Novel<i>E. coli</i>mutants deficient in blosynthesis of 5-methylamlnomethyl-2-thiouridine

Elseviers, Dirk; Petrullo, Lynn A.; Gallagher, Patricia J.

doi:10.1093/nar/12.8.3521

Cited by 111 publications

(121 citation statements)

References 41 publications

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“…␤-Galactosidase assays were performed on SDS-chloroform-permeabilized cells as described (21). Special precautions for very low ␤-galactosidase activities were taken (2,5).…”

Section: Methodsmentioning

confidence: 99%

“…Null mnmE mutants are defective in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U 34 ) 1 (2), which is found in the wobble position (position 34) of tRNAs that read codons ending with A or G, in the mixed codon family boxes, specifically tRNAs for lysine and glutamic acid (3). The synthesis of mnm 5 s 2 U 34 is complex and requires several enzymatic reactions (4,5), shown below in Reaction 1.…”

mentioning

confidence: 99%

“…The synthesis of mnm 5 s 2 U 34 is complex and requires several enzymatic reactions (4,5), shown below in Reaction 1. The mnmA gene product (6) carries out thiolation in the 2-position of the wobble uridine (U 34 ), whereas mnmE controls the first step of the modification in the 5Ј-position, but it is unclear how many steps precede the formation of cmnm 5 s 2 U 34 (2,4). Several data support that a second gene, named gidA, trmF, or mnmG, is also involved in the cmnm 5 group addition and that the MnmE activity precedes the activity of GidA (2,5).…”

mentioning

confidence: 99%

“…The mnmA gene product (6) carries out thiolation in the 2-position of the wobble uridine (U 34 ), whereas mnmE controls the first step of the modification in the 5Ј-position, but it is unclear how many steps precede the formation of cmnm 5 s 2 U 34 (2,4). Several data support that a second gene, named gidA, trmF, or mnmG, is also involved in the cmnm 5 group addition and that the MnmE activity precedes the activity of GidA (2,5). The mnmC gene product has two enzymatic activities that transform the cmnm 5 intermediate into the final mnm 5 modification (4).…”

mentioning

confidence: 99%

“…The mnmC gene product has two enzymatic activities that transform the cmnm 5 intermediate into the final mnm 5 modification (4). The 2-thio group, but not the cmnm 5 group, is important for aminoacylation of tRNA Glu and tRNA Lys (2,3), whereas modification in both 2-and 5-positions function in the codon recognition process (3,5,(7)(8)(9)(10)(11).…”

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confidence: 99%

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The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Yim¹,

Martínez-Vicente²,

Villarroya³

et al. 2003

Journal of Biological Chemistry

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The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.The evolutionarily conserved MnmE (TrmE) protein of Escherichia coli is a GTPase that differs extensively from regulatory GTPases such as p21 (1). Thus, MnmE exhibits a very high intrinsic GTPase hydrolysis rate and low affinity for GTP and GDP, and it can form self-assemblies. MnmE has a molecular mass of 50 kDa and is organized as a multidomain protein (see Fig. 1) consisting of an ϳ220-amino acid N-terminal domain, probably required for self-assembly, a middle GTPase domain, of about 160 residues, and an ϳ75-amino acid C-terminal domain, which contains the only Cys residue present in the protein. Strikingly, the isolated GTPase domain roughly conserves the guanine nucleotide binding and GTPase activities of the intact MnmE molecule (1).Null mnmE mutants are defective in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U 34 ) 1 (2), which is found in the wobble position (position 34) of tRNAs that read codons ending with A or G, in the mixed codon family boxes, specifically tRNAs for lysine and glutamic acid (3). The mnmA gene product (6) carries out thiolation in the 2-position of the wobble uridine (U 34 ), whereas mnmE controls the first step of the modification in the 5Ј-position, but it is unclear how many steps precede the formation of cmnm 5 s 2 U 34 (2, 4). Several data support that a second gene, named gidA, trmF, or mnmG, is also involved in the cmnm 5 group addition and that the MnmE activity precedes the activity of GidA (2, 5). The mnmC gene product has two enzymatic activities that transform the cm...

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“…␤-Galactosidase assays were performed on SDS-chloroform-permeabilized cells as described (21). Special precautions for very low ␤-galactosidase activities were taken (2,5).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Yim¹,

Martínez-Vicente²,

Villarroya³

et al. 2003

Journal of Biological Chemistry

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Bacterial wobble modifications of NNA‐decoding tRNAs

Nilsson

Alexander

2019

IUBMB Life

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Nucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A‐ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A‐ending codons are not read by U34‐containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A‐ending codons. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1158–1166, 2019

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Crystal structure of the bifunctional tRNA modification enzyme MnmC from Escherichia coli

et al. 2011

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Post-transcriptional modifications of bases within the transfer RNAs (tRNA) anticodon significantly affect the decoding system. In bacteria and eukaryotes, uridines at the wobble position (U34) of some tRNAs are modified to 5-methyluridine derivatives (xm 5 U). These xm 5 U34-containing tRNAs read codons ending with A or G, whereas tRNAs with the unmodified U34 are able to read all four synonymous codons of a family box. In Escherichia coli (E.coli), the bifunctional enzyme MnmC catalyzes the two consecutive reactions that convert 5-carboxymethylaminomethyl uridine (cmnm 5 U) to 5-methylaminomethyl uridine (mnm 5 U). The C-terminal domain of MnmC (MnmC1) is responsible for the flavin adenine dinucleotide (FAD)-dependent deacetylation of cmnm 5 U to 5-aminomethyl uridine (nm 5 U), whereas the N-terminal domain (MnmC2) catalyzes the subsequent S-adenosyl-L-methionine-dependent methylation of nm 5 U, leading to the final product, mnm 5 U34. Here, we determined the crystal structure of E.coli MnmC containing FAD, at 3.0 Å resolution. The structure of the MnmC1 domain can be classified in the FAD-dependent glutathione reductase 2 structural family, including the glycine oxidase ThiO, whereas the MnmC2 domain adopts the canonical class I methyltransferase fold. A structural comparison with ThiO revealed the residues that may be involved in cmnm 5 U recognition, supporting previous mutational analyses. The catalytic sites of the two reactions are both surrounded by conserved basic residues for possible anticodon binding, and are located far away from each other, on opposite sides of the protein. These results suggest that, although the Aya Kitamura and Toru Sengoku contributed equally to this work. MnmC1 and MnmC2 domains are physically linked, they could catalyze the two consecutive reactions in a rather independent manner.

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NovelE. colimutants deficient in blosynthesis of 5-methylamlnomethyl-2-thiouridine

Cited by 111 publications

References 41 publications

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

The GTPase Activity and C-terminal Cysteine of the Escherichia coli MnmE Protein Are Essential for Its tRNA Modifying Function

Bacterial wobble modifications of NNA‐decoding tRNAs

Crystal structure of the bifunctional tRNA modification enzyme MnmC from Escherichia coli

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