Proc. Nat. Acad. Sci. USAVol. 71, No. 1, pp. 225-229, January 1974

Activation of Transcription of hut DNA by Glutamine Synthetase(histidine utilization (hut) operons/Salmonella typhimurium/control of glutamate-forming enzymes/cyclic AMP)

BONNIE TYLER, ALBERT B. DELEO, AND BORIS MAGASANIK

Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139

Contributed by Boris Magasanik, October 31, 1973

ABSTRACT The correct transcription of the hut(histidine utilization) operon DNA of Salmonella typhi-murium requires activation either by 3':5'-cyclic aden-osine monophosphate and catabolite-activating proteinor by nonadenylylated glutamine synthetase (EC 6.3.1.2.).Thus, glutamine synthetase plays a regulatory role dis-tinct from its enzymatic function.

The synthesis of many degradative enzymes in bacteria issubject to catabolite repression. In the case of the entericbacteria Escherichia coli, Salmonella typhimurium, and Kleb-siella aerogenes, this repression can be reversed by exogenous3': 5'-cyclic adenosine monophosphate (cAMP) (1-3); andstrains of bacteria lacking the ability to make cAMP do notsynthesize most catabolite-sensitive enzymes (4). In addition,studies of transcription of the DNA of certain catabolic oper-ons with purified RNA polymerase have shown that cAMPand a cAMP-binding protein [catabolite-activating protein-(CAP) ] are required for transcription of these operons (5-7).These observations have led to a model of catabolite repres-sion based on the intracellular level of cAMP (2).

Prival and Magasanik (3) showed that the control of somecatabolic enzymes depends on more than just the intra-cellular level of cAMP. Their results with K. aerogenes showthat the degradative enzymes 3-galactosidase (EC 3.2.1.23),histidase (-histidase ammonia-lyase, EC 4.3.1.3), and prolineoxidase (iproline: 02 oxidoreductase EC 1.4.3.2) are allsubject to catabolite repression in the presence of anadequate supply of nitrogen and that this repression isrelieved by cAMP. However, under conditions of nitrogenlimitation the catabolite-sensitive enzymes that degrade thenitrogenous compounds zhistidine and -proline are relievedof catabolite repression even in a strain unable to synthesizecAMP, while 0-galactosidase remains strongly repressedunder conditions of nitrogen limitation unless exogenouscAMP is present. Further studies suggested that a cytoplasmicfactor other than cAMP can stimulate transcription of thehistidine utilization (hut) genes of both K. aerogenes and S.typhimurium.

In this study we have examined the molecular basis of con-trol of transcription of hut DNA from S. typhimurium byRNA polymerase from E. coli.

Abbreviations: hut, histidine utilization; CAP, catabolite-activat-ing protein; H, the heavier DNA strands in CsCI; L, the lighterDNA strands in CsCl; SDS, sodium dodecyl sulfate; Eo throughE12, adenylylation values corresponding to progressively greateradenylylation.

MATERIALS AND METHODS

M1edia and Solutions. X agar, X broth, and LB broth aredescribed by Signer (8). LBT broth is LB broth containing0.1 M Tris (pH 7.6). SBT broth contaiiis 32 g of tryptone, 20g of yeast extract, and 5 g of NaCl in one liter of 0.1 M Tris(pH 7.6). SSC is 0.015 M sodium citrate and 0.15 M NaCl.Minimal medium (8) and W medium (3) have been describedpreviously.

Chemicals and Reagents. [8H]UTP was obtained from NewEngland Nuclear Corp. Polyuridylieguanylic acid (lot nos.6801 and 7001) was from Schwarz/Mann. RNA polymerasecontaining sigma factor was purified by the method of Burgess(9). cAMP was from P-L Biochemicals. CAP protein waseither a gift from D. McGeoch at the Harvard MedicalSchool or was purified in this laboratory by the methodof Anderson et al. (10). All other compounds were commer-cially available.

