Proc. Natl. Acad. Sci. USAVol. 74, No. 12, pp. 5529-5533, December 1977Biochemistry

In vitro degradation of guanosine 5'-diphosphate,3'-diphosphate(stringent factor/relaxed control/ribosomes)

JOSE SYThe Rockefeller University, New York, New York 10021

Communicated by Fritz Lipmann, October 7, 1977

ABSTRACT The degradation of guanosine 5'-diphos-phate,3'-diphosphate (ppGpp) by the "crude" ribosomal fractionof Escherichia coli CP 78 (rel+, spoT+) was demonstrated andcharacterized. When the 3'-pyrophosphoryl group of ppGpp washydrolyzed, the primary degradation product was 5'-GDP.PhospboryIation of ppGpp to guanosine 5'-triphosphate,3'-diphosphate (pppGpp) prior to degradation was not necessary.The degradation process required Mn2+ and was inhibited byEDTA. Levallorphan, an inhibitor of in vivo ppGpp degrada-tion, also inhibited ppGpp degradation by the crude ribosome.Thiostrepton and tetracycline did not have any inhibitory effect,indicating that the reaction is not a reversal of pyrophosphor-ylation catalyzed by the stringent factor/ribosome complex.Crude ribosome fractions from E. coli NF161 and NF162, bothspoT, contained little degrading activity, but similar fractionsof E. coli CP79, a reL4- and spoT+ strain, contained ppGppdegrading activity.

Guanosine 5'-diphosphate,3'-diphosphate (ppGpp) is a pleo-tropic effector that regulates various metabolic pathways (1)as well as the transcription of certain operons during nutritionalstress (2) in bacterial cells. Bacteria have therefore developeda sensitive balance of biosynthesis and degradation in control-ling the cellular concentration of ppGpp (3). The biosynthesisof this important nucleotide during amino acid deprivation wasfound to be a ribosome-dependent process (4), the basicmechanism of which is that the stringent factor, ATP:GTP(GDP) 3'-pyrophosphotransferase, is activated by a ribo-some-mRNA complex when the aminoacyl-tRNA site of theribosome is occupied by a codon-specified, uncharged tRNA.A nonribosome-dependent synthesis of ppGpp has also beenfound (5, 6).The degradation process of ppGpp has not been well defined

due to the lack of an in vitro cell-free system. In vivo studieshave shown that it is very rapid (7, 8) and is controlled by theavailability of an energy source (9), but studies with spoT-mutant, which has a 10-fold slower degradation rate, have ledto conflicting theories as to its degradation pathway (10-13).I report here on the characterization of an in vitro cell-freeppGpp degradation system.

METHODSMaterials. Escherichia coli CP78 (arg-, his-, leu -, thr,

thi-, relA+, spoT+), CP79 (arg-, his-, leu-, thr-, thi-,relA-, spoT+), NF161 (met-, arg-, relA+, spoT-), andNF162 (met , arg-, relA , spoT-), kindly supplied by N. Fiilof the University of Copenhagen, were grown in a yeast ex-tract/phosphate medium (14). [3H]ppGpp (8.1 Ci/mmol) wasprepared as described (15). [3H]GDP and [3H]GTP were pur-chased from New England Nuclear. Levallorphan tartrate wasa gift from W. Scott, Hoffman-LaRoche, Nutley, NJ. Boric acid

gel (particle size 0.1-0.4 mm) was obtained from AldrichChemical Co. and was pretreated with acetone as described(16). Nucleotides other than ppGpp were obtained from P-LBiochemicals.

