Proc. Nati. Acad. Sci. USAVol. 80, pp. 5554-5558, September 1983Biochemistry

Isolation of the nitrogen assimilation regulator NRI, the product ofthe ginG gene of Escherichia coli

(nitrogen regulation/transcription in vitro/glnL promoter)

LAWRENCE J. REIWZER AND BoRis MAGASANIK*Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Contributed by Boris Magasanik, June 13, 1983

ABSTRACT The product of the ginG gene, a member of thecomplex glnALG operon, is an essential component in the re-sponse of Escherichia coli K-12 and other enteric bacteria to ni-trogen-limited growth. We have purified this protein which wepropose to call "NRI," for nitrogen regulator I, to about 95% pu-rity from an overproducing strain. Purified NRI was identified asa dimer by gel filtration. NRB specifically inhibited initiation oftranscription from a DNA fragment containing the glnL promoterbut was without effect on lacZ transcription. We determined theintracellular concentration of NRI under different growth con-ditions by using immunological techniques. The ratio of glutaminesynthetase polypeptides, the product of the ginA gene, to NRIpolypeptides was about 80:1. NRB was not rapidly degraded afterammonia shock, even though the ability to activate nitrogen-con-trolled systems was lost.

Enteric bacteria respond to growth rate limitation by the sourceof nitrogen by increasing the intracellular concentration of glu-tamine synthetase as well as that of many enzymes that degradenitrogen compounds, such as histidase, and of some amino acidpermeases (Ntr, nitrogen-regulated systems) (1-4). The prod-ucts of the ginF (ntrA) and ginG (ntrC) genes are necessary foractivation of the synthesis of glutamine synthetase and of Ntr(1, 5-9). The ginG gene is a member of the complex ginALGoperon whose two promoter-operators, ginAp and ginLp, aresubject to regulation by the product of ginG. Genetic and phys-iological studies have shown that products of ginG and glnF areresponsible for activating ginAp during nitrogen-limited growth(5-9) and that the product of ginG exerts repression at ginApduring growth with an excess of nitrogen and is capable of ex-erting repression at glnLp under all conditions (5, 10, 11). TheginG gene product, which we propose to name "NRI" for ni-trogen regulator I, has been identified as a polypeptide with asubunit Mr of 54,000 (12, 13).

In this paper, we describe the purification, characterizationas a transcriptional repressor, and concentration of NR1 inEscherichia coli.

MATERIALS AND METHODSAll E. coli strains used were derivatives of strain YMC9 (thi,endA, hsr, AlacU169). Strain YMC10 is a hutC derivative ofstrain YMC9 (12). Strain TH17 contains a temperature-sensi-tive A repressor. Strain YMC9 was transduced to resistance tophage A with a lysate of strain N4830 (14), resulting in strainTH17 whose genotype is Gal'A8(AcI857 ABAM, /Hl).

All plasmids were derivatives of pBR322. Plasmid pgln53 (15)contains a fusion of the ginG gene to the glnA promoter. Plas-mid pTH806 contains A PL fused to ginG and was derived from

pgln4O (15) which contains the ginG fragment of pglnS3 withoutthe ginA promoter. The glnG-containing fragment, bounded byEcoRI and HindIII sites, was rendered flush by DNA poly-merase I and ligated into the Hpa I site of pKC30 (16). pGL101was obtained from Gail Lauer (Harvard University) and con-tains the promoter-operator region of the lac operon, whichdoes not require activation by cAMP and the cAMP-bindingprotein (17).The enzymes for endonuclease digestion and other cloning

procedures were from New England BioLabs. Growth condi-tions for cells, the minimal medium (W salts) (18), and brothmedium (3) have been described. Medium fully activating forthe glnALG operon and Ntr containedW salts, vitamin B1, 0.4%D-glucose, and 0.2% L-glutamine; medium repressing for thesesystems was identical but also contained 0.2% (NH4)2SO4.