Strains of Bacteria and Bacteriophage. The A phage usedhere carry the c1857 thermo-inducible repressor allele (8) andthe Ssus7 lvsis-defective allele (11). E. coli strain M5107[gal-htrRTTRXR (AcI857Ssus7) ] from Dr. E. Signer served as asource of wild-type X DNA. E. coli strain GS-148, lysogenicfor Xphut36cI857Ssus7, was isolated from strain MR134 aspreviously described (12); it carries the wild-type hut operonof S. typhimurium, NE. Derivatives of Xhut phage in whichthe b2 region was deleted were prepared by a standard cross ofXb2 and Xphutcl857Ssus7. Strain QD5003 from Dr. E. Signercarries the SuIII suppressor which allows Ssus7 strains toform plaques (11). E. coli strain WD5045 (gal-) lysogenic forXpgalcI857 was from Dr. E. Signer.Phage 481 which has an attachment site closely linked to

the X attachment site (13) was obtained from Dr. E. Signer.A +81 lysogen of E. coli strain GS-9 (hutQ222) described bySmith (12) was isolated and mitomycin-C or UV-inducedlysates of this strain vielded hut+ transductants of E. coli.Hft 081hut lysates obtained by mitomvyin-C induction fromone of these transductants (T1006) contained both 481 anddefective-081 hut transducing particles which were of greaterdensity than the 081 phage.K. aerogenes strain MKI53 from this laboratory has been

described previously (3).

Growth of Bacteriophage and Isolation of DNA. Lysates ofwild-type X, Xhut, and Agal phages were prepared by thermalinduction of the phages from strains M5107, GS-148, and

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Proc. Nat. Acad. Sci. USA 71 (1974)

L5 A~~ att PL PRL5' A J bb2[- M I G CPUH I N OL OR RH3 U 53

FIG. 1. Structure of Xphut-transducing phage. The relativepositions of the genes are not drawn to scale. See Smith (12) forconstruction of the phage and Roberts (21) for identification ofsymbols on the phage genome. The region underscored by heavyline represents the hut genes of S. typhimurium. Histidine isdegraded by S. typhimurium in four enzymatic steps to gluta-mate, ammonia, and formamide. HutMIGC constitutes; the"left-hand" operon and codes for the third enzyme, 4-imid-azolone-.-propionate amidohydrolase (I) [EC 3.5.2.7); the fourthenzyme, N-formimino-i-glutamate formiminohydrolase (G);and the repressor protein, the C-gene product. HutPUH con-stitutes the "right-hand" operon and codes for the secondenzyme, urocanase (U) and the first, enzyme, ihistidine am-monia-lyase (histidase, H) (EC 4.3.1.3). M is the promoter ofthe left-hand operon and P is the promoter of the right-handoperon. P- mutants lack U and H enzymes. Rho (p) is the RNAtranscription termination factor described by Roberts (21).The open arrows indicate the direction and extent of RNAtranscription in the presence of rho.

WD5045XR. Lysates of X phage carrying the b2 deletion wereprepared by growing plate stocks on strain QD5003. Lysatesof 481 were prepared by lytic infection (multiplicity of infec-tion = 0.1) of strain WD5021 growing in LBT broth at 37°.Lysates of 081hut were prepared by addition of mitomycin (2,4g/ml) to cultures of T1006 at 6 X 108 cells per ml in SBTbroth.

All phage were concentrated with polyethylene glycol (14)and purified by CsCl block and equilibrium gradients; theDNA for template was extracted as described by Adesnikand Levinthal (15).

Strand Separation of DNA. Phage DNA strands were sep-arated according to the method of Hradenca and Szybalski(16). The separated strands were treated with 0.3 M KOHovernight at room temperature, dialyzed against twice-concentrated SSC and heated at 660 for 5 hr. The final con-centration of the strands was estimated from A260nm (36 ,g/ml = 1 unit A860n0,). 811H or XHDNA is used to denote theheavier DNA in CsCl, and 481L or XLDNA denotes the lighterDNA.

Hybridization. Liquid hybridization with separated strandsof DNA was carried out in 60 M1 of twice-concentrated SSCcontaining 50,000-100,000 cpm of [3H ]RNA from the phenol-extracted reaction mixture. For this quantity of RNA, 4 ug/ml of DNA was in excess for the hybridization reaction.Using such conditions, the annealing reaction reached comple-tion in 2-3 hr at 660. DNA -RNA hybrids were isolated andpurified as described in Gillespie and Spiegelman (17), exceptthat 15-ml washes were substituted in each case for 100 ml.Liquid hybridizations for each in vitro reaction were routinelyperformed in duplicate using two different DNA concentra-tions in excess of 5 Mug/ml. Assays were only considered sig-nificant when the cpm hybridized at these two concentrationsagreed within 10%, indicating that DNA was in excess overRNA. The RNA made in vitro hybridized in liquid to theseparated stral ids of the homologous DNA with an efficiency

