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The wobble bases of bacterial tRNAs responsible for NNR codons are modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U). 2-thio modification of mnm5s2U is required for accurate decoding and essential for normal cell growth. We identified five genes yhhP, yheL, yheM, yheN, and yccK (named tusA, tusB, tusC, tusD, and tusE, respectively) that are essential for 2-thiouridylation of mnm5s2U by a systematic genome-wide screen ("ribonucleome analysis"). Efficient 2-thiouridine formation in vitro was reconstituted with recombinant TusA, a TusBCD complex, TusE, and previously identified IscS and MnmA. The desulfurase activity of IscS is stimulated by TusA binding. IscS transfers the persulfide sulfur to TusA. TusE binds TusBCD complex and stimulates sulfur transfer from TusA to TusD. TusE also interacts with an MnmA-tRNA complex. This study revealed that 2-thiouridine formation proceeds through a complex sulfur-relay system composed of multiple sulfur mediators that select and facilitate specific sulfur flow to 2-thiouridine from various pathways of sulfur trafficking. 相似文献
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Isma?l Moukadiri Silvia Prado Julio Piera Adrián Velázquez-Campoy Glenn R. Bj?rk M.-Eugenia Armengod 《Nucleic acids research》2009,37(21):7177-7193
The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE•GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE•GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins. 相似文献
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Novel methyltransferase for modified uridine residues at the wobble position of tRNA 总被引:1,自引:0,他引:1
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We have identified a novel tRNA methyltransferase in Saccharomyces cerevisiae that we designate Trm9. This enzyme, the product of the YML014w gene, catalyzes the esterification of modified uridine nucleotides, resulting in the formation of 5-methylcarbonylmethyluridine in tRNA(Arg3) and 5-methylcarbonylmethyl-2-thiouridine in tRNA(Glu). In intact yeast cells, disruption of the TRM9 gene results in the complete loss of these modified wobble bases and increased sensitivity at 37 degrees C to paromomycin, a translational inhibitor. These results suggest a role for this potentially reversible methyl esterification reaction when cells are under stress. 相似文献
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We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin. 相似文献
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The uridine at the 34th position of tRNA, which is able to base pair with the 3′-end codon on mRNA, is usually modified to influence many aspects of decoding properties during translation. Derivatives of 5-methyluridine (xm5U), which include methylaminomethyl (mnm-) or carboxymethylaminomethyl (cmnm-) groups at C5 of uracil base, are widely conserved at the 34th position of many prokaryotic tRNAs. In Gram-negative bacteria such as Escherichia coli, a bifunctional MnmC is involved in the last two reactions of the biosynthesis of mnm5(s2)U, in which the enzyme first converts cmnm5(s2)U to 5-aminomethyl-(2-thio)uridine (nm5(s2)U) and subsequently installs the methyl group to complete the formation of mnm5(s2)U. Although mnm5s2U has been identified in tRNAs of Gram-positive bacteria and plants as well, their genomes do not contain an mnmC ortholog and the gene(s) responsible for this modification is unknown. We discovered that MnmM, previously known as YtqB, is the methyltransferase that converts nm5s2U to mnm5s2U in Bacillus subtilis through comparative genomics, gene complementation experiments, and in vitro assays. Furthermore, we determined X-ray crystal structures of MnmM complexed with anticodon stem loop of tRNAGln. The structures provide the molecular basis underlying the importance of U33-nm5s2U34-U35 as the key determinant for the specificity of MnmM. 相似文献
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Jungwook Kim Hui Xiao Junseock Koh Yikai Wang Jeffrey B. Bonanno Keisha Thomas Patricia C. Babbitt Shoshana Brown Young-Sam Lee Steven C. Almo 《Nucleic acids research》2015,43(9):4602-4613
Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons. 相似文献
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Tomoyuki Numata 《Bioscience, biotechnology, and biochemistry》2013,77(3):347-353
Bacteria and archaea have 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm2C or agmatidine), respectively, at the first (wobble) position of the anticodon of the AUA codon-specific tRNAIle. These lysine- or agmatine-conjugated cytidine derivatives are crucial for the precise decoding of the genetic code. L is synthesized by tRNAIle-lysidine synthetase (TilS), which uses l-lysine and ATP as substrates. Agm2C formation is catalyzed by tRNAIle-agm2C synthetase (TiaS), which uses agmatine and ATP for the reaction. Despite the fact that TilS and TiaS synthesize structurally similar cytidine derivatives, these enzymes belong to non-related protein families. Therefore, these enzymes modify the wobble cytidine by distinct catalytic mechanisms, in which TilS activates the C2 carbon of the wobble cytidine by adenylation, while TiaS activates it by phosphorylation. In contrast, TilS and TiaS share similar tRNA recognition mechanisms, in which the enzymes recognize the tRNA acceptor stem to discriminate tRNAIle and tRNAMet. 相似文献
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The title oligoribonucleotides UpCpA; U = s2mcm5U, s2mnm6U, s2U have been synthesized by the condensations of the dimer 10 with phosphodiesters 4, 5 and 6, followed by the two-step deprotection of the fully blocked oligomers 11, 12 and 13, respectively. 相似文献
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A conformational rationale for the wobble behaviour of the first base of the anticodon triplet in tRNA 总被引:1,自引:0,他引:1
We present a conformational rationale for wobble behaviour of the first base in the anticodon triplet of tRNA and hence for the well-known degeneracy of the genetic code. The U-turn hydrogen bond plays an important role in the structure of the anticodon arm and particularly for the anticodon triplet to be in a geometry suitable for the process of recognition in the adaptor-mediated synthesis of proteins. This hydrogen bond in turn precludes a hydrogen bond between the first two sugars of the anticodon triplet, allowing the first base to wobble, while it facilitates one between the second and third sugars of the triplet, positioning these bases for the standard base-pairing with the codon. This neatly explains why there is a degeneracy in the code and why a RNA happens to be the adaptor for protein synthesis. Relevent conformational calculations are presented in support of the theory. 相似文献
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Identification of a gene involved in the generation of 4-thiouridine in tRNA. 总被引:6,自引:0,他引:6
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E G Mueller C J Buck P M Palenchar L E Barnhart J L Paulson 《Nucleic acids research》1998,26(11):2606-2610
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Hen lysozyme single-disulfide variants were constructed to characterize the structures associated with the formation of individual native disulfide bonds. Circular dichroism spectra and the effective concentration of protein thiol groups showed that the propensity for structure formation was relatively high for Cys-6–Cys-127 and Cys-30–Cys-115 disulfides. The urea concentration dependence of individual effective concentrations showed that the apparent sizes of the structures were 14–50% of the whole molecule. The intrinsic stability of each submolecular structure in a reduced form of protein, obtained by subtracting the entropic contribution of cross-linking, was highest for Cys-64–Cys-80 and lowest for Cys-76–Cys-94 disulfide bonds. 相似文献
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Role of modified nucleosides in tRNA: effect of modification of the 2-thiouridine derivative located at the 5''-end of the anticodon of yeast transfer RNA Lys2. 总被引:4,自引:2,他引:2
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Yeast tRNA Lys2 codes preferentially for AAA and contains a 2-thiouridine derivative (U) at the 5'-position of the anticodon. Removal of the 2-thio group from U by treatment with CNBr did not affect the amino acid accepting activity of the modified tRNA Lys2. CNBr treated tRNA Lys2 was active in protein synthesis but with a much reduced efficiency. Although the modified tRNA Lys2 was recognized by elongation factor (EF) T, the EFT dependent binding to ribosomes to tRNA Lys2 (CNBr) was markedly decreased. 相似文献