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1.
Nat Struct Mol Biol ; 24(9): 778-782, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28783151

ABSTRACT

The genetic code is not frozen but still evolving, which can result in the acquisition of 'dialectal' codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non-universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3' adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.


Subject(s)
Genetic Code , Mitochondria/metabolism , RNA Processing, Post-Transcriptional , RNA, Transfer, Lys/metabolism , Sea Urchins/genetics , Sea Urchins/metabolism , Animals , Asparagine/metabolism , Hydroxylation , Lysine/metabolism
2.
Proc Natl Acad Sci U S A ; 114(19): 4954-4959, 2017 05 09.
Article in English | MEDLINE | ID: mdl-28439027

ABSTRACT

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.


Subject(s)
Bacterial Proteins , Iron-Sulfur Proteins , Ligases , Thermus thermophilus , Thiouridine/analogs & derivatives , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalysis , Crystallography, X-Ray , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Ligases/chemistry , Ligases/metabolism , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Thermus thermophilus/chemistry , Thermus thermophilus/metabolism , Thiouridine/chemistry , Thiouridine/metabolism
3.
Biochem J ; 474(6): 957-969, 2017 03 07.
Article in English | MEDLINE | ID: mdl-28130490

ABSTRACT

Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor- and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegansC. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e.g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aa-tRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems.


Subject(s)
Drosophila Proteins/chemistry , Drosophila melanogaster/genetics , Mitochondrial Proteins/chemistry , Peptide Elongation Factor Tu/chemistry , Protein Biosynthesis , RNA, Transfer, Amino Acyl/chemistry , Amino Acid Sequence , Animals , Biological Evolution , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cloning, Molecular , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation , Kinetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Nucleic Acid Conformation , Peptide Elongation Factor Tu/genetics , Peptide Elongation Factor Tu/metabolism , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Transfer, Amino Acyl/genetics , RNA, Transfer, Amino Acyl/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Trichinella/genetics , Trichinella/metabolism
4.
FEBS Lett ; 590(24): 4628-4637, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27878988

ABSTRACT

Incorporation of a sulfur atom into 2-thioribothymidine (s2 T or 5-methyl-2-thiouridine) at position 54 in thermophile tRNA is accomplished by an elaborate system composed of many proteins which confers thermostability to the translation system. We identified ttuD (tRNA-two-thiouridine D) as a gene for the synthesis of s2 T54 in Thermus thermophilus. The rhodanese-like protein TtuD enhances the activity of cysteine desulfurases and receives the persulfide generated by cysteine desulfurases in vitro. TtuD also enhances the formation of thiocarboxylated TtuB, the sulfur donor for the tRNA sulfurtransferase TtuA. Since cysteine desulfurases are the first enzymes in the synthesis of s2 T and other sulfur-containing compounds, TtuD has a role to direct sulfur flow to s2 T synthesis.


Subject(s)
Bacterial Proteins/metabolism , RNA, Transfer/metabolism , Sulfurtransferases/metabolism , Thermus thermophilus/chemistry , Thiouridine/analogs & derivatives , Amino Acid Sequence , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Molecular Mimicry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Transfer/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Sulfurtransferases/genetics , Thermus thermophilus/enzymology , Thiosulfate Sulfurtransferase/genetics , Thiosulfate Sulfurtransferase/metabolism , Thiouridine/metabolism
5.
Genes Cells ; 21(7): 740-54, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27238446

ABSTRACT

TrmFO is a N(5) , N(10) -methylenetetrahydrofolate (CH2 THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5) U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 °C). In T. thermophilus, modified nucleosides in tRNA and modification enzymes form a network, in which one modification regulates the degrees of other modifications and controls the flexibility of tRNA. To clarify the role of m(5) U54 and TrmFO in the network, we constructed the trmFO gene disruptant (∆trmFO) strain of T. thermophilus. Although this strain did not show any growth retardation at 70 °C, it showed a slow-growth phenotype at 50 °C. Nucleoside analysis showed increase in 2'-O-methylguanosine at position 18 and decrease in N(1) -methyladenosine at position 58 in the tRNA mixture from the ∆trmFO strain at 50 °C. These in vivo results were reproduced by in vitro experiments with purified enzymes. Thus, we concluded that the m(5) U54 modification have effects on the other modifications in tRNA through the network at 50 °C. (35) S incorporations into proteins showed that the protein synthesis activity of ∆trmFO strain was inferior to the wild-type strain at 50 °C, suggesting that the growth delay at 50 °C was caused by the inferior protein synthesis activity.


