Mechanism of repression of methionine biosynthesis in Escherichia coli

Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases. The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone. Now, twodimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo. In E. coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo. These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E. coli but, in general, establish the importance of error-editing mechanisms in living cells. Aminoacylation of tRNA is a two-step reaction. In the first step, an amino acid (AA) is activated to form enzyme (E)-bound aminoacyl adenylate.In the second step, the amino acid is transferred from the adenylate to tRNA.E-AA-AMP + tRNAAA -* E + AAAtRNAAA + AMP Extraordinary fidelity of the tRNA aminoacylation reaction is due to enzymatic proofreading or editing activities of aminoacyl-tRNA synthetases (5, 6). It has been proposed that aminoacyl-tRNA synthetases can exercise kinetic proofreading of incorrect substrates (7,8). Editing can occur by the hydrolysis of the noncognate aminoacyl adenylate (9-11) or the aminoacyl-tRNA (12-15). Both editing mechanisms have been directly demonstrated in vitro. Although it has been inferred from the in vitro studies that editing is also important in vivo, direct evidence for this was missing.The aminoacyl-adenylate proofreading pathway originally discovered with valyl-tRNA synthetase (9, 10) has subsequently been established in vitro to be of major importance with several other synthetases (11). In particular, methionyl-, isoleucyl-, and valyl-tRNA synthetases edited misactivated homocysteine by the adenylate pathway with the formation of homocysteine thiolactone. This feature of the homocysteine editing reaction provides a means to assay for editing in vivo by looking for a special product of editing, i.e., homocysteine thiolactone. I now report that homocysteine thiolactone is in fact synthesized in vivo by the bacterium Escherichia coli. The data indicate that the thiolactone synthesis in E. coli is due to efficient in vivo editing of homocysteine by methionyl-tRNA synthetase, thus establishing the existence and importance of proofreading in vivo. MATERIALS AND METHODSBacterial Strains and Plasmids. Wild-type E. coli K-38 was obtained from N. Zinder (Rockefeller University). E. coli K-12 metE was from N. Brot (Roche Institute of Molecular Biology). The following strains were obtained from the E. coli Genetic Stock Center (Yale University): RK4536 (metE metH); CS50 (metG); CA274 (trp49); AB2575 (ilvD); Hfr3000YA73 (thrB). CR147 was from M. Cashel (National In...

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“…In contrast to what was expected, the methioninestarved metE mutant did not accumulate homocysteine ( Fig. 1 B-D (20). As shown in Fig.…”

contrasting

confidence: 56%

“…Previous attempts to genetically map mutations leading to prototrophy of the metG-strain failed due to the lack of markers in the metG region of the E. coli chromosome (20) and therefore were not repeated here.…”

Section: Methodsmentioning

confidence: 99%

Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli.

Jakubowski

1990

Proc. Natl. Acad. Sci. U.S.A.

132

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“…The metK gene codes for SAM synthetase (1,6,8,9,21), and the metJ gene codes for the aporepressor for the methionine pathway (1,3,8,9). The current model for regulation of the methionine pathway is the interaction of SAM with the MetJ protein to form an activated repressor complex.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine

Plamann

Stauffer

1989

J Bacteriol

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The methionine component of glyA gene regulation in Escherichia coli K-12 was investigated. The results indicate that the glyA gene is positively controlled by the metR gene product. Activation of glyA by the MetR protein requires homocysteine, an intermediate in methionine biosynthesis. The positive-acting metR regulatory system functions independently of a regulatory system shown previously to control glyA gene expression.In Escherichia coli, the glyA gene product, serine hydroxymethyltransferase (SHMT), is responsible for the conversion of serine to glycine and 5,10-methylenetetrahydrofolate. This reaction is the only source of glycine (15) and the major source of one-carbon (C1) units (13) The metJ gene encodes a repressor protein for the methionine biosynthetic genes, and the metK gene product, SAM synthetase, catalyzes the production of SAM, the corepressor for the MetJ protein (17). In E. coli, mnetK mutations result in elevated SHMT levels (7, 12). In both E. co/i and Salmonella typhimurium, metK and metJ mutations prevent complete repression of SHMT synthesis (7,12,21

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“…Methionine auxotrophs were tentatively classified as described previously (1). Cells were grown overnight in LB and washed twice in GM medium, and 0. mutant.…”

Section: Methodsmentioning

confidence: 99%

“…2). The digestion products were run on a 1% low-melting-temperature agarose gel, and the approximately (1). Thus, an initial series of transductions was carried out to determine the status of the metE, metF, and metH genes in strain GS244.…”

Section: Methodsmentioning

confidence: 99%

A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium

Urbanowski¹,

Stauffer²,

Plamann³

et al. 1987

J Bacteriol

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We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that (i) the mutant carries normal metE, metH, and metF genes; (ii) the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and (iii) the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmidborne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.The methylation of homocysteine to form methionine can be carried out by either of two transmethylases in Salmonella typhimurium and Escherichia coli (for a review, see reference 15). The first is a vitamin B12-independent enzyme, the product of the metE gene; the second is a vitamin B12-dependent enzyme, the product of the metH gene. The methyl donor for both enzymes is 5-methyltetrahydrofolate, produced by the metF gene product at a point of convergence of two major pathways, the methionine biosynthetic pathway and the C1 pathway (Fig. 1). The cell regulates the flow of C1 units through this convergence point on several levels to balance the requirements for protein synthesis, methylation reactions, and nucleic acid synthesis.The genes in the nonfolate branch of the methionine pathway (metA, metB, metC, and metK) and those in the folate branch of the pathway (metF, metE, and, to a small extent, metH) are all negatively controlled by the metJ repressor system. In addition, the metH gene product is involved in repression of the metE and metF genes when the cells are grown in medium containing vitamin B12. We report here the finding of a third regulatory mechanism at the methionine-C, convergence point, namely, the positive activation of the metE and metH genes. MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. All bacterial strains used are derivatives of E. coli K-12 and are described in Table 1. Plasmids pGS47 and pGS69, and their metE::Tn5 derivatives have been described previously (16). Plasmid pMC1403 (4) was from M. Casadaban. Bacteriophage Xgt2 (13) was from R. Davis. Plasmid pBR322 has been described previously (9). Plasmid pGS191, the lacZ * Corresponding author. fusion plasmids, and la...

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Mechanism of repression of methionine biosynthesis in Escherichia coli

Cited by 60 publications

References 37 publications

Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli.

Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli.

Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine

A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium

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