Filters for hybridization were loaded with DNA as de-scribed by Adesnik and Levinthal (15), with the modificationthat 3.6 jug of H or L single stranded DNA, previously sep-

arated on CsCl gradients, were loaded onto each large filterso that each small filter contained 0.54 ,ug of DNA. Hybridiza-tions were carried out in 50 ul of RNA in four-times concen-

trated SSC plus 0.2% sodium dodecyl sulfate (SDS). Filterscontaining 081 or 081hut DNA were first hybridized with un-

labeled X RNA (generated in an in vitro reaction) by incu-

bating the vials at 660 for 20 hr, followed by extensive wash-ing with twice-concentrated SSC, standard RNase treatment,

and more rinsing with twice-concentrated SSC plus 0.2%SDS. This procedure effectively "covered" the 081 DNA so

that the amount of in vitro-made X RNA which hybridized to

081H-strand DNA was reduced by more than 75%. The DNA-containing filters were then hybridized to [3H ]RNA from invitro reactions using the same procedure except that incuba-tion was at 560 for 18 hr. Finally, the filters were dried andcounted in toluene scintillation fluid.For filter hybridizations which utilized a fixed concentra-

tion of DNA in excess over the [3H]RNA being assayed, allhybridizations were performed in duplicate using two differentamounts of RNA (25,000 and 50,000 cpm), and assays were

considered significant only when the cpm hybridized were

linear with input RNA. Using this procedure, the efficiency ofhybridization of in vitro-made X RNA to noncovered filterscontaining XH and XL strands of DNA was 70-75%.

In Vitro Transcription and Purification of RNA. The re-

action mixture for transcription contained 0.01 M Tris (pH7.9), 0.08 M KCl, 0.2 mM K2HPO4 (pH 7.0), 1 mM dithio-threitol, 5 mM Mg(OAc)2, and 25 Mg/ml of bovine-serumalbumin. Template DNA was present at about 50 Mlg/ml forX or Xb2hut DNA and at 10-12 Mg/ml for Xpgal DNA. Forsynthesis of unlabeled RNA, the reaction contained all nu-

cleotide triphosphates at 0.2 mM; whereas for synthesis ofradioactive RNA, [3H]UTP was present at 0.04 mM (22.2Ci/mM) for X or Xb2hut transcription and at 0.2 mM (5 Ci/mM) for Xpgal transcription.The reaction mixture was incubated for 5 min at 370 before

the addition of RNA polymerase at 60 Mg/ml for X or Xb2huttranscription and 9 Mg/ml for Xpgal transcription; 20 minlater the reaction was terminated by chilling. Reactions to beassayed by liquid hybridization immediately received one-

tenth volume of 1 M sodium acetate (pH 5.5) and were twiceextracted at 660 with freshly distilled phenol saturated with0.1 M sodium acetate (pH 5.5). The final aqueous phase was

stored over phenol until used for hybridization, usually within24 hr. Reactions to be assayed by filter hybridizations received1-2 ml of buffer ST [0.1 M Tris (pH 7.4), 0.2% SDS; and400 ,g/ml of yeast tRNAJ and were then generally frozen at-20° for phenol extraction at a later time. Immediately be-fore phenol extraction, one-tenth volume of 1 M sodium ace-

tate (pH 5.5) was added and the RNA was isolated as

described by Adesnik and Levinthal (15), before finally beingdissolved in four-times concentrated SSC which contained0.2% SDS.

Total incorporation of [iH ]uridine into RNA was estimatedfrom the radioactivity precipitated by cold 5% trichloro-acetic acid using 100 Mg of yeast tRNA as carrier.

Purification and Assay of Glutamine Synthetase. Glutamine

of100-110%.y

226 Biochemistry: Tyler et al.

synthetase was obtained from K. aerogews strain MK-53

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Glutamine Synthetase Activation of Transcription 227

TABLE 1. Assay of hut RNA

cpm bound to hut-specific cpm

Total (k81hut-t081Template template DNA Correct IncorrectDNA 081hutH '81lH 081hUtL 081L XH + XL strand strand

X 2240 3250 1100 1250 53,600 -Xhut 4830 2120 7350 2250 52,000 2710 5100

RNA was synthesized in a purified transcription system, as described in Materials and Methods, with X template DNA at 65 ,ug/ml andXhut template D)NA at 60 ,g/ml. Hybridizations were carried out in liquid as described in Materials and Methods, with 50,000 cpm of inputRNA.