During the purification of NRI, its presence was assessed ateach step by testing for the presence of a Mr 55,000 band whichwas not found in cell extracts of a strain deleted for the ginALGoperon after a sample had been subjected to electrophoresis inan NaDodSO4/polyacrylamide gel and staining (19). Protein wasdetermined as described (20).

For large-scale preparations of NRI, an overnight culture ofTH17/pTH806 was grown in broth plus ampicillin at 30TC. Thesecells were inoculated into a high-density fermenter (Lab-Line)with 4 liters of medium containing 10.75 g of tryptone (Difco)and 22.5 g of yeast extract (Difco) per liter in W salts (18) and1% glycerol. The initial cell density was 5 x 107/ml and in-creased to 3 x 109/ml with forced aeration. The temperatureof the surrounding water was raised rapidly to 420C. Four hourslater, the cells were collected by centrifugation and frozen. From100 g of a wet paste, 10 mg of purified NRI was obtained. Thisprocedure has not been optimized.

All steps in the purification were performed at 0-40C. Thebuffer used throughout was 20 mM Hepes, pH 7.5/1 mM di-thiothreitol. The frozen cells were thawed in 0.4 liter of buffer,disrupted with five 1-min bursts of sonication, and centrifugedat 6,000 rpm in a Sorvall centrifuge (GSA rotor). The pellet wasresuspended in 200 ml of buffer, sonicated, and centrifugedagain. Streptomycin sulfate (Calbiochem or Sigma; 12 g) wasadded to the pooled supernatants, the suspension was slowlystirred for 30 min, and the pellet was discarded after centrif-ugation. (NH4)2SO4 (Schwarz/Mann, ultrapure; 0.20 g/ml) wasadded to the supernatant, stirred slowly, and centrifuged. Thepellet, which contained 79 mg of protein, was resuspended inbuffer and loaded onto 50 ml of phosphocellulose (P11-What-man). After the column was washed with buffer, the proteinwas eluted with a linear gradient of KCl from 0 to 0.75 M. Theeluted protein (13.5 mg) was concentrated and loaded onto 50

Abbreviation: Ntr, nitrogen-regulated systems.* To whom reprint requests should be addressed.

5554

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Proc. Natl. Acad. Sci. USA 80 (1983) 5555

ml of DNA-agarose (21), washed, and eluted with 0.3 M KC1.The eluted protein was precipitated by (NH4)2SO4, resus-pended in buffer with 20% glycerol, and frozen at -700C. Theability to inhibit transcription in vitro specifically (described inResults) was unaffected by repeated cycles of freezing andthawing. The final yield was 6.6 mg of NRI.

For determination of molecular weight, a Sephadex G-200column (Pharmacia) was calibrated with bovine serum albumin(68,000) (Sigma), pig heart glutamic-oxaloacetate transaminase(90,000), yeast alcohol dehydrogenase (148,000), and pig heartfumarase (194,000) (the last three enzymes were from Boeh-ringer Mannheim).

For transcription of glnL promoter containing DNA, a 270-base-pair fragment of pgln92 (11) was obtained after HindIIIdigestion. The lacZ DNA template came from digestion ofpGL101 with EcoRI and Rsa I. The DNA was purified from a5% polyacrylamide gel and quantitated photographically (22).NRI was purified further (see Results) before use as an an-

tigen. Denatured NRI (20 Ag) was injected at multiple sitessubcutaneously into a New Zealand White rabbit. One monthlater, the rabbit was given another 20 pug of NRI. One weeklater, blood was drawn. The blood was centrifuged and the serumheated was for 1 hr at 560C. The immunoglobulin fraction wasprecipitated by adding (NH4)2SO4 to 50% saturation. The an-tibodies were resuspended in 20 mM Hepes, pH 7.5/150 mMNaCl and frozen.

RESULTSPurification of NRI. In order to overproduce NRI, we grew

strain TH17/pTH806 which contains a fusion of the A PL pro-moter to glnG in a strain carrying a temperature-sensitive re-pressor. Small-scale batch cultures produced 2-5% of cellularprotein as NRI, but this has not been achieved during large-scalegrowth.

