Proc. Nad. Acad. Sci. USAVol. 86, pp. 4407-4411, June 1989Biochemistry

Methionine synthesis in Escherichia coli: Effect of the MetR proteinon metE and metH expressionXIAO-YAN CAI*, MARY E. MAXON*, BETTY REDFIELD*, ROBERT GLASSt, NATHAN BROT*,AND HERBERT WEISSBACH**Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110; and tDepartment of Biochemistry, Queen's Medical Centre, Nottingham,NG7 2UH England

Contributed by Herbert Weissbach, March 15, 1989

ABSTRACT Studies by Urbanowski et al. [Urbanowski,M. L., Stauffer, L. T., Plamann, L. S. & Stauffer, G. V.(1987) J. Bacteriol. 169, 1391-1397] have identified a regula-tory locus, called metR, required for the expression of the metEand metH genes. We recently purified the MetR protein fromEscherichia coli and showed that it could stimulate the in vitroexpression of the metE gene and autoregulate its own synthesis.In the present study, the purified MetR protein has been shownto stimulate the in vitro expression of the metH gene. Also, thein vitro synthesized MetE, MetH, and MetR proteins wereshown to be biologically active. The transcription start sites forthe metE and metR genes have been determined, and DNAfootprinting experiments have identified regions in the metE-metR intergenic sequence that are protected by either the MetRor MetJ proteins.

The terminal reaction in the biosynthesis of methionine inEscherichia coli involves the transfer of a methyl group fromN5-methyltetrahydrofolic acid to homocysteine. This reac-tion can be catalyzed by either oftwo enzymes with distinctlydifferent characteristics (for a review, see ref. 1). The first isthe product of the metE gene and is a vitamin B12-in-dependent enzyme, whereas the second enzyme containsvitamin B12 as a prosthetic group and is the product of themetH gene. Both enzymes have been purified (2, 3) and theirrespective genes cloned (4-7). It is well documented that theexpression of the metE gene, and other met genes with theexception of metH, are repressed by the addition of highlevels of methionine to the growth medium (8-14). Thisrepression requires the MetJ protein and a corepressor,S-adenosylmethionine (AdoMet) (15-19). In addition, it isknown that vitamin B12 can repress in vivo the synthesis ofboth the MetE and MetF enzymes (8, 9), and this is believedto be mediated by the MetH holoenzyme (9). Recently, amethionine regulatory locus, metR, has been described inboth E. coli and Salmonella typhimurium (14). The results ofthese genetic experiments indicated that metR encodes aprotein that is required for the expression of the metE geneand to a lesser extent, the metH gene. We have recentlypurified the MetR protein to near homogeneity and showed,in vitro, that the MetR protein can markedly increase thesynthesis of MetE from a plasmid template and autoregulateits own synthesis (20). The present study describes in vitroexperiments, which show that the MetR protein is also atransactivator ofmetH expression and that the MetE, MetH,and MetR proteins that are synthesized in vitro are biologi-cally active. In addition, the transcription start sites for themetE and metR genes have been identified as well as the

DNA binding sites in the metE-metR intergenic region for theMetR and MetJ proteins.

MATERIALS AND METHODSE. coli strain RK4536 (metE, metH) was obtained from the E.coli Genetic Stock Center (Yale University) and GS244(metR, pheA905, thi) from G. Stauffer (14). The constructionof plasmids pRSE562 (metE and metR genes), and pQN1011,(metHgene) have been described (7, 20). The MetJ and MetRproteins were purified as described (15, 20) and AdoMet wasobtained from Sigma and further purified (21). Methyl-B12was synthesized from vitamin B12 (Sigma) as describedelsewhere (22). Pteroyl-y-glutamyl-y-glutamic acid (folate-Glu3) was kindly provided by L. Ellenbogen (Lederle Lab-oratories). DL-H4folate-Glu3-and DL-N5-[14C]methyl-H4folate-Glu3 were prepared as described (23, 24). DL-N5-[14CJmethyl-H4folate-Glul was purchased fromAmersham. Protein was determined by the method ofLowryet al. (25).DNA-Directed in Vitro Protein Synthesis and Biological