grown at 300 in WV medium containing 1.5 mM 'ammoniumsulfate for nonadenylylated enzyme, or 20 mM ammoniumsulfate for adenylylated enzyme. Glutamine synthetase was

purified from cell-free extracts by modification of a methodof Miller and Stadtman (personal communication) to greaterthan 95% purity as judged by the appearance of a singlemajor band and two trace bands on SDS-acrylamide gels.The purification procedure involved treatment of the crudeextract with streptomycin sulfate followed by addition of0.75 mMI Zn++ in the presence of 50 mMI Mg++ to precipitatethe glutamine synthetase. The precipitate was suspended in2.5 mMI Mg++ and 10 mM\ imidazole (pH 7.0) and the solu-bilized enzyme was again precipitated with 70% ammoniumsulfate in the presence of 10 mM MIn++. The enzyme was

then resuspended in 2.5 mMX1 Mg++ and 10 mM imidazole(pH 7.0) to give a highly purified preparation. Glutaminesynthetase was assayed and assigned an adenylylation value(Eo through E,2, with higher number corresponding to greateradenylylation) as discussed by Ginsburg and Stadtman (18).

RESULTS

Earlier work from this laboratory has shown that the hutgenes in S. typhimurium are clustered in two adjacent oper-

ons: hutMIIGC hutPUH (19, 20). These genes of S. typhi-murium were transferred into E. coli and Xphut transducingphages were isolated (12) (Fig. 1). We used such a phage as a

source of template DNA for transcription and assayed thehut-specific RNA produced by using as a reagent for hybrid-ization the DNA from k81hut, another specialized transducingphage carrying the same hut genes (see Materials and Mleth-ods). We have shown (Cooper, Tyler, and Magasanik, inpreparation) that the hut genes in the cell, whether in thechromosome or in Xhut phage, are transcribed into an mRNAcomplementary to only the H-strand of 081hut DNA. If thehut-specific RNA made in the purified system is produced byRNA polymerase binding to the promoter active in the intactcell, then the hut RNA made in the purified system must alsobind to the H-strand of k81hut DNA.

Therefore, we used the separated strands of 481 and 081-hut DNA to assay by hybridization for hut RNA made in thecell-free system. We found (Table 1) that about 3-5% of theRNA transcribed from X DNA will hybridize to each of theseparated strands of i81; but in each case less RNA hy-bridizes to the corresponding 081hut strand. In contrast,when RNA from the transcription of Xhut is used for the hy-bridizations, much more RNA binds to each strand of k81-hut DNA than to the corresponding strand of 081 DNA. Thus,in spite of some homology between X and q81 DNA, thissystem successfully assays for hut-specific RNA. In fact, the

data in Table 1 show that with Xhut DNA as template about5% of the total RNA made is hut RNA transcribed from thecorrect strand, or H strand, and about 8% is hut RNA tran-scribed from the incorrect strand, or L strand. This transcrip-tion from the incorrect strand is predicted from the structureof the phage genome (Fig. 1), since some transcription shouldinitiate at the X "N" gene and proceed onto the incorrectstrand of the hut region in the absence of RNA terminationfactor rho (p) (21).On the other hand, the extent of correct strand hut trans-

cription was somewhat unexpected, since results from intactcells had suggested (3) that activators are necessary for trans-cription of this operon. However, with Xphut DNA as tem-plate, this correct strand hut transcription could also be dueto "read through" by the RNA polymerase from the b2 regionof the X genome, which is known to be transcribed by the puri-fied RNA polymerase (21); and, in keeping with this idea,transcription of hut from this phage was not stimulated fur-ther by CAP protein and cAMP.