Subject(s)
RNA, Transfer/genetics , tRNA Methyltransferases/genetics , Flavin-Adenine Dinucleotide/genetics , Flavin-Adenine Dinucleotide/metabolism , Folic Acid/genetics , Folic Acid/metabolism , Guanosine/analogs & derivatives , Guanosine/genetics , Mutation , Temperature , Thermus thermophilus/enzymology , Thermus thermophilus/genetics , Uridine/analogs & derivatives , Uridine/genetics , tRNA Methyltransferases/metabolism
6.
Seikagaku ; 86(5): 547, 2014 Oct.
Article in Japanese | MEDLINE | ID: mdl-25509319

Subject(s)
Motivation , Humans
7.
J Biol Chem ; 288(11): 7645-7652, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23362261

ABSTRACT

Non-universal genetic codes are frequently found in animal mitochondrial decoding systems. In squid mitochondria, four codons deviate from the universal genetic code, namely AUA, UGA, and AGA/AGG (AGR) for Met, Trp, and Ser, respectively. To understand the molecular basis for establishing the non-universal genetic code, we isolated and analyzed five mitochondrial tRNAs from a squid, Loligo bleekeri. Primary structures of the isolated tRNAs, including their post-transcriptional modifications, were analyzed by mass spectrometry. tRNA(Met)(AUR) possessed an unmodified cytidine at the first position of the anticodon, suggesting that the AUA codon is deciphered by CAU anticodon via non-canonical A-C pairing. We identified 5-taurinomethyluridine (τm(5)U) at the first position of the anticodon in tRNA(Trp)(UGR). τm(5)U enables tRNA(Trp) to decipher UGR codons as Trp. In addition, 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) was found in mitochondrial tRNAs for Leu(UUR) and Lys in L. bleekeri. This is the first discovery of τm(5)U and τm(5)s(2)U in molluscan mitochondrial tRNAs.


Subject(s)
Loligo/genetics , Mitochondria/metabolism , Amino Acids/chemistry , Animals , Anticodon/genetics , Codon , Cytidine/genetics , Genetic Code , Mass Spectrometry/methods , Models, Genetic , Nucleic Acid Conformation , Nucleosides/metabolism , Protein Structure, Secondary , RNA/metabolism , RNA, Mitochondrial , RNA, Transfer/metabolism
8.
J Nucleic Acids ; 2011: 623095, 2011.
Article in English | MEDLINE | ID: mdl-22007289

ABSTRACT

In animal mitochondria, six codons have been known as nonuniversal genetic codes, which vary in the course of animal evolution. They are UGA (termination codon in the universal genetic code changes to Trp codon in all animal mitochondria), AUA (Ile to Met in most metazoan mitochondria), AAA (Lys to Asn in echinoderm and some platyhelminth mitochondria), AGA/AGG (Arg to Ser in most invertebrate, Arg to Gly in tunicate, and Arg to termination in vertebrate mitochondria), and UAA (termination to Tyr in a planaria and a nematode mitochondria, but conclusive evidence is lacking in this case). We have elucidated that the anticodons of tRNAs deciphering these nonuniversal codons (tRNA(Trp) for UGA, tRNA(Met) for AUA, tRNA(Asn) for AAA, and tRNA(Ser) and tRNA(Gly) for AGA/AGG) are all modified; tRNA(Trp) has 5-carboxymethylaminomethyluridine or 5-taurinomethyluridine, tRNA(Met) has 5-formylcytidine or 5-taurinomethyluridine, tRNA(Ser) has 7-methylguanosine and tRNA(Gly) has 5-taurinomethyluridine in their anticodon wobble position, and tRNA(Asn) has pseudouridine in the anticodon second position. This review aims to clarify the structural relationship between these nonuniversal codons and the corresponding tRNA anticodons including modified nucleosides and to speculate on the possible mechanisms for explaining the evolutional changes of these nonuniversal codons in the course of animal evolution.

9.
J Biol Chem ; 286(41): 35494-35498, 2011 Oct 14.
Article in English | MEDLINE | ID: mdl-21873425

ABSTRACT

Variations in the genetic code are found frequently in mitochondrial decoding systems. Four non-universal genetic codes are employed in ascidian mitochondria: AUA for Met, UGA for Trp, and AGA/AGG(AGR) for Gly. To clarify the decoding mechanism for the non-universal genetic codes, we isolated and analyzed mitochondrial tRNAs for Trp, Met, and Gly from an ascidian, Halocynthia roretzi. Mass spectrometric analysis identified 5-taurinomethyluridine (τm(5)U) at the anticodon wobble positions of tRNA(Met)(AUR), tRNA(Trp)(UGR), and tRNA(Gly)(AGR), suggesting that τm(5)U plays a critical role in the accurate deciphering of all four non-universal codes by preventing the misreading of pyrimidine-ending near-cognate codons (NNY) in their respective family boxes. Acquisition of the wobble modification appears to be a prerequisite for the genetic code alteration.