Preparation of Crude Ribosomdl Fraction. E. coli CP78cells from the late logarithmic phase were harvested andwashed once with buffer A (10mM Tris-OAc, pH 7.8/14 mMMg(OAc)2/60 mM KCI/1 mM dithiothreitol). The washed cellswere then resuspended with 1 volume of buffer A containingelectrophoretically pure DNase (Worthington) at 1 Mg/ml anddisrupted by passing them through a French press at 18,000 psi(124 mPa). The crude extract was centrifuged at 30,000 X g for30 min, and the supernatant was centrifuged at 100,000 X g for4 hr in a Spinco 40 rotor. The supernatant solution (S-100) wassaved and the pelleted crude ribosomes were resuspended inan equal volume of buffer A. The crude ribosome- and' S-100fractions were stored in liquid nitrogen until used. The cruderibosome fraction prepared in this manner contained variousinhibitors of the stringent factor/ribosome-dependent reactionand therefore carried out a minimal synthesis of ppGpp. Toobtain maximal ppGpp synthesis, the fraction was further pu-rified by centrifugation over a cushion of 40% sucrose and thenremoval of the brownish fluffy layer that sedimented on topof the clear ribosomal pellet. The sucrose-washed ribosomeswere then'highly active in ppGpp synthesis which, in previousexperiments, appeared largely to have overshadowed degra-dation.Assay for ppGpp Degradation. Reaction mixtures of 20 ,l

containing 40mM Tris-OAc (pH 8.1), 1 mM dithiothreitol, 10mM Mg(OAc)2, 5 mM ATP, 9 M [3H]ppGpp (0.3 Ci/mmol),and 1.8 OD2M0 units of crude ribosomes were incubated for 30min at 300. The reactions were stopped' by the addition ofHCOOH. The precipitated proteins and ribosomes were cen-trifuged and the supernatant solutions were applied to stripsof polyethyleneimine-cellulose thin-layer sheets. The degra-dation products were resolved with 0.75 M KH2PO4 (pH 3.45),and the developed chromatograms were scanned with a Varianradioscanner. A high concentration of ATP was routinely addedto the reaction mixtures as a substrate to saturate any nonspe-cific hydrolytic activities catalyzed by nucleotidases andphosphatases.DEAE-Cellulose Chromatographic Separation of Degra-

dation Products. A 10-fold scaled-up version of the standarddegradation experiment was used as the source for the isolationof the various degradation products by DEAE-cellulose chro-matography. The supernatant solution containing the variousnucleotides was diluted with 5 ml of 50 mM triethylamineHC03- (pH 8.0) and applied to a column of DEAE-cellulose(0.6 X 18 cm) previously equilibrated with the same buffer. Thenucleotides were then eluted with 100 ml of a 50-450mM (50ml:50 ml) triethylamine-HCO3 (pH 8) linear gradient at a rate

Abbreviations: ppGpp, guanosine 5'-diphosphate,3'-diphosphate;pppGpp, guanosine 5'-triphosphate,3'-diphosphate.

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The costs of publication of this article were defrayed in part by thepayment of page charges This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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ppGpp GTP GDP GMPi 4 i I

b

d

cm cm

FIG. 1. Degradation of ppGpp by the crude ribosomal system.The incubation mixture for the degradation of ppGpp was as de-scribed in Methods, with the following modifications: (q) no furtheraddition; (b) plus 1 mM EDTA; (c) plus 1 mM EDTA and 2 mMMnCl2; (d) plus 2mM MnCl2, but the enzyme source, crude ribosome,was omitted. After incubation for 30 min at 300, the reactions werestopped by the addition ofHCOOH and the nucleotide products wereseparated by polyethyleneimine-cellulose chromatography.

of 4.5 ml/hr. Fractions (2.5 ml) were collected and 50-Aul ali-quots from each were assayed for radioactivity with Aquasol-2(New England Nuclear) in a Packard scintillation counter.

Boric Acid Gel Column Chromatography. Nucleotidescontaining-free 2'- and 3'-OH groups were separated from 2'-or -3'-substituted nucleotides by a modified boric acid gelchromatographic method (16). The.boric acid gel column (0.6X 18' cm) was preequilibrated with 0.95 M triethylamine-HCO3, pH 9/50 mM Mg(OAc)2. Nucleotides to be separatedwere diluted to a final volume of 2 ml containing 0.875 M tri-ethylamine-HCOs (pH 9) and 50 mM Mg(OAc)2 and were

applied to the column, which was then washed with theequilibration buffer at a rate of 10 ml/hr; 2.5-ml fractions werecollected. After the 10th fraction, the column was furtherwashed with distilled water at the same flow rate to elute anybound nucleotides. Aliquots (0.5 ml) from each fraction wereassayed for radioactivity by liquid scintillation. The recoveryof bound and unbound nucleotides was greater than 95%. Thecolumn was reequilibrated and reused after thorough washingwith water.