Activity of in Vitro Synthesized Products. An S-30 extract wasprepared from E. coli RK4536 as described by Zubay et al.(26) and used for the DNA directed in vitro protein synthesissystem. Each reaction mixture (35,l) contained S-30 (260,ugof protein), other components as described (27), and eitherplasmid pRSE562 (1.5 ttg) or pQN1011 (1.2 ,ug).[35S]Methionine (15,Ci; 1100 Ci/mmol; 1 Ci = 37 GBq) wasused in those experiments in which the radioactive productwas separated by gel analysis and unlabeled methionine wasused when the product was assayed for biological activity.For the latter studies, 20-,ul aliquots of the incubation mix-tures were assayed for MetE or MetH enzymatic activity asdescribed (2,3), except that methyl-B12 was used in the MetHassay. The biological activity ofthe in vitro synthesized MetRwas based on its ability to stimulate the expression of metHin vitro. For these experiments, two consecutive proteinsynthesis incubations were used. In the first incubation,MetR was synthesized from plasmid pRSE562 (1.5 ,ug) in areaction mixture (35 ,ul, 20 min, 37°C) containing the S-30protein synthesis system described above. The contents ofthis incubation were then transferred to a second 35-,lI S-30protein synthesis reaction mixture containing 2.4 ,ug of plas-mid pQN1011 (metH). After incubation for an additional 20min at 37°C, a 20-IAl aliquot was removed for the determina-tion of MetH enzyme activity.

Transcription Start Site Mapping and DNA Footprinting.Total cell RNA was prepared from either GS244 cells that hadbeen transformed with pRSE562 or from E. coli B as de-scribed (28). Deoxynucleotide primers were chemically syn-thesized (Applied Biosystems 380B) complementary to the 5'

Abbreviation: AdoMet, S-adenosylmethionine.

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Proc. Natl. Acad. Sci. USA 86 (1989)

ends of the metE and metR mRNAs and end-labeled with[y-32PJATP. The transcriptional start sites for the metE andmetR genes were determined by the primer-extension pro-

cedure as described by Berthiolet et al. (29). DNA for MetRfootprinting was prepared from pRSE562. The plasmid was

digested with restriction enzymes Sma I and Bgl II to liberatea 710-base-pair (bp) restriction fragment that contained themajority of the metR gene and 162 bp of upstream DNA. Thisfragment was isolated from an agarose gel and further di-gested with Sty I to yield a smaller fragment of 290 bpcontaining 162 bp of upstream DNA and 128 bp of DNAencoding the 5' end of metR. Reverse transcriptase was usedto end label the Sty I 5' overhang. For a calibration ladder,an aliquot of the labeled DNA was treated by the Maxam-Gilbert cleavage method (30). DNA for MetJ footprinting wasprepared from plasmid pMM450, which contains the metE-metR intergenic sequence upstream of the 83-galactosidasecoding sequence. To obtain pMM450, the vector pMC1403(31) was digested with Sma I to allow the insertion of theintergenic sequence from pRSE562. This construction placedthe intergenic sequence between two unique restriction sites,BamHI and EcoRI. For footprinting, the BamHI/EcoRIfragment was purified from an agarose gel. Reverse tran-scriptase was used to end label the fragment, and a secondarycleavage was performed with Nru I to generate an -292-bpfragment, labeled at the BamHI (metR) end. This fragmentcontains 50 bp of DNA encoding metE, the metE-metRintergenic region, and 6 bp of DNA encoding metR. Thelabeled DNA fragments for MetR and MetJ binding were

used for DNase I protection footprinting experiments by a

modified procedure of Galas and Schmitz (32).

RESULTSEffect of MetR Protein on metE and metH Expression. Fig.