Subject(s)
Anticodon/metabolism , Mitochondria/metabolism , RNA/metabolism , Taurine/metabolism , Uridine/metabolism , Urochordata/metabolism , Animals , Anticodon/genetics , Mitochondria/genetics , RNA/genetics , RNA, Mitochondrial , Taurine/genetics , Uridine/genetics , Urochordata/genetics
10.
Article in English | MEDLINE | ID: mdl-20075606

ABSTRACT

In animal mitochondria, several codons are non-universal and their meanings differ depending on the species. In addition, the tRNA structures that decipher codons are sometimes unusually truncated. These features seem to be related to the shortening of mitochondrial (mt) genomes, which occurred during the evolution of mitochondria. These organelles probably originated from the endosymbiosis of an aerobic eubacterium into an ancestral eukaryote. It is plausible that these events brought about the various characteristic features of animal mt translation systems, such as genetic code variations, unusually truncated tRNA and rRNA structures, unilateral tRNA recognition mechanisms by aminoacyl-tRNA synthetases, elongation factors and ribosomes, and compensation for RNA deficits by enlarged proteins. In this article, we discuss molecular mechanisms for these phenomena. Finally, we describe human mt diseases that are caused by modification defects in mt tRNAs.


Subject(s)
Genetic Code/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Protein Biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Evolution, Molecular , Humans , Molecular Sequence Data , RNA, Transfer/genetics
11.
Nucleic Acids Res ; 37(5): 1616-27, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19151083

ABSTRACT

Mitochondrial (mt) tRNA(Met) has the unusual modified nucleotide 5-formylcytidine (f(5)C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNA(Met) carrying f(5)C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNA(Met) lacking f(5)C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNA(Met)(m) with the anticodon ac(4)CAU (ac(4)C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNA(Met) (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f(5)C modification in mt tRNA(Met) plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f(5)C-G and f(5)C-A based on the chemical properties of f(5)C are presented.


Subject(s)
Codon/chemistry , Cytidine/analogs & derivatives , Mitochondria/genetics , Protein Biosynthesis , RNA, Transfer, Met/chemistry , RNA/chemistry , Animals , Anticodon/chemistry , Base Pairing , Base Sequence , Cattle , Codon, Initiator/chemistry , Cytidine/chemistry , Escherichia coli/genetics , Methionine/metabolism , Molecular Sequence Data , RNA/metabolism , RNA, Mitochondrial , RNA, Transfer, Met/metabolism , Ribosomes/metabolism
12.
EMBO J ; 27(24): 3267-78, 2008 Dec 17.
Article in English | MEDLINE | ID: mdl-19037260

ABSTRACT

2-Thioribothymidine (s(2)T), a modified uridine, is found at position 54 in transfer RNAs (tRNAs) from several thermophiles; s(2)T stabilizes the L-shaped structure of tRNA and is essential for growth at higher temperatures. Here, we identified an ATPase (tRNA-two-thiouridine C, TtuC) required for the 2-thiolation of s(2)T in Thermus thermophilus and examined in vitro s(2)T formation by TtuC and previously identified s(2)T-biosynthetic proteins (TtuA, TtuB, and cysteine desulphurases). The C-terminal glycine of TtuB is first activated as an acyl-adenylate by TtuC and then thiocarboxylated by cysteine desulphurases. The sulphur atom of thiocarboxylated TtuB is transferred to tRNA by TtuA. In a ttuC mutant of T. thermophilus, not only s(2)T, but also molybdenum cofactor and thiamin were not synthesized, suggesting that TtuC is shared among these biosynthetic pathways. Furthermore, we found that a TtuB-TtuC thioester was formed in vitro, which was similar to the ubiquitin-E1 thioester, a key intermediate in the ubiquitin system. The results are discussed in relation to the mechanism and evolution of the eukaryotic ubiquitin system.


