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Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes
ANALYTICAL
BIO(‘liKMISTHY
Preparation and
57,
100-107
(1974)
of Guanosine
Tetraphosphate
(ppGpp)
Guanosine Pentaphosphate (pppGpp) from Escherichia co/i Ribosomes
co/i has thv c:tp:wity to syttthcsizc two unusual nucleotides as well as the ability to pwciscly regulate the ccllulnr concentrations of these cotnpouttd~ in respottw to phpkologically stressful conditions. A close correlation exista heta.~~cti the opcratiott of the stringent cellular response to amino acid st:trv:ttioti atitl the :tccutnul:ttion of tliesc conlpounds in stringent sttxittr lw:trittg tlw wl allele of the R?;h C’ontrol gent ; a tlistinctly clitictwit rc~llulnr reqxnse 1)y sitnihrly stnrl-cad ~1~ mutants is nssoci:~ted with the f:Glrtw to ncct!tnul:~W tlicse cornpounds (1.2). Howvvcr the :tcctttnul:ttiott of at Icast OII(’ ol tltcw cottt1~outtds is for sourrw uf erokcrl in both wl ant1 wl strains during tlryrivatioti carl,oti or nitrogen such that growth raks arc liinitccl by tii~ans other than simple miiiio acid stnrmtioti (3.4 1. (he of tlicw c~~rnpouticl~ , :L gu:m:rsinc t~,tr:~l)liosl)li:rtc, has heti isolated from acid extracts ol siznlAc fmncvttor culturc>s :tt1(1 chnrnctwizcd as gu:~nosinc 5’ tlipliospli:ttc~ 2’ or 3’ (li~ho.q~li:~to (lqK;pl) I (5 1. B ~ccond nuclrotidc. u~unlly hut not, inr-arinhly nssocint,cd with pp(:pp, diffrrs from pp(+pp only in tltnt it, posscssrs an ndditional l~liosplinte residue on tltc rihow 5’ position nn~l thus is guatiosinc I)c~tit~ty)liosl)li:tte (ppp( :pp 1 (5,6’1. \‘cry rwrntly, thr 2’ or 3’ I’yrol)liosl)tt:ttc, twiduc of pp(:pp was low lized to tlic 3’ rihsotnc~ position on tlic Im bpis of 3’-spwific phosplioEscherichia
100
102
MICHAEL
CBSHEL
of the reaction during their subsequent izc the nucleotide lwoducts purifirat,ion. (‘02~~7~ ch~oi~~wfog~nphz~. Column cl~ron~atogr:~l~l~ic isolnt,ion of the rcnction products was performed as before Ilsing I)EAE-Sephadcx, A-25 (Pharmacia 1 in 2.5 X 40 cm columns and eluting with linear I,iC’I gradients (0.1-0.5 NJ in 0.05 M Tris-(‘1 (pH 7.4) (5). C’hewicnI nssn~s. Determinations for ribosc. phosphate, and uv spectral propertics of the reaction products were carried out as describccl (5 1. Chemicals nil isotopes. The GDP, GTP, and ATP uwd in the reactions were purchnscd from t,he Sigma Chemical (‘ompan;v. Thwe subst,ratcs used for preparative purposes were I~sunlly not further purified unless contamination with 5’ linear tetrn1~l~osl~hates was present, as indicated from cl~ron~atograpl~p of 2.0 /moles of nucleoticle. The (:TP analog, p-7 methylrnpl (;TP was purcl~nst~d from Miles Laboratories. P;vruvic kinase was obtained in Iyophilized form from \T’orthington Biochcmicals and the sodium salt of I)lioaplloerlolI)~rnT-atc was uaetl as it.s suhtmte. Carrier-free H”Pi was purchased from TCN-Tracer Labs and [“H]GTP from Yew England Nuclear. Radioactive counting and autoradiographic methodologies have been described (5).
Preparation of ribosomes. Frown cells are mixed wit,112 vol of buffer and thawd by blending at low speedswith a rheostated blcndor. RKasefree DNase Worthington) is added (5 ,,g/ml) and the cells broken by two passagesin n French Press or a Gaulin homogenizer Press with a maximal t’empernture of 15°C. The l.i,OOO~/,60 min supernatnnt from this lpsate is sedimented at 30,000 rlnn in a Spinco 30 rotor for 6 hr. The pellets are resuspended in standard buffer containing O.Sy&,Triton X-100 ancl sedimcntcd for 12 hr at 30,OOOrpm. The rcrulting Ivllct is again wsuspendedand similarly pellctcd. Usually in the second or third pc~llc~ting, t,he rihosomcs separate out into a clear lower layer and a superficial layer of brown m:rtjerinl; n-hen this occurs t’hr brown layer is wmorcd with a spatula and discarded. The ribosomnl lwllct is finally rcwspendcd in st’andard lnlffer (lacking Triton) and pelleted twice at 30,OsOO rlun for 6 hr. These .5X pclletcd ribosomcs arc colorless and are rcsuapenrle~lin standard buffer at an A,,;, of 600-1000 and constitute the low salt washed riboPomc>s. Ribsomal ppGpp synthetic activity semis best preservrd by fast freezing in liquid nitrogen. In this manner low salt, washed ribosomes have maintained the pp(;pp synthetic act’ivitics ohserrrd at tile time of
W and 3pP Activity
TABLE Remaining 0 Mill
0.0.5 \f:Tris-Cl (pH 0.4 M sodium formate (pH 3.4) 1 M formic arid 0.:; M KOTT
Sj
1 in pp(+pp
aftar
Hydrolgsis
30 Ylrin
60 klirl
3H cpm
32P rprn
VT cpm
3?P cpm
3IT cprn
S?P cpm
6000 6200
3120 41 no
5901) 6SOO
2800 :3050
6550 .57.50
3100 L’n4n
6000 7200
3090 3100
:%l60 141
lS70 6”
1370 :is
770 27
Acid and alkali lability uf ppGpp made in rilv and in ~,i/ro. A preparation of (“2P]ppGpp (1000 epm/nmolei from amino acid starved E. coli (.i I was mixed with [3H]ppGpp 11.500 cpm/nmule) synthesized from [“H]GTP in the irk zsi/,ro rilwsc,mal reart.ion together with lullabeled ~~p(;pp. After irlclthntions at 60°C under the cI,nditiorrs indicated, 3 ~1 aliqklots wntainiq 2.5 Iunoles ppGpp were c,hlomatogra.phetl itI I ..i M KH2P04 and t,he residrxtl 3zP and 3lT activities in ppGpp mrasured. Altholtgh Iuk shown, the vast majority of both “H ;IIK~ ?:I’ :ic+iviticx in the hydrolysis prothlrt cc unigratc~d u-ith ppGp.