Therefore, we constructed derivatives of the Xhut phage inwhich the b2 region is deleted. Using this phage as template(Table 2), the correct strand hut transcription decreased 10-fold so that the hut-specific RNA constituted only about 0.5%of the total RNA synthesized. This synthesis was independentof the ratio of RNA polymerase to DNA over a wide rangeand, therefore, presumably represents initiation at the hutpromotors (hutM and hutP) rather than any read-through

TABLE 2. Regulation of hut transcription from Xb2hut DNAby CAP protein and cAMP

Additions

CAP % hut-specific IINAprotein cAMP Correct strand Incorrect strand

_ _ 0.5 12.0+ 0.6 12. 5

+ - 0.9 11.5+ + 3.4 13.4

RNA was synthesized in a purified system as described inMaterials and Methods, with Xb2hut template DNA at 50 ,4g/ml,cAMP at 5 X 10-4 MI, and CAP protein at 5 ,g/ml. Hybridiza-tions were carried out in liquid as described in Materials andMethods and hut-specific RNA was obtained, as in Table 1, bythe difference between RNA hybridizing to q081hut DNA and081 DNA. Approximately 3% of the total hybridizable RNAbinds to each strand of <81 DNA. Percent hut-specific RNA isthe percent of total hybridizable RNA which is specific for hutby this assay.

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TABLE 3. Effect of glutamine synthetase on hut transcription

Additions

Glutamine Synthetase

Adenylylation EDTA % hut-specific RNAAg level (10-4 M) correct strand

- 0.4-- + 0.4

17 Eo - 2.835 Eo - 3.260 Eo - 3.225 El0.5 - 0.417 E2.5 - 2.217 E12.5 + 0.3

Conditions were as described for Table 2. Glutamine syn-thetase was added in the indicated amounts per reaction mixture(100 Al).

from residual b2 DNA (21). Using the Xb2hut DNA as tem-plate, we found (Table 2) that addition of cAMP and CAPprotein (together but not separately) results in a 6- to 8-foldincrease in the level of hut RNA transcribed from the correctstrand but does not change total transcription or hut tran-scribed from the incorrect strand. Moreoever, these factorsstimulate correct hut transcription less than half as well whenthe template DNA contains a hutP- mutation (see Table 4).These are the expected results from the studies on hut trans-cription in the intact cell (Cooper et al., in preparation) andindicate that our system is subject to the controls which oper-ate in the intact cell. We then asked what other element(s)can stimulate hut transcription.Both genetic and physiological studies have demonstrated a

close relation between the level of glutamine synthetase inthe cell and the ability of hut enzymes to escape from catabo-lite repression during nitrogen limitation, suggesting thatglutamine synthetase might also play a role in regulation ofthe hut operon (22). Biochemical studies on glutamine syn-thetase have shown that the enzyme can exist in an "activeform," which is not adenylylated, and an "inactive form,"which is adenylylated. The "active" or nonadenylylatedenzyme is the major form present in the cell during nitrogenlimitation (18). Therefore, we added purified nonadenylylatedglutamine synthetase to the system. We found (Table 3, lines3-5) that this protein does, in fact, stimulate hut transcrip-tion. Our results show that the rate of hut transcription is thesame whether nonadenylylated glutamine synthetase orCAP plus cAMP stimulates transcription. This result is incomplete agreement with observations on histidase levels inthe intact cell measured under conditions where, presumably,the two different elements are responsible for activating huttranscription (3). In addition, we found (Table 3, line 6)relatively little stimulation of hut transcription by the adenyl-ylated or "inactive form" of glutamine synthetase which isprimarily present in the cell during nitrogen excess whencAMP and CAP regulate hut transcription. Biochemicalstudies on purified glutamine synthetase have also shownthat the conformation of the enzyme is altered by EDTA(18). We found (Table 3, line 8) that addition of EDTA tothe system decreases the effectiveness with which nonadenyl-

ylated glutamine synthetase stimulates hut transcription, al-though it did not alter the enzymatic activity of the protein.Taken together, these results strongly imply that the

stimulation of hut transcription from the correct strand is dueto the highly purified glutamine synthetase in the proteinpreparation and not to a trace contaminant. In this case wewould predict that the stimulation should be specific forcatabolite-sensitive operons whose enzymes degrade nitroge-nous compounds. In fact, we found no stimulation in overalltranscription of XDNA or in transcription of those segmentsof XDNA which are homologous to qs81 DNA as judged byhybridization. In addition, we found no significant increase intranscription of Xpgal DNA upon the addition of glutaminesynthetase, although addition of CAP and cAMP (togetherbut not separately) increased total transcription of this tem-plate 2-fold. This CAP protein and cAMP-stimulation ofXpgal transcription has been shown to be due to increasedtranscription from the gal promoter (7), and so we interpretthe lack of stimulation by glutamine synthetase as evidencethat the enzyme does not stimulate transcription of the catab-olite-sensitive gal operon.