RESULTSCell-free extracts of E. colt CP78 were capable of degrading[3H]ppGpp when it was present at a low concentration. Onfractionation, both the ribosome-free S-100 and the cruderibosome fractions were active in ppGpp degradation but thecell wall membranous fraction was not. The degradative ac-

tivity of the crude ribosome fraction usually was 2- to 4-foldhigher than that of the S-100 fraction. The difference variedwith the strains used, the growth phase of the harvested cells,and possibly other unknown factors.When ppGpp was digested with the crude ribosomal fraction,

two prominent products corresponding to guanosine nucleotidescontaining three and two phosphate groups and a minorproduct corresponding to guanosine monophosphate could be

0 5000

ppGpp

2500

pG

10 20 30 40

Fractions

FIG. 2. DEAE-cellulose chromatographic separation of nucleo-tide products. The reaction mixture contained in 200 Al: 25 mMTris.OAc, pHl 8.1; 1 mM dithiothreitol; 10 mM Mg(OAc)2; 5 mM ATP;9 gM [5HlppGpp (0.6 Ci/mmol); and 18 OD20 units of crude ribo-somes. Incubation was for 30 min at 300, and the reaction was stoppedby the addition of 5 Al of 88% HCOOH. The degradation productswere separated by DEAE-cellulose chromatography and elution witha linear gradient of triethylamine HCO3- buffer. Arrows indicatewhere nucleotide markers eluted.

detected (Fig. la). The ratio of the two major products de-pended on the amount of ATP added to the incubation mixture;the'triphosphate predominated when a high concentration ofATP was present. The crude ribosome-degrading system wasroutinely assayed in the presence of 10 mM Mg2+; no degra-dation occurred when Mg2+ was omitted from the reactionmixture (data not shown). It was interesting to find that, in thepresence of 10mM Mg2+, the degradation was inhibited by 1mM EDTA (Fig. lb). Less than 15% of ppGpp was degradedin the presence of EDTA as compared to nearly completedegradation in its absence. The inhibition of EDTA on degra-dation could be completely reversed by the addition of a molarexcess of Mn2+ (Fig. 1c) and was actually greater with 2 mMMn2+ plus 1 mM EDTA but no reversal of EDTA inhibition wasfound when Ca2+ or Fe2+ was added instead of Mn2+; additionof Co2+ or Zn2+ resulted in partial reversal. Mn2+ did not cat-alyze nonenzymatic hydrolysis of ppGpp under the assayconditions used (Fig. id).When 5'-[3H]GDP instead of [3H]ppGpp was incubated with

the crude ribosome system under identical conditions, little orno degradation of [3H]GDP to [3H]GMP was found (data notshown). There was, however, a phosphorylation of GDP to GTPby ATP that was. due to nucleoside diphosphate kinase.. Thesame results were observed when incubations were done in thepresence of EDTA and/or Mn2 , indicating that their effecton the degradation of ppGpp is specific and is not a generalphenomenon of nucleotide degradation.To analyze for the structure of the nucleotide products, a

larger sample of [3H]ppGpp was incubated with the crude ri-bosomal fraction and the various nucleotide products wereseparated by DEAE-cellulose column chromatography. Asshown in Fig. 2, two major products appeared that corre-

ppGpp GTP GDP GMPI I I I

40CECL

200

E

10

T I 9 0

a

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I

So0

E0.

0

-E

cL

ci

Q.0.