1 is an autoradiogram showing the effect of the purified MetRprotein on the expression of both the metE and metH genes

in a DNA-directed protein synthesis system. As shownpreviously (20), using plasmid pRSE562 as template, theMetR protein markedly stimulates the synthesis of MetE

MetE

MetR-w

Metil

GuiX

* ff: s

fim::'::,

1 2 3 4 5 6 7 8

FIG. 1. DNA-directed in vitro synthesis of MetE and MetHproteins. Plasmids containing the metE and metH genes were incu-bated in an S-30 extract containing [35S]methionine as described.MetR protein (2 qg), MetJ protein (0.4 Jug), and AdoMet (50 ,uM)were added as indicated. Aliquots were subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiographed. Lanes 1-4, plasmid pRSE562 (metE); lane 1, no addition; lane 2, MetJ proteinand AdoMet; lane 3, MetR protein; lane 4, MetR protein, MetJprotein, and AdoMet; lanes 5-8, plasmid pQN1011 (metH); lane 5,no addition; lane 6, MetJ protein and AdoMet; lane 7, MetR protein;lane 8, MetR protein, MetJ protein, and AdoMet. Lanes 1-4 are

similar to results reported previously (20).

(lane 1 vs. lane 3), and the MetJ protein and AdoMet inhibitthe expression of the metE and metR genes both in theabsence and presence of MetR (lanes 2 and 4). Similar to theresults of metE expression, MetR greatly stimulates thesynthesis of MetH using plasmid pQN1011 as template (lane5 vs. lane 7). However, in contrast to metE expression, theMetJ protein and AdoMet have no effect on MetH synthe-sized either in the absence or presence of MetR (lanes 6 and8). Other experiments showed that the MetR protein had noeffect on the in vitro synthesis of MetF (data not shown). Itshould be noted (lanes 1-4) that although plasmid pRSE562directs the synthesis of the MetR protein there is still a largestimulation of metE expression by added MetR protein. Itappears that the amount of MetR synthesized in vitro is toolow to have a marked effect on metE expression (see below).

Activity of in Vitro Synthesized MetE, MetH, and MetR. Inan attempt to simplify the assay and to quantify the amountsofMetE and MetH synthesized in vitro, advantage was takenof the catalytic properties of these proteins. Table 1 showsthe results of a typical experiment in which an S-30 extractprepared from a metE metH mutant was incubated witheither plasmid pQN1011 (metH) or pRSE562 (metE + metR)and other components, as indicated, and then assayed forMetE and MetH activity (2, 3). It is shown in Table 1 that theMetE and MetH proteins synthesized in vitro are enzymat-ically active (line 1). When purified MetR protein is added tothese incubations, there are 16- and 8-fold increases in thelevels ofMetE and MetH activity, respectively (line 1 vs. line2). Although MetJ and AdoMet have little effect on thesynthesis of MetH (line 1 vs. line 3), they inhibit the expres-sion of the metE gene by 75% (line 1 vs. line 3). The additionof MetJ and AdoMet to incubations containing MetR alsoresults in a 75% inhibition of metE expression but no effecton MetH expression (line 2 vs. line 4). The data in Table 1confirm the results obtained by gel analysis (Fig. 1). Usingthis quantitative enzymatic assay, the effect of MetR con-centration on MetE and MetH synthesis was studied. Fig. 2shows that 0.8-1.0 ,.g of purified MetR protein is sufficientto give maximal MetH synthesis, whereas 4-5 times moreMetR protein is required for MetE synthesis.To determine whether the MetR synthesized in vitro was

biologically active as a transactivator of metH expression, atwo-step incubation was used as described in Materials andMethods. Table 2 shows that when pRSE562 containing themetR gene is present in the first incubation, there is about a4.5-fold increase in the level of MetH synthesized from themetHplasmid during the second incubation (line 2 vs. line 1).This can be compared to a 7-fold increase in activity when asaturating amount of purified MetR protein is present in thefirst incubation (line 3). If the MetR protein synthesized frompRSE562 was responsible for the observed increase in MetHsynthesis, the MetJ protein and AdoMet, if added to the firstincubation, should inhibit the increase by preventing MetRsynthesis. This is the case, as shown in line 4. These results