Subject(s)
Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , Coenzymes/biosynthesis , RNA, Transfer/metabolism , Thermus thermophilus/enzymology , Thermus thermophilus/metabolism , Thiouridine/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Gene Deletion , Metalloproteins/biosynthesis , Models, Biological , Molybdenum Cofactors , Pteridines , Thermus thermophilus/genetics , Thiamine/biosynthesis
13.
J Biol Chem ; 283(27): 18801-11, 2008 Jul 04.
Article in English | MEDLINE | ID: mdl-18456657

ABSTRACT

Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (taum(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (DeltamnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U.G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing taum(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the taum(5)U.G base pair does not have classical U.G wobble geometry. These structures provide help to explain how the taum(5)U modification enables efficient decoding of UUG codons.


Subject(s)
Anticodon/chemistry , Base Pairing , Escherichia coli K12/chemistry , RNA, Transfer, Arg/chemistry , Uridine/analogs & derivatives , Uridine/chemistry , Animals , Anticodon/genetics , Anticodon/metabolism , Base Pairing/genetics , Crystallography, X-Ray , Escherichia coli K12/genetics , Escherichia coli K12/metabolism , Mitochondria/chemistry , Mitochondria/genetics , Mitochondria/metabolism , Nucleic Acid Conformation , RNA, Transfer, Arg/genetics , RNA, Transfer, Arg/metabolism , Ribosomes/chemistry , Ribosomes/genetics , Ribosomes/metabolism , Uridine/genetics , Uridine/metabolism
14.
Biochemistry ; 45(36): 10920-7, 2006 Sep 12.
Article in English | MEDLINE | ID: mdl-16953577

ABSTRACT

In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Mitochondrial Proteins/metabolism , Peptide Elongation Factors/metabolism , Serine/metabolism , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans Proteins/genetics , Mitochondrial Proteins/genetics , Molecular Sequence Data , Mutation , Peptide Elongation Factors/genetics , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Substrate Specificity , Thermus thermophilus/genetics
15.
Biochem J ; 399(2): 249-56, 2006 Oct 15.
Article in English | MEDLINE | ID: mdl-16859488

ABSTRACT

Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.


Subject(s)
Ascaris suum , Mitochondria/genetics , Mitochondria/metabolism , Peptide Elongation Factor Tu/metabolism , RNA, Helminth/metabolism , RNA, Transfer, Met/chemistry , RNA, Transfer, Met/metabolism , Amino Acid Sequence , Animals , Ascaris suum/cytology , Ascaris suum/genetics , Base Sequence , Binding Sites , Caenorhabditis elegans/metabolism , Cross-Linking Reagents , Ethylnitrosourea , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Nucleic Acid Conformation , Peptide Elongation Factor Tu/chemistry , RNA, Helminth/chemistry , RNA, Helminth/genetics , RNA, Transfer, Met/genetics , Sequence Alignment , Sequence Deletion
16.
Am J Hum Genet ; 79(2): 291-302, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16826519

ABSTRACT

The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.


Subject(s)
Deafness/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Mutation , Phenotype , RNA, Ribosomal/genetics , RNA, Transfer/metabolism , RNA/genetics , tRNA Methyltransferases/genetics , Amino Acid Sequence , Female , HeLa Cells , Humans , Male , Mitochondrial Proteins/physiology , Molecular Sequence Data , Pedigree , RNA Processing, Post-Transcriptional/genetics , RNA, Mitochondrial , tRNA Methyltransferases/physiology
18.
J Biol Chem ; 281(20): 14296-306, 2006 May 19.
Article in English | MEDLINE | ID: mdl-16547008

ABSTRACT

Thermostability of tRNA in thermophilic bacteria is effected by post-transcriptional modifications, such as 2-thioribothymidine (s2T) at position 54. Using a proteomics approach, we identified two genes (ttuA and ttuB; tRNA-two-thiouridine) that are essential for the synthesis of s2T in Thermus thermophilus. Mutation of either gene completely abolishes thio-modification of s2T, and these mutants exhibit a temperature-sensitive phenotype. These results suggest that bacterial growth at higher temperatures is achieved through the thermal stabilization of tRNA by a 2-thiolation modification. TtuA (TTC0106) is possibly an ATPase possessing a P-loop motif. TtuB (TTC0105) is a putative thio-carrier protein that exhibits significant sequence homology with ThiS of the thiamine synthesis pathway. Both TtuA and TtuB are required for in vitro s2T formation in the presence of cysteine and ATP. The addition of cysteine desulfurases such as IscS (TTC0087) or SufS (TTC1373) enhances the sulfur transfer reaction in vitro.