E

1000EQ.u-

H20

I0.40NCO0

E0.C)

5 10 15

FractionsFIG. 3. Boric acid gel column chromatographic separation of

nucleotides. (a) A 2-ml sample containing 0.875 M triethylamineHCO3- (pH 9), 50 mM Mg(OAc)2, 50 AM GTP, and [3H]ppGpp(70,000 cpm) was chromatographed in a boric acid gel column. Frac-tions (2.5 ml) were collected and their absorbance at 260 nm was

measured. A 100-Al aliquot from each fraction was assayed for ra-

dioactivity with Aquasol-2. (b and c) A 150-il aliquot of fraction 28from the DEAE-cellulose chromatography step (Fig. 2) containing27,000 cpm (b) and a 150-Al aliquot of fraction 23 containing 14,000cpm (c) were diluted separately to final volumes of 2 ml containing0.875 M triethylamine HCO3- (pH 9) and 50 mM Mg(OAc)2 andchromatographed in a boric acid gel column. The arrows indicate theposition where water replaced the triethylamine HCO3- buffer.

sponded to guanosine nucleotides with three and two phosphategroups. No product corresponding to guanosine 5'-triphos-phate,3'-diphosphate (pppGpp) was detected, indicating that5'-phosphorylation of ppGpp did not occur. Hydrolysis of a

single phosphate group from ppGpp should yield products thathave phosphate groups substituted at the 2'- or 3-position(pGpp, ppGp, or ppG>p), whereas hydrolysis of two phosphatesof ppGpp from either both 8'- and 5'-diphosphate or only 5'-diphosphate should yield products having 2'- or 3'-substitution(pGp or Gpp) or no substitution at the 2'- and 3'-positions (ppG)if 3'-diphosphate is hydrolyzed. Moreover, because phospho-

b

5 5 10 15

cm FractionsFIG. 4. Degradation of ppGpp in the presence of GDP. (a) The

reaction mixture contained in 20 gl: 25mM Tris-OAc (pH 8.1), 1 mMdithiothreitol, 10 mM Mg(OAc)2, 5 mM GDP, [3H]ppGpp (120,000cpm), and 1.8 OD260 units of crude ribosomes. Incubation was at 300for 30 min and the reaction was stopped by the addition of HCOOH.The resulting nucleotides were then resolved by polyethyleneimine-cellulose chromatography. The radioactive nucleotides found asso-

ciated with the GDP position (indicated by the horizontal line) wereeluted from the chromatogram by two successive extractions with 1ml of 1 M triethylamine HCO3-, pH 9. (b) The combined eluates weremade to 50 mM Mg2+ and chromatographed in a boric acid gel col-umn.

rylation of ppG to pppG occurs under our assay conditions, thelatter could also be present at the triphosphate region.The peak fractions of nucleotide triphosphate (fraction 28)

and diphosphate (fraction 23) from the DEAE-cellulose sepa-ration were subjected to boric acid gel chromatography todistinguish the presence of free 2'- and 3'-OH groups in thenucleotide products. Fig. Sa demonstrates the resolving powerof boric acid gel chromatography and shows that ppGpp iscompletely separated from GTP. Under the same conditions,the nucleotide products in fraction 28 (triphosjhate) andfraction 23 (diphosphate) were found to be completely boundto the boric acid gel (Fig. 3b and c), indicating that the tri- anddiphosphate products have no substitution at their 2'- and 3'-positions and that the products of ppGpp degradation are GTPand GDP.Two possible degradative mechanisms can be offered to

explain the finding of GTP as one of the products of degrada-tion. One is that ppGpp is first phosphorylated to pppGpp be-fore degradation (10, 11) and GTP is then formed when the3'-pyrophosphate group is hydrolyzed from pppGpp. The otherpossibility is that ppGpp is directly degraded to GDP which issubsequently phosphorylated to GTP. To distinguish betweenthese two mechanisms, degradation experiments were per-formed in the absence of a phosphoryl donor but with 5 mMGDP instead of ATP. As shown in Fig. 4a, the major productof degradation was GDP which was further shown to be 5'-GDPby boric acid gel chromatography (Fig. 4b). These results in-dicate that the primary product of ppGpp degradation is GDPand that prior phosphorylation of ppGpp to pppGpp is un-necessary in the crude ribosome system.