Table 1. Enzymatic activity of MetE and MetH synthesizedin vitro

Addition MetE, units MetH, units x l0o3None 43 0.83MetR 707 6.7MetJ, AdoMet 12 0.65MetR, MetJ, AdoMet 170 6.5

Plasmids pRSE562 and pQN1011 were incubated in an S-30 proteinsynthesis system. Where indicated, MetR protein (2 /Lg), MetJprotein (0.4 pg), and AdoMet (50 ,uM) were added. At the end of theincubation, aliquots were removed and assayed for MetE and MetHactivity (2, 3). The values represent total units of enzymatic activityper 35 ttl of incubation mixture. A unit is defined as pmol ofmethionine formed in a 60-min (MetE) or 30-min (MetH) incubation.

4408 Biochemistry: Cai et al.

Proc. NatL. Acad. Sci. USA 86 (1989) 4409

-

-._

I0.

,Ug MetR

FIG. 2. Effect of MetR concentration on the expression of themetE and metH genes. Various amounts of purified MetR proteinwere added to the S-30 protein synthesis system containing plasmidspRSE562 (metE) or pQN1011 (metH). After 20 min at 37°C, aliquotswere removed and assayed for MetE and MetH activity. The valuesrepresent total enzyme units per 35-,1u incubation mixture.

indicate that the MetR protein synthesized in vitro fromplasmid pRSE562 is biologically active.

Transcription Start Sites. The in vivo transcription startsites for both metE and metR were determined by using aprimer-extension method (29). Fig. 3 shows that a single startsite was found for metE (lane E) located 169 nucleotidesupstream of the translation start site of the protein anddesignated + 1 in Fig. 5 (see below). In contrast, two startsites (RP1 and RP2) of equal intensity were found for metR(lane R), located at -29 and -47, respectively, upstream ofthe metE transcription start site (see Fig. 5). The metE andmetR start sites are located very close to those previouslyreported for S. typhimurium (33), although in the latterorganism the two start sites for metR were separated by onlytwo bases.DNA Footprinting. DNA footprinting experiments were

performed to identify regions that could be protected by theMetR or MetJ proteins against DNase I digestion (see Ma-terials and Methods). As shown in Fig. 4, a stretch of ::w24nucleotides is protected by 350 ng of the MetR protein (lane1 vs. lane 2), corresponding to positions -49 to -72 in themetE-metR intergenic region as summarized in Fig. 5. Fig. 4also shows that when 400 ng of MetJ protein is used, a regionof -35 nucleotides (-8 to +27) ofDNA is protected (lane 3

Table 2. Ability of in vitro synthesized MetR protein to stimulatemetH expression

Addition to first incubation MetH, units x 10-3None 0.74pRSE562 (metR gene) 3.3MetR protein 5.2pRSE562, MetJ, AdoMet 0.76

The reaction was carried out using two consecutive incubations.pRSE562 (1.5 ug), purified MetR protein (1.4 j&g), purified MetJprotein (0.4 ,.g), and AdoMet (50 IAM) were added to the firstincubation, as indicated, in addition to the S-30 extract and othercomponents in a total volume of 35 ILI (see Materials and Methodsand ref. 26). After a 20-min incubation at 37TC, the contents of thisincubation were added to a second S-30 reaction mixture (35 IL1)containing plasmid pQN1011 (metH). After further incubation at370C for 20 min, aliquots were removed and assayed for MetHactivity. The values represent total activity per 70 jA of incubationmixture. Other details are described in Materials and Methods.

--EP0-._

w04)

--RP

-- RP

E R

FiG. 3. Primer-extension mapping of the 5' ends of the metE andmetR mRNA. Total cellularRNA extracted from GS244 transformedwith pRSE562 or E. coli B was used for mapping ofthe 5' ends of themetR and metE transcripts, respectively. After hybridization andreverse transcription, the reaction mixtures were extracted withphenol/chloroform, precipitated with ethanol, and subjected toelectrophoresis (29). Left lane, sequencing ladder; lane E, localiza-tion of the 5' end of metE mRNA (EP); lane R, localization of the 5'ends of the metR mRNA (RP1, RP2).

vs. lane 4). However, in the presence of AdoMet, anotherarea becomes protected (lane 5 vs. lane 4) at positions +102to +137 (see Fig. 5). Although not shown, when the amountof MetJ protein used for protection was decreased to 50 ng,

--7'I.

