthetic machinery to render this process resistant to the tetracycline in vivo and in vitro (V. Burdett, J. Biol. Chem. 266:2872-2877, 1991). To understand this process more completely, a mutant of Escherichia coli which is altered in the ability of Tet(M) to confer resistance has been identified. This mutation maps to miaA and displays phenotypes characteristic of previously isolated miaA mutations. The miaA gene product modifies A37 adjacent to the anticodon of several tRNA species. Both the mutant isolated in this work and previously isolated miaA mutants confer tetracycline sensitivity in the presence of functional Tet(M), both share a slow growth phenotype, and in neither case is a wild-type phenotype restored in trans by F'112 carrying the 89- to 98-min region of the chromosome. These similar phenotypes further substantiate the assignment of the mutation described here to the miaA locus.

Bacterial Proteins↗
PubMed2001

A primordial tRNA modification required for the evolution of life?

The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

Amino Acid Sequence↗
PubMed2000

The properties of a tRNA-specific adenosine deaminase from Drosophila melanogaster support an evolutionary link between pre-mRNA editing and tRNA modification.

Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system.

5' Untranslated Regions↗
PubMed1993

Modification of tRNA as a regulatory device.

Our knowledge of the different biological roles of tRNA modification has increased considerably in recent years. Not only have we learned about how modified nucleosides affect the performance of tRNA in translation, but also how they influence regulation of intermediary metabolism, antibiotics production, gene expression in eukaryotic viruses, cell division, cell-cycle control, u.v. sensitivity, and mutation frequency. This review summarizes our current understanding of the role of tRNA modification.

Cell Cycle↗