Addition of levallorphan to E. coli cell cultures has beenshown to result in a reduction in the rate of ppGpp degradation(17). Fig. 5 a and b shows that 2-5 mM levallorphan tartrateinhibited the degradation of ppGpp in the crude ribosomesystem. Tartrate at the same concentration did not exhibit anysuch activity (Fig. 5 c and d). The ribosome stringent factor-dependent ppGpp synthetic reaction was not affected by similarconcentrations of levallorphan (data not shown). However, thespecificity of levallorphan inhibition remains to be determinedbecause the compound tends to precipitate at neutral pH.

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ppGpp GTP GDPI I i

ECL

400

E

200

400

E

200

GMP

a

C

I

B~~~~~~~e

ppGpp GTP GDPXI I

GMP

b

d

'p-,

5 10 5 10

cm cm

FIG. 5. Effect of levallorphan tartrate, tartrate, thiostrepton, andtetracycline on the degradation of ppGpp. Degradation experimentswere performed as described in Methods with the following additions:(a) 2mM and (b) 5mM lavallorphan tartrate; (c) 2mM and (d) 5mMNaK tartrate; (e) 50 gM thiostrepton; and (I) tetracycline at 0.22mg/ml. The reactions were carried out at 300 for 30 min and theproducts formed were analyzed as described in Methods.

The degradation of ppGpp by the crude ribosome system wasunaffected by the addition of thiostrepton and tetracycline (Fig.5 e and f), indicating that the degradation process is differentfrom that of the reverse reaction which is catalyzed by thestringent factor-ribosome mRNA-uncharged tRNA complexand is inhibited by thiostrepton and tetracycline (15). The spoTgene has been shown to govern the in vipo rate of ppGpp deg-radation (10, 11). The relationship of the observed in vitro de-gradative activity with the spoT gene was ascertained bycomparing the degradative activity of crude ribosome fractionsfrom spoT+ strains CP78 and CP79 and spoT- strains NF161and NF162. Crude ribosome fractions from the spoT+ strains,irrespective of the condition of the relA locus, contained de-gradative activity (Table 1). On the other hand, crude ribosomefractions from the spoT- strains contained little degradativeactivity. This loss of activity is not the result of a diffusible in-hibitor in the crude ribosome fractions of the NF strains becausea mixture of spoT+ and spoT- ribosomes produced a-high rateof ppGpp degradation (data not shown). The parallel loss of invitro and in vivo degradative activity in the spoT- strainssuggests that the crude ribosome-mediated ppGpp degradativeactivity studied here is the major pathway of ppGpp degrada-tion. Table 1 also shows that the degrading activity of the spoT-strains can be stimulated severalfold by excess'Mn2+. A Mn2+requirement for the degradation of ppGpp in permeable cellpreparations of spoT strains was previously shown by Raue

Table 1. Degradative activities of the variousribosome preparations

OD % ppGpp hydrolyzedSourceof units No + EDTA + Mn2+ribosome added addition (2 mM) (2 mM)

CP79 (relm spoT+) 1.6 44 1 65CP78 (rel+, spoT+) 1.5 49 3 36NF162 (relb spoT-) 1.6 4 0 17NF161 (rel+, spoT-) 1.3 6 5 13

E. coli CP78, CP79, NF161, and NF162 were grown in yeast ex-tract/glucose/phosphate medium (14) supplemented with casaminoacids (10 g/liter) and were harvested at midlogarithmic phase. Cruderibosomal fractions were then prepared and assayed for ppGpp de-grading activity. All assays were done at 300 for 30 min.

and Cashel (18). Recently, a Mn2+ requirement for the in vvodegradation of ppGpp in spoT+ strains was also indicated(19).A low concentration of [3H]ppGpp was used throughout the

experiments reported here; however, it was subsequently foundthat, at higher concentrations of ppGpp, excess Mn2+ and thepresence of K+ gave considerably higher activity. For example,crude ribosomes (1.6 OD units) from CP79 in the standard re-action mixture containing Mn2+ (2 mM), K+ (100 mM), and a[3HlppGpp concentration of 590 AM instead of 9 ,uM degraded74% of [3H]ppGpp after a 60-min incubation.