-

ft_6 -.--49

_ '-f

im

!

.WA__

1 2

E_,| +137

-t102

- #27

118EEuB

3 4 5

FIG. 4. DNase I footprint of the binding of MetR and MetJproteins to the metE-metR intergenic region. The DNA fragments forthe footprinting experiments were isolated and labeled as describedin Materials and Methods. The DNA was incubated with eitherMetRor MetJ protein, treated with DNase I, and subjected to electropho-resis on an 8% polyacrylamide sequencing gel. Lane 1, MetRomitted; lane 2, 350 ng of MetR; lane 3, MetJ omitted; lane 4, 400 ngof Med; lane 5, 400 ng of Med, 50 ,uM AdoMet.

Biochemistry: Cai et al.

Proc. Natl. Acad. Sci. USA 86 (1989)

< - metR RP2 _-

TTCG AT-72 MetR Biniang

GAATAATTTG

r- EP

RP, m

CGCTTGAQGA-29

ATATACAGTA ACCGCCAAI

X TCGTCT TTTAAATTTA+27

TCACTTACTT CAGTAAGCTC

AAATTCAAAA TCC TAGGAT+1t

ATGGATGTGT AAACATCTGGB+1 Met) Binding

ACGGCTAAAA

TGGTGCGTTG GCTGCGTTTC TCCACCCCGG

CCGGGG GA ATAAACTTGC CGCCTTCCCT+102 MetJ BindingmTtECT

TTACATATAA TTAGAGGAAG AAAAAATGACAATA

FIG. 5. Summary of regulatory regions for metE and metR genes. The metE-metR intergenic nucleotide sequence is shown. The nucleotidesare numbered using the metE transcription start site EP as + 1. The two metR transcription start sites are noted as RP1 and RP2. The overlinedareas represent potential regulatory met boxes (19), with at least 50% homology to the consensus sequence AGACGTCT, and the underlinedsequences show the start of translation of the two proteins. The MetR and MetJ binding regions are indicated as hatched or open boxes,respectively.

the region at positions -8 to +27 was protected only in thepresence ofAdoMet, and there was no protection ofthe +102to +137 region. These data suggest that the metE-metRintergenic space contains two distinct regions where MetJbinds, one of which (region -8 to +27) has a much higheraffinity for MetJ.

DISCUSSIONPrevious in vivo data suggested that the MetR protein is atransactivator of both metE and metH expression (14). Re-cent results from this laboratory have provided direct evi-dence that purified MetR protein can stimulate the synthesisof MetE in a DNA-directed in vitro protein synthesis system(20). The results of the present study extend these observa-tions and show that the MetR protein also stimulates the invitro expression of metH and that both the MetE and MetHproteins synthesized in vitro are enzymatically active. En-zymatic activity provides a rapid and quantitative assay forthe amounts of both proteins synthesized in the in vitrosystem. Thus, it was found that the MetR protein stimulatedmetE expression -16-fold and metH expression -8-fold.This is consistent with in vivo data that showed a greaterstimulation by the MetR protein of metE expression thanmetH expression (14). In addition, while the MetJ protein andAdoMet significantly inhibited the expression ofmetE (eitherin the presence or absence of the MetR protein), they hadlittle or no effect on the expression of metH. These resultsagree with in vivo results in E. coli that showed that highlevels of methionine can repress metE expression but notmetH expression (6, 8-10). Also, a recent report (34) showedthat the promoter region of the metH gene from S. typhimur-ium lacks the MetJ repressor recognition sequence (met box)that is present in the other methionine biosynthetic genes(19). It was also found that =5-fold more MetR was requiredto give maximal expression of the metE gene compared to themetH gene. The reason for this difference is not clear but itmay be related to the observation that S-30 preparationsappear to contain a specific inhibitor ofMetE synthesis (datanot shown). Alternatively, another yet unidentified factormay be required for MetR transactivation of metE, but notmetH, expression. In this regard, Stauffer and coworkers (38,39) have results from in vivo experiments indicating thathomocysteine may be involved in the regulation of expres-sion ofboth metE and metR. We have preliminary results thatsuggest homocysteine can stimulate both metE expressionand metR autoregulation in the presence of the MetR proteinin vitro.