The NRI purification was based on the presence of a Mr 55,000subunit that was not found in cells of a strain bearing a deletionof the glnALG operon. The purification involved disruption ofthe cells by sonic oscillation, removal of nucleic acids by strep-tomycin sulfate, precipitation at 35% ammonium sulfate satu-ration, and binding to and elution from phosphocellulose andDNA-agarose. NRI was about 95% pure as determined by vi-sual inspection of a Coomassie blue-stained NaDodSO4/poly-

2 3 4

FIG. 1. NaDodS04/polyacrylamide gel analysis of purified NRI, theglnG gene product. Purified NRI was subjected to electrophoresis in a7.5% gel as described (19). Lanes 1, 2, and 3 contained 30, 10, and 2 ,ugof protein, respectively. Lane 4 contained about 1 pLg of glutamine-de-pendent asparagine synthetase which has a Mr of 57,000 (23). The sumof the contaminating bands in lane 1 are approximately equal to theNRI in lane 3. We therefore estimate 95% purity.

acrylamide gel (Fig. 1). Gel filtration chromatography gave aMr of 110,000-120,000 (data not shown); NRI therefore is a di-mer in the buffer and salt conditions used.

Transcription from the glnL Promoter. The only known reg-ulation of transcription from the glnL promoter is repression byNRI (10, 11). Therefore, we chose to study the action of NRIon transcription in vitro initiated at the glnL promoter. We fo-cused our attention on a 270-base-pair fragment from pgln92which contains the glnL promoter, hereafter called "Hin-270."

The pattern observed when Hin-270 was transcribed in vitrois shown in Fig. 2, lanes 1-5. Transcript a, the major transcript,is about 130 nucleotides long.- A minor transcript, a' is about10 nucleotides shorter. Digestion of Hin-270 with Cla I, whichcleaves 30 bases from the glnL side of the promoter, also de-creased the lengths of transcripts a and a' (Fig. 2, lanes 11 and12). These transcripts were therefore transcribed in the correctdirection. Transcript b is about 270 bases long and probablyresulted from transcription starting at one end of Hin-270 andproceeding to the other end. Transcript c was about 400 baseslong, the sum of transcripts a and b. By a number of criteria,transcript c started at the same site as transcript a. First, theformation of transcripts a and c was salt-sensitive, unlike theformation of transcript b. Second, the formation of transcriptc was repressed by NRI to the same extent as that of transcripta, whereas formation of transcript b was unaffected (see Figs.2 and 3). We conclude that transcript c and longer transcriptsresult from hybridization of the single-strand tails created byrestriction enzymes and are artifacts. Note that in Fig. 2 sucha phenomenon was not apparent for transcription of lacZ DNA.When NRI was added to the transcription systems the for-

mation of transcripts from the glnL promoter fragment (tran-scripts a and c) was inhibited (Fig. 2, lanes 1-5). Very low levelsof purified NRI were effective (Table 1), whereas high levelsof NRI did not affect formation of transcripts from lacZ DNA(Fig. 2, lanes 6-10). NRI prevented the initiation of transcrip-tion: when NRI was added to Hin-270 before RNA polymerase,complete inhibition of transcription occurred (Fig. 3, lanes 1and 2). Less inhibition was observed when RNA polymerasewas added first (Fig. 3, lanes 3-8). The experiments shown inFigs. 2 and 3 rule out the possibility that NRI is a RNase or anonspecific inhibitor of transcription. Excess NRI did not de-grade lacZ RNA (Fig. 2, lane 10). Furthermore, incubation ofRNA initiated from the glnL promoter with purified NRI (es-sentially the experiment shown in Fig. 3, lanes 7 and 8) waswithout effect.

