共查询到20条相似文献,搜索用时 31 毫秒
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Isma?l Moukadiri M.-José Garzón Glenn R. Bj?rk M.-Eugenia Armengod 《Nucleic acids research》2014,42(4):2602-2623
In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In and , however, cmnm5 appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm5) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm5 into nm5, whereas MnmC(m) converts nm5 into mnm5, thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that and are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm5s2U by MnmEG-MnmC in vivo avoids build-up of intermediates in . We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions. 相似文献
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The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm5s2U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) in tRNA specific for Gln. The sulfur of (c)mnm5s2U may be exchanged by selenium (Se)–a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s2 group of mnm5s2U and of cmnm5s2U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm5ges2U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH+ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm5s2U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA. 相似文献
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Transfer RNA (tRNA) contains a number of complex ‘hypermodified’ nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient ‘assembly line’ that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm5s2U nucleoside in Escherichia coli tRNAGlu, which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a Km value of 600 nM and kcat 0.34 s−1 for the first step, and Km 70 nM and kcat 0.31 s−1 for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm5(s2)U while avoiding build-up of the nm5(s2)U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes. 相似文献
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In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyluridine (mcm5U), 5-carbamoylmethyluridine (ncm5U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um) nucleosides in the anticodon at the wobble position (U34). Earlier we showed that mutants unable to form the side chain at position 5 (ncm5 or mcm5) or lacking sulphur at position 2 (s2) of U34 result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm5 and s2 side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors. 相似文献
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Grazyna Leszczynska Piotr Leonczak Agnieszka Dziergowska Andrzej Malkiewicz 《Nucleosides, nucleotides & nucleic acids》2013,32(11):599-616
In this paper, we discuss the usefulness of reductive amination of 5-formyl-2′,3′-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc)3) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm5(s2)U) and 5-taurinomethyl(-2-thio)uridine (τm5(s2)U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2′,3′-O-isopropylidene-2-thiouridine is also reported. 相似文献
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Christopher D Katanski Christopher P Watkins Wen Zhang Matthew Reyer Samuel Miller Tao Pan 《Nucleic acids research》2022,50(17):e99
Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas in mammals the substrate for Q-modification in tRNA is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing from biological RNA samples. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method periodate-dependent analysis of queuosine and sulfur modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs. 相似文献
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The wobble hypothesis revisited: uridine-5-oxyacetic acid is critical for reading of G-ending codons
According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo5U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo5U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo5U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo5U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo5U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo5U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34. 相似文献
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Debabrata Mandal Caroline K?hrer Dan Su I. Ramesh Babu Clement T.Y. Chan Yuchen Liu Dieter S?ll Paul Blum Masayasu Kuwahara Peter C. Dedon Uttam L. RajBhandary 《RNA (New York, N.Y.)》2014,20(2):177-188
Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes. 相似文献
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Summary The trmC gene, responsible for the formation of 5-methylaminomethyl-2-thiouridine (mnm5s2U) from 2-thiouridine, present in the first position in the anticodon of some tRNAs, has been located at 50.5 min on the Escherichia coli K12 chromosome. Results from transductional mapping suggest that the trmC gene is located counter-clockwise of aroC. A ColE1 hybrid plasmid carrying the aroC
+, trmC
+ and hisT
+ genes was isolated, and the gene order was established, by subcloning, to be hisT-trmC-aroC. The trmC gene is located 1.9 kb from the aroC gene. Two mutations (trmC1 and trmC2) were shown to be recessive, suggesting that the trmC gene is the structural gene for the tRNA-(mnm5s2U)methyltransferase. 相似文献
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Howard M. Laten Jane Harris Cramer Robert H. Rownd 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(1):1-6
By culturing Saccharomyces cerevisiae in growth medium containing Mg35SO4, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)1 is the major, if not only, thiolated derivative in S. cerevisiae tRNA. In addition, a comparison of the chromatographic mobility of mcm5s2Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification. 相似文献
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Marie-Hélène Mazauric Léon Dirick Suresh K. Purushothaman Glenn R. Bj?rk Bruno Lapeyre 《The Journal of biological chemistry》2010,285(24):18505-18515
The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm5U34 and mcm5s2U34), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm5U34 and mcm5s2U34. When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm5U34 on tRNA in vitro but not mcm5s2U34. An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell. 相似文献
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Summary Two mutants of Escherichia coli, trmC1 and trmC2, which are both defective in the synthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U) were utilized to study the function of this complex modified nucleoside. Transfer RNAs specific for glutamine, glutamic acid and lysine as well as a specific ochre suppressor derived from lysine tRNA (tRNA
UAA
lys
encoded by the supG allele), contain this modified nucleoside at position 34 (the wobble position). It was found that two different undermodified derivatives of mnm5s2U were present in the two trmC mutants, which suggests that the two mutations affect two different enzymatic activities. Using the lacI-Z fusion system (Miller and Albertini 1983), we found that the efficiency of supG-mediated suppression was reduced to 30%–90% of the wild-type value in the trmC mutants. The modificationdeficient supG-tRNA in the mutants showed a higher sensitivity to codon context than the normal tRNA
UAA
lys
. 相似文献
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Transfer RNA (tRNA) modifications enhance the efficiency, specificity and fidelity of translation in all organisms. The anticodon modification mcm5s2U34 is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. We determined the in vivo ribosome distributions at single codon resolution in yeast strains lacking mcm5s2U. We found accumulations at AAA, CAA, and GAA codons, suggesting that translation is slow when these codons are in the ribosomal A site, but these changes appeared too small to affect protein output. Instead, we observed activation of the GCN4-mediated stress response by a non-canonical pathway. Thus, loss of mcm5s2U causes global effects on gene expression due to perturbation of cellular signaling. 相似文献