DISCUSSIONWith the finding that degradation of ppGpp is mediated by thecrude ribosome fraction, the following metabolic pathway ofppGpp in E. coli may be constructed:

AMP

ATP (1)

pppG

(4)~pi\

pppGpp

(2) P

ppGpp

(3) PPi or 2PjppG

This scheme is similar to schemes proposed by Chaloner-Larsson and Yamazaki (12), Fiil et al. (13), and Weyer et al.(20), all of which were derived from in vivo studies. In thescheme, pppGpp is assumed to be the primary product of thestringent factor-ribosome complex-dependent reaction (re-action 1) during the stringent response because the intracellularconcentration of GTP is much higher than that of GDP. Thein vivo conversion of pppGpp to ppGpp (reaction 2) may becatalyzed by a specific enzyme or, more probably, by severalenzymes that have GTPase activity. In vitro, the hydrolysis ofpentaphosphate to tetraphosphate has been shown with cell-freeextracts (4), and hydrolysis dependent on EF-G and EF-T hasalso been shown (21). The degradation of ppGpp by the cruderibosome fraction results in the hydrolysis of the 3'-pyrophos-phate group and the formation -of 5'-GDP (reaction 3). Theprocess is quite specific for ppGpp because high concentrationsof ATP (5-10 mM) or GDP-(5 mM) do not inhibit. It is notknown at present whether the 3'-pyrophosphate moiety is hy-drolyzed as a unit or is removed one phosphate at a time. Studies

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Proc. Natl. Acad. Sci. USA 74 (1977) 5533

with purified enzyme will be required to distinguish betweenthese two alternatives. However, the inability to find any 2'-or 3'-substituted nucleotides suggests that the pyrophosphateis directly removed as a unit (Figs. 3 and 4). When ppGppdegradation experiments were done in the presence of ATP,the GDP formed was readily phosphorylated to GTP (reaction4). Thus, in vvo, the coupling of the degrading enzyme andnucleoside diphosphate kinase could rapidly transform ppGppinto GTP. Preliminary experiments indicate that the degradingactivity can be separated from the ribosome by a high-salt wash(0.5 M NH4Cl) and that, once separated, the degrading activityis independent of the ribosomes (data not shown). This is alsoshown by the presence of degradation in the ribosome-freesupernatant mentioned earlier. Thus, the significance of theassociation of degrading activity with the ribosome is currentlyuncertain. Crude ribosome fractions prepared from osmoticallyshocked cells (22) still retain ppGpp degrading activity, indi-cating that this activity is not due to any of the phosphatases inthe periplasmic space.

In vivo, the rate of ppGpp degradation depends on theavailability of an energy source (9, 19). How the energy sourcecontrols the activity of ppGpp degrading enzyme(s) will be ofparticular interest.

The author is very grateful to Dr. Lipmann, in whose laboratory thiswork was performed, for his encouragement and constructive criticism.The work was supported by grants from the National Institutes ofHealth (GM-13972 to F. Lipmann) and from the National ScienceFoundation (BMS74-17303 to J.S.).

1. Cashel, M. & Gallant, J. (1974) "Cellular regulation of guanosinetetraphosphate and guanosine pentaphosphate," in Ribosomes,eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring HarborLaboratory, Cold Spring Harbor, NY), pp 733-745.

2. Yang, H. L., Zubay, G., Urm, E., Reiness, G. & Cashel, M. (1974)"Effects of guanosine tetraphosphate, guanosine pentaphosphate,and 3,'y-methylguanosine pentaphosphate on gene expressionof Escherichia coli in vitro," Proc. Natl. Acad. Sci. USA 71,63-67.

3. Gallant, J. & Lazzarini, R. (1976) "The regulation of ribosomalRNA synthesis and degradation in bacteria," in Protein Synthesis,ed. McConkey, E. H. (Marcel Dekker, New York), Vol. 2, pp.309-359.