Measurement of the Intracellular Concentration of NRI.Antibodies were raised against NRI after further purification.The purest NRI preparation was subjected to electrophoresis ina long (20 cm) NaDodSO4/polyacrylamide gel, localized by KC1staining, and eluted by electrophoresis before injection into arabbit. Preimmune serum did not react with NRI as deter-mined by either the immunoblotting technique (24) or by elec-trophoresis of an immunoprecipitate from a [35S]methionine-labeled extract. Fig. 4 shows an autoradiograph of immuno-precipitates from an extract of a wild-type strain when the strainhad been grown in medium derepressing for the glnALG op-eron. There were two crossreacting bands (although only oneis apparent) of unknown identity with subunit Mrs of approx-imately 37,000. These bands were immunoprecipitated despitepreabsorption of the antiserum with an extract prepared froman isogeneic strain with a deletion of the glnALG operon. Thefailure of the preabsorption probably was due to loss of theseprotein species during solubilization of the deletion extract. Thesebands were crossreacting because unlabeled purified NRI com-peted with these peptides for anti-NRI antibodies (Fig. 4).

Fig. 5 shows the immunoprecipitation of [35S]methionine-la-

Biochemistry: Reitzer and Magasanik

5556 Biochemistry: Reitzer and Magasanik

X*dI4:I

C.? 4

2~~~~~ ~~ ~~~~~~~~~~~~~3

Proc. Natl. Acad. Sci. USA 80 (1983)

8 9 10

FIG. 2. Effect ofNRj on in vitro transcription ofDNA fragments containing theglnL and lacZ promoters. The concentrations ofRNA polymeraseandDNA template were 50 and 10 nM, respectively. The DNA template in lanes 1-5 was a 270-base-pair fragment from plasmid pgln92 containingthe glnL promoter; the concentrations of NR1 were 50, 20, 10, 5, and 0 nM, respectively. In lanes 6-10, a 312-base-pair fragment containing thelacZ promoter from plasmid pGL101 was transcribed; NR- was 0, 20, 50, 100, and 200 nM, respectively. Lanes 11 and 12 show transcription of theglnL promoter fragment with and without.prior digestion with Cla I. The reaction mixtures contained 50mM Tris at pH 7.5, 10 mM MgCl2, 1 mMdithiothreitol, 0.1 mM EDTA, 50mM KCl, 20 pM CTP, and 10 ,uCi of [a-32P]CTP, DNA, and NRI or buffer. This mixture was incubated for 5 minat 3000 before RNA polymerase was added. The bindingreaction proceeded for 5 min in 20 pul. Then, 2 pl of a mixture containing ATP, GTP, andUTP each at 2.5 mM and heparin (Sigma) at 0.4 mg/ml.was added to initiate the reaction. The reaction proceeded for 15 min and was terminatedby adding 100 jtl of phenol; then, 80 ,ul of yeast tRNA (1 mg/ml) was added. After centrifugation, 80 ul of the aqueous phase was removed, 10 /ulof 20% Na acetate and 270 1ul of 95% ethanol were added, and the mixture was chilled at -20TC. After-centrifugation the pellet was washed in 70%ethanol, dried, redissolved in 15 1Ld ofdeionized formamide, and heated for 2-3 min at 9000. A tracking dye was added and the samples were subjectedto electrophoresis in a 7 M urea/7,5% polyacrylamide gel until the xylene cyanol had run 10 cm. The gel was then exposed to x-ray film.

beled extracts from haploid and plasmid-bearing strains grownin medium repressing and derepressing for NRI. Note that theMr 37,000 crossreacting bands serve as internal controls. Thefraction of [35S]methionine incorporated into NRI relative tototal [3S]methionine incorporated into protein in cells grownin nitrogen-excess and nitrogen-limited media is-given in Table2.

Stability of NRB. When enteric bacteria grown in mediumderepressing for the glnALG operon (nitrogen-limited growth)are subjected to "ammonia shock," the synthesis of glutaminesynthetase is arrested immediately (25). The degradation of NRIcould account for this observation. However, we observed no

Table 1. Effect of NRI on transcription of glnL and lacZRatios

RNATranscription polymerase/

template DNA

glnL 2

5

lacZ 5

NR1/DNA0.51.02.05.0

2.05.0

251020

decrease in NRI 1 hour after ammonia shock (data not shown).We should have been able to detect a 50% decrease in NRI overthe fluctuations in densitometer tracings of similar samples.