Subject(s)
Mutation , RNA, Transfer/chemistry , Sulfhydryl Compounds/chemistry , Amino Acid Motifs , Amino Acid Sequence , Base Sequence , Carrier Proteins/chemistry , Cell Proliferation , Escherichia coli Proteins/chemistry , Hot Temperature , Molecular Sequence Data , Nucleic Acid Conformation , Phenotype , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Thermus thermophilus/metabolism
19.
Hum Mol Genet ; 15(6): 897-904, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16446307

ABSTRACT

The A3243G mutation in the mitochondrial gene for human mitochondrial (mt) tRNA(Leu(UUR)), responsible for decoding of UUR codons, is associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). We previously demonstrated that this mutation causes defects in 5-taurinomethyluridine (taum(5)U) modification at the anticodon first (wobble) position of the mutant mt tRNA(Leu(UUR)), leading to a UUG decoding deficiency and entraining severe respiratory defects. In addition, we previously identified a heteroplasmic mutation, G12300A, in the other mt leucine tRNA gene, mt tRNA(Leu(CUN)), which functions as a suppressor of the A3243G respiratory defect in cybrid cells containing A3243G mutant mtDNA. Although the G12300A mutation converts the anticodon sequence of mt tRNA(Leu(CUN)) from UAG to UAA, this tRNA carrying an unmodified wobble uridine still cannot decode the UUG codon. Mass spectrometric analysis of the suppressor mt tRNA(Leu(CUN)) carrying the G12300A mutation from the phenotypically revertant cells revealed that the wobble uridine acquires de novo taum(5)U modification. In vitro translation confirmed the functionality of the suppressor tRNA for decoding UUG codons. These results demonstrate that the acquisition of the wobble modification in another isoacceptor tRNA is critical for suppressing the MELAS mutation, and they highlight the primary role of the UUG decoding deficiency in the molecular pathogenesis of MELAS syndrome.


Subject(s)
MELAS Syndrome/genetics , MELAS Syndrome/metabolism , Point Mutation , RNA, Transfer, Leu/genetics , RNA/genetics , Suppression, Genetic , Uridine/genetics , Adenosine/genetics , Anticodon/genetics , Base Sequence , Cell Line, Tumor , Guanosine/genetics , Humans , MELAS Syndrome/prevention & control , Molecular Sequence Data , Nucleic Acid Conformation , RNA/metabolism , RNA, Mitochondrial , RNA, Transfer, Leu/metabolism , Uridine/analogs & derivatives
20.
J Biol Chem ; 281(4): 2104-13, 2006 Jan 27.
Article in English | MEDLINE | ID: mdl-16317006

ABSTRACT

2-Thioribothymidine (s(2)T) is a modified nucleoside of U, specifically found at position 54 of tRNAs from extreme thermophilic microorganisms. The function of the 2-thiocarbonyl group of s(2)T54 is thermostabilization of the three-dimensional structure of tRNA; however, its biosynthesis has not been clarified until now. Using an in vivo tRNA labeling experiment, we demonstrate that the sulfur atom of s(2)T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s(2)T in vitro, using a cell extract of Thermus thermophilus. Specific 2-thiolation of ribothymidine, at position 54, was observed in vitro, in the presence of ATP. Using this assay, we found a strong temperature dependence of the 2-thiolation reaction in vitro as well as expression of 2-thiolation enzymes in vivo. These results suggest that the variable content of s(2)T in vivo at different temperatures may be explained by the above characteristics of the enzymes responsible for the 2-thiolation reaction. Furthermore, we found that another posttranscriptionally modified nucleoside, 1-methyladenosine at position 58, is required for the efficient 2-thiolation of ribothymidine 54 both in vivo and in vitro.


Subject(s)
RNA, Transfer/genetics , Thermus thermophilus/metabolism , Thiouridine/analogs & derivatives , Adenosine/analogs & derivatives , Adenosine/chemistry , Adenosine Triphosphate/chemistry , Bacterial Proteins/genetics , Binding Sites , Carbon-Sulfur Lyases/genetics , Chromatography, High Pressure Liquid , Culture Media , Cysteine/chemistry , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Lyases/genetics , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oligonucleotides/chemistry , Protein Binding , RNA/chemistry , RNA Processing, Post-Transcriptional , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Recombination, Genetic , Sequence Analysis, RNA , Sulfates/chemistry , Sulfurtransferases/genetics , Temperature , Thiouridine/chemistry , Thiouridine/metabolism , Uridine/analogs & derivatives , Uridine/chemistry , tRNA Methyltransferases/genetics
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