In addition to the observation that the in vitro synthesizedMetE and MetH proteins were biologically active, it wasfound that the MetR protein synthesized in vitro couldstimulate the in vitro expression of metH (Table 2). Prelim-inary calculations indicate that the in vitro synthesized MetRprotein is much more active in stimulating metH expressionthan an equivalent amount of the purified MetR protein. Oneexplanation for this discrepancy is that the purified MetRprotein (20) may be partly inactive so that the amount neededfor activation of metE and metH expression in vitro appearshigher than actually required. Further experiments areneeded to investigate the differences in activity between thein vitro synthesized MetR protein and the purified MetRprotein.DNA footprinting experiments were carried out to obtain

more information on the mechanism of regulation by MetJand MetR on the expression ofmetE and metR. MetR proteinwas found to protect only one area of the DNA and this areais located close to the beginning ofthe transcription start sitesfor metR and =49 nucleotides from the metE start site (Fig.5). It should be mentioned that Plamann and Stauffer (35)noted that a deletion in this area resulted in a loss of MetRactivation of the in vivo synthesis of a metE-lac fusionprotein. The finding of only one MetR binding site suggeststhat the binding ofMetR to this region may be responsible forboth the activation of expression of metE as well as theautoregulation ofmetR (20, 36). In addition, the E. coli metRgene has been sequenced (data not shown) and the deducedamino acid sequence reveals the presence of a helical regioncontaining four leucine residues seven amino acids apart.This motif, called the leucine zipper, has been found to be acharacteristic of several eukaryotic DNA binding proteins(37). The leucine zipper has been proposed to play a role inprotein dimerization, which is required for DNA binding.Preliminary cross-linking experiments have shown that theMetR protein exists as a dimer (data not shown).

Since it is now clear that the MetR protein is a transacti-vator of both metE and metH expression, it would bepredicted that the control region of the metH gene wouldhave a nucleotide sequence homologous to that of the MetRbinding region of the metE gene. Urbanowski and Stauffer(34) observed a short region of dyad symmetry, upstream ofboth the S. typhimurium metE and metH genes, that wassimilar. This region ofdyad symmetry (TGAA ... TTCA) isalso found in the E. coli metE upstream region and iscontained within the MetR binding sequence. It would bepredicted that the E. coli metH gene, when sequenced, willprove to have a similar region of dyad symmetry in the regionwhere MetR binds.

4410 Biochemistry: Cai et al.

Proc. Natl. Acad. Sci. USA 86 (1989) 4411

The results with the MetJ protein are somewhat morecomplicated. Two areas of the DNA were protected by MetJin the presence ofAdoMet. Both of these areas contain DNAsequences that are at least 50% homologous with putativemet boxes (19). However, the region located between -8 to+27 (Fig. 5) was protected by MetJ at 10 times lowerconcentrations than those needed to protect the area at + 102to + 137 (data not shown). These results suggest that althoughMetJ can bind to two regions ofthe DNA, it has a much higheraffinity for the sequences at -8 to +27. It is ofinterest to notethat the -8 to +27 area encompasses both the -35 region forthe RP1 transcription start signal and the transcription startsite for metE. Thus, binding of MetJ to this area could serveto simultaneously repress both metR and metEexpression. Inaddition, it has recently been reported that mutations withinthe -8 to +27 region result in a partial loss of repression bymethionine ofthe synthesis ofmefE-lac fusion protein in vivo(35). These latter results, together with the experimentsdescribed here showing that MetJ binds to this region ofDNA, strongly suggest that the -8 to +27 region is involvedin metE repression.

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