DISCUSSIONWe have' purified NRI, the ginG gene product, to about 95%purity. The specific inhibition of transcription initiation from

ow

c.S-I_. r

__..o''',t'-..~' e e

*...W.O. ..

7M

inhibition29477491

3587

-5-10-525

2 3 4 5 6 7 8

FIG. 3. Effect of NRI on transcription initiation at the glnL pro-moter: The concentrations of DNA.template, RNA polymerase, andNRI were 10, .50,. and 50 nM, respectively. At time zero, RNA poly--merase was added. Nucleotides and heparin were added at 5 min. NRIwas added at -.3, 1, 3, and 7 min in lanes 2, 4, 6, and 8, respectively.Buffer was added at the same times in lanes 1, 3, 5, and 7. Note thattranscript b serves as an internal control. Fig. 2 legend gives more de-tails of the methods.

The transcription was performed as described in the legend to Fig. 2.The concentration of the-DNA template was 10 nM. After the locationof the transcript was determined, it was cut out and its radioactivitywas determined after elution with 0.5 M NaCl.

Proc. Natl. Acad. Sci. USA 80 (1983) 5557

37,C00 --M r

2 3 4 5 6

FIG. 4. Iinmunoprecipitation of NRI. Strain YMC1O (wild type) cells(20 ml) growing exponentially in medium derepressing for NRI were

labeled with L-[35S]methionine (20 ,uCi/ml; Amersham) without car-

rier for 10 min and chased with 0.2 ml of 1% methionine for 5 min.- Themedium was removed after centrifugation and the-cells were frozen.Extracts were made with three cycles of suspension in-0.4ml of 20mMTris, pH 8.1/1% NaDodSO4, brief sonication, heating at 65TC, and cen-

trifugation. The immunoprecipitations were performed in 0.2 ml of 20mM Tris, pH 8.1/1% NaDodSO4/1% Nonidet P-40 (Sigma) containingbovine serum albumin at 10 mg/ml.-Antiserum was absorbed first withan unlabeled extract ofstrainYMC11 which has adeletionoftheglnALGoperon, then with purified NR1, and finally, with 100 Al of labeled ex-

tract. After incubation for at least 1 hour atroom temperature, the IgGswere precipitated with protein A-Sepharose CL-4B (Pharmacia). Afterprecipitation at 40C for 1 hour, the pellet was washed three times with1 ml ofthe immunoprecipitation buffer without bovine serum albumin.The precipitate was dissolved in gel sample buffer, boiled for 5 min, cen-

trifuged, and subjected to electrophoresis in a NaDodSO4/7.5% poly-acrylamide gel (19). The gel was dried and exposed to x-ray film. Lanes1 and 6 contained 14C-labeled standards (New England Nuclear): bo-vine serum albumin (69,000), ovalbumin (45,000), and carbonic an-

hydrase (30,000). In the experiments shown in lanes 2-5, 0, 4, 13, and33 ,g, respectively, of purified NRI was added to the immunoprecipi-tation reactions.

the ginL promoter provides direct evidence that NRI is a DNA-binding protein, a hypothesis strongly suggested by the geneticevidence (1). Furthermore, NRI protects from DNase digestiona region of DNA that overlaps the RNA polymerase binding site(unpublished data). A mutant of the glnL promoter unable torespond to NR1 in vivo has been isolated. When this DNA was

used in the transcription system, NRI was no longer inhibitory,proving that transcription in vitro accurately reflected tran-scription in vivo (unpublished data).

NRI -

37,000-Mr

2 3 4 5 6 7 8 9 10

FIG. 5. Measurement of the concentration of NRI by immunopre-cipitation. [35S]Methionine-labeled extracts were immunoprecipitatedandthe precipitate was dissolved in sample buffer and subjected to elec-trophoresis as described in Fig. 4. Lanes 1-3 contained 25, 50, and 100g1, respectively, of a-labeled strain YMC10 extract grown in nitrogen-excess medium. Lanes 4-6 contained 25, 50, and 100 pl, respectively,ofan extract from the same strain grown in nitrogen-limiting medium.Lanes 7-10 contained .10, 2.5, 0.62, and 0.16 p1, respectively, of a la-beled extract of strain YMC10/pgln53 grown in nitrogen-limiting me-dium. Note that the Mr 37,000 crossreacting band serves as an internalcontrol.