4. Cashel, M. (1975) "Regulation of bacterial ppGpp and pppGpp,"Annu. Rev. Microbiol. 29,301-318.

5. Sy, J. (1976) "A ribosome-independent, soluble stringent fac-tor-like enzyme isolated from a Bacillus brevis," Biochemistry15,606-609.

6. Nishino, T. & Murao, S. (1974) "Purification and some properties

of ATP: Nucleotide pyrophosphotransferase of Streptomycesadephospholyticus," Agric. Biol. Chem. 38, 2491-2496.

7. Lund, E. & Kjeldgaard, N. 0. (1972) "Metabolism of guanosinetetraphosphate in Escherichia coli," Eur. J. Biochem. 28,316-326.

8. Cashel, M. (1969) 'The control of ribonucleic acid synthesis inE. coli," J. Biol. Chem. 244, 3133-3141.

9. Gallant, J., Margason, G. & Finch, B. (1972) "On the turnoverof ppGpp in Escherichia coli," J. Biol. Chem. 247, 6055-6058.

10. Laffler, T. & Gallant, J. (1974) "SpoT, a new genetic locus in-volved in the stringent response in E. coli," Cell 1, 27-30.

11. Stamminger, G. & Lazzarini, R. A. (1974) "Altered metabolismof the guanosine tetraphosphate, ppGpp, in mutants of E. coli"Cell, 1, 85-90.

12. Chaloner-Larsson, G. & Yamazaki, H. (1976) "Synthesis ofguanosine 5'-triphosphate,3'-diphosphate in a spoT strain of E.coli," Can. J. Biochem. 54, 935-940.

13. Fiil, N. P., Willbmsen, B. M., Friesen, J. D. & von Meyenburg,K. (1977) "Interaction of alleles of the relA, relC and spoT genesin E. coli: Analysis of the interconversion of GTP, ppGpp, andpppGpp," Mol. Gen. Genet. 150, 87-101.

14. Zubay, G., Chambers, D. A. & Cheong, L. C. (1970) "Cell-freestudies on the regulation of the lac operon," in The LactoseOperon, eds. Beckwith, J. R. & Zipser, D. (Cold Spring HarborLaboratory, Cold Spring Harbor, NY), pp. 375-391.

15. Sy, J. (1974) "Reversibility of the pyrophosphoryl transfer fromATP to GTP by E. coli stringent factor," Proc. Natl. Acad. Sci.USA 71, 3470-3473.

16. Schott, H., Rudloff, E., Schmidt, P., Roychoudhury, R. & Kossel,H. (1973) "A dihydroxyl-substituted methacrylic polymer. forthe column chromatographic separation of mononucleotides,oligonucleotides, and transfer RNA," Biochemistry 12, 932-938.

17. Boquet, P. L., Devynck, M., Monnier, C., Fromageot, P. & Ros-chenthaler, R. (1973) "Inhibition of stable RNA synthesis by le-vallorphan in Escherichia coli," Eur. J. Biochem. 40, 31-42.

18. Raue, H. A. & Cashel, M. (1975) "Regulation of RNA synthesisin E. coli. III. Degradation of ppGpp in cold-shocked cells,"Biochim. Biophys. Acta 383,290-304.

19. De Boer, H. A., Bakker, A. J. & Gruber, M. (1977) "Breakdownof ppGpp in spoT+ and spoT- cells of E. coli," FEBS Lett. 78,19-24.

20. Weyer, W. J., de Boer, H. A., de Boer, J. G. & Gruber, M. (1976)"The sequence of pppGpp and ppGpp in the reaction schemefor magic spot synthesis," Biochim. Biophys. Acta 442, 123-127.

21. Hamel, E. & Cashel, M. (1973) "Role of guanine nucleotides inprotein synthesis. Elongation factor G and ppGpp," Proc. Natl.Acad. Sci. USA 70,3250-3254.

22. Brockman, R. W.-& Heppel, L. A. (1968) "On the localizationof alkaline phosphatase and cyclic phosphodiesterase in E. coli,"Biochemistry 7,2554-2562.

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