Finally, we decided to re-examine the stimulation of huttranscription very carefully using a "cover assay" (Cooper,T., Whitney, P., and Magasanik, B., in preparation) in whichmost of the regions of 481 DNA homologous to X DNAare covered by the corresponding X RNA which is un-labeled so that very few counts are bound to 481 DNA duringthe final step of the assay with [3H]RNA. The data obtainedin this way completely substantiate the previous observa-tions: the addition of purified nonadenylylated glutaminesynthetase results in a very significant stimulation of huttranscription, EDTA decreases the efficiency of this molecule,and adenylylated glutamine synthetase is substantially lesseffective. In addition, we used this assay to examine the effectof glutamine synthetase on transcription of DNA from ahutP- promoter mutant whose histidase is not derepressed bynitrogen limitation (Gerson, S. and Magasanik, B., unpub-lished observations). We found (Table 4) that glutaminesynthetase stimulates the Xb2hutP- template less than the X62 hut template. The small observed stimulation was compar-

TABLLE 4. Effect of hutP38 mutation on transcriptionof hut operon

Additions % hut-specifichutP character %htseliof template CAP Glutamine RNA correct

DNA protein cAMP synthetase strand

hutP+ - - - 0.4+ + _ 2.7- - + 2.8

hutP38 - - - 0.4+ + - 1.2_ _ + 1.6

[3H]RNA was synthesized under the conditions described forTable 2, except that cAMP was present at 10-3 M; 45 ug ofglutamine synthetase (E2,5) was added where indicated. Hybrid-izations were carried out on filters using the cover assay, asdescribed in Materials and Methods, which reduced the amount of[3HJRNA binding to +81 DNA to less than 0.2% of the input[3H]RNA. Percent Hut-specific RNA is the percent of input[3HI RNA.

228 Biochemistry: Tyler et al.

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Glutamine Synthetase Activation of Transcription 229

able to that elicited by cAMP and CAP protein with this tem-plate (Table 4). This residual stimulation is presumably dueto transcription from the hutM promoter.

DISCUSSIONWe have presented evidence that transcription of the catab-olite-sensitive hut operon of S. typhimurium can be stimu-lated in two ways: by cAMP in the presence of CAP proteinand by glutamine synthetase. Our observation that cAMPand CAP protein increase transcription of hut DNA by puri-fied RNA polymerase was predicted from experiments onwhole cells (3) and is consistent with the results obtained withpurified RNA polymerase and the DNA of other catabolite-sensitive operons (5-7).Our data showing that highly purified preparations of glu-

tamine synthetase also stimulate transcription of hut DNA isin keeping with earlier speculation (22). Experiments with in-tact cells (22) correlated the degree to which histidase syn-thesis escaped from catabolite repression with the amount ofglutamine synthetase in the cell and showed that cells un-able to synthesize glutamine synthetase cannot escape fromcatabolite repression. The growth conditions which increasethe level of glutamine synthetase in the cell also decrease thelevel of adenylylation of the enzyme. Similarly, our resultswith purified RNA polymerase and Xb2hut DNA show thatthe stimulation of hut transcription by glutamine synthetaseis inversely related to the degree of adenylylation of the en-zyme. Experiments with intact cells which have a mutationin the hutP promoter show that histidase is not synthesizedduring nitrogen limitation. This same hutP mutation dramat-ically decreases the effectiveness of glutamine synthetase instimulating hut transcription.We estimate that a cell of K. aerogenes grown under condi-

tions of nitrogen limitation contains approximately 10,000molecules of glutamine synthetase, corresponding to a concen-tration of 11 jiM (DeLeo, A. and Magasanik, B., unpublishedobservations). In our purified-transcription system we findthat 350 molecules of glutamine synthetase (Eo) per moleculeof hut DNA are sufficient for maximal stimulation of huttranscription. We estimate this concentration of glutaminesynthetase to be 0.4 MM, comparable to the concentration ofcAMP binding protein (0.1 uM) used in the studies reportedin this paper. Therefore, in our system glutamine synthetase

is not present in excess of the amount found in normal intactcells.On the basis of these results we propose that glutamine syn-

thetase serves as a positive control element in the regulationof the transcription of hut DNA.

We are grateful to Jeffrey Felton for expert technical assistance.A.B.D. wns the recipient of an N.I.H. postdoctoral fellowship(GM50356). This work was supported by Public Health ServiceResearch Grants GM 07446 and AM 13894 and National ScienceFoundation Grant GB-5322.

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