When we. measured the intracellular concentration of NRIantigen (Table 2), we found 70 molecules-of NRI dimers per cellin nitrogen-limiting medium and 5 molecules per cell in nitro-gen-excess medium, a ratio of 14:1, which agrees well with theindirect estimates based on evidence from gene fusions (9). Thus,we have now shown directly that growth of the cells on a poorsource of nitrogen results in an increase of the intracellular con-centration of NRI.

In cells of E. coli activated for expression of the glnALG op-eron, when the transcription of both ginA and ginG is initiatedat the ginA promoter, the ratio of monomers of glutamine syn-thetase to those of NRI is approximately 80: 1. [The specific ac-tivity of purified glutamine synthetase is 90 pumol/min per mgof protein (26); in cells fully activated for glutamine synthetaseproduction, the specific activity is 1 ,umol/min per mg of pro-tein, indicating that the enzyme accounts for 1.1% of the cellprotein.] At least two factors determine the magnitude of this

Table 2. Intracellular concentration of NRI

% NIRI* Dimer,moleculesStrain Growth condition Mean Individual values per cell_

YMC10 Nitrogen-excess 0.001 0.0014, 0.0007 5Nitrogen-limiting 0.014 0.0092, 0.011, 0.021 70

YMC10/pgln53 Nitrogen-excess 0.082 0.082 410Nitrogen-limiting 0.71 0.52, 0.71, 0.90 3,550

*% NRI refers to the quantity of [35Slmethionine incorporated intoNRI relative to total [35S]methionineincorporated into cellular protein. The total [355]methionine incorporated into protein was determinedby placing an aliquot of an extract to be immunoprecipitated into 10% trichloroacetic acid, collectingthe precipitate on a nitrocellulose filter, and washing it with more acid and then 70% ethanol. The filterswere dried and the radioactivity was determined in a scintillation counter. The concentration of NRIfrom strain YMC10/pgln53 grown in medium derepressing for NR1 was determined directly and usedas a standard. After immunoprecipitation from an extract-of this strain, an aliquot was removed fromgel sample buffer and the radioactivity was measured. Fig. 5 lanes 7-10 shows that NRI is nearly 100%of the immunoprecipitated protein. The intensity of the Mr 55,000 bands from cultures with less NRIwere compared quantitatively by scanning with a densitometer and compared to the standards from thesame gel. The legend to Fig. 4 gives more details of the methods.

tThis calculation is based on the assumption that 1 g (wet weight) is 1012 cells and contains 0.1 g of pro-tein.

Biochemistry: Reitzer and Magasanik

5558 Biochemistry: Reitzer and Magasanik

ratio. There is less ginG mRNA, probably due to terminationof transcription between ginA and ginG (10); however, themagnitude of this effect is estimated to be only 3- to 4-fold (10).The additional reduction therefore must be post-transcrip-tional.

In conclusion, we have shown that purified NRI is biologi-cally active by demonstrating the ability of this protein to pre-vent specifically the initiation of transcription at the glnL pro-moter. The intracellular concentrations of NRI in cells grownwith nitrogen limitation or excess are compatible with the cur-rent hypothesis of the role of NR1 in the regulation of the glnALGoperon and the Ntr (1).The authors thank Aaron Mitchell for helpful discussions and assis-

tance with the immunological techniques and Thomas Hunt for con-struction of TH17 and pTH806. This research was supported by Re-search Grants GM-07446 from the National Institute of General MedicalSciences and AM-13894 from the National Institute of Arthritis, Dia-betes and Digestive and Kidney Diseases and by Grant PCM78-08576from the National Science Foundation.

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Proc. Natl. Acad. Sci. USA 80 (1983)