IO4
MICHAEL
C4SHEL
FIN. 1. The effect, of pyruvic kinasc on t)ppGpp and ppGpp synthesis reaction mixtures contained the indicated amounts of fcu-“‘PIGTP (10,000 cpm/nmole) and twice these concentrations of unlnheltd ATP as well as 60 AZ,:,, units of low salt washed ribowmes. Each tancl shows tltc concrntmlions of I)hosphoc,noll,~rl1~:ltc~ ; when present, such mixtures also contained 36 units/ml pvruvic kinasr. after incttbat,ion (1 hr. 37~‘C) 5 ~1 aliquots were chromnloyrnI~llrt1 with 1.5 RI RH2P0, aud t11c acti\ritics of pppGpp (-0-j and ppGpp (-0-j mearurc~d. Prrincubation of mixtures lacking ribosomes with phospl~ocnolp~rl~v:~te and I,>-ruvic kinasc ,virlded even lrw ppGpp synt.hesized after ribosome addition.
rate of the reaction (left panel). Higher levels of GTP t,and hTP) inhibit reaction rates, although detailed analyses of the extent of the reaction at severely inhihikd ratw have not been performed. The presencc of a tn-o-fold excess of ATP over C~+TPis suflicicnt for a ctuantitative conversion of the C+TP or C;DP substrate into product. As has been shown elscwherc (161, elongation factor G (EF C;I may not he a requirement for the reaction as initially proposed. Hoacwr the proportion of ppGpp and pppC+p produced by the reaction tloes depend upon the EF-G activity i 16). M’ith the low $alt washed ribosomes described here the ratios of pppC:pp to [)pGp~? are about I : 1 (Fig. 1, left panel). Ribosomes wnshcd fewer times in ion- salt mnkc prerlominatel~ ppCrpp (see 6,7). Attempts to phosphorylatc preparations of ppC:pl) with pyruvic kinasc (36 IT/ml ) in the absence of ribosonws with phosphoenolpvruvate concentrations from 2 to 10 mM were unsuccessful. Accordingly the preparative feasibility of utilizing pyrur-ate kinase to maintain high levels of both ATP and C$TP subskates has been explored. As indicated in Fig. 1, the presence of pyruvic kinnsc together with phos~~l~oenol pyruvate greatly augments the amount. of pppGpp produced at the cxpcnse of ppGpp. This study also indicates that optimal pliosplioenolpyruvatr conrent~rations rnngc from 2 to 5 ni>r. Preparntinn of ppGpp. Ribosomnl preparations woe first titr:~te(t in
106
MICHAEL
CASHEL
somes in the presence of l,21oe1’21ocnolpq’rLlvate and pyruvic kinase. A variable amount of ppGpp depending upon the length of incubation is recovered as well. The ribore and spectral analysis of pppGpp pwparations is identical with ppGpp while the phosphate content ranges from 4.9 to 5 moles ~~liosphate/rnolc ribosc. l-EPrepa.ra fion of pcppCpp. As demonetrutcd in analytical action mixtures, /3-y mcthylcnyl analog of (:TP can serve as a pyrophosphatc acceptor in the riboeomal reaction I61. Thus. in order to prepare the correspondin g guanosine pcntnphoaphatc analog (pcpp(;pp) , pcppC$ was substitutccl for GTY in the reaction at 1 mzx with ATP at 2 trill. The subsequent isolation of pcppGpp was carried out as abow; yield 74p.. The product was subjected to hydrolysis in the prcscnce of cndonuclease-free phosphomonoestcr:~s~,from E. coli (\\‘orthington alkaline phosphataseI (5 I which produced an ultrariolct fluorescing q)ot comigrating with pcpp(:, as cspccted hp the relative resistance of the met,hylcnyl bond to hydrolysis. AS reported elsewhere, prpp(;pp and pp(:pp aw relatively stable in crude $30 extracts capable of coupled transcription ant1 translation while pppGpp is unstable ( 17). So far, conditions have not been found which allow pyro1~lios1~liatr analog donation to pp( :pp as might hc cslwted with pcp~)A or pn~)pA. Re-use ribosomes for prepnrcrfille ~rencfions. Since the preparation of ribosomes for the synthesis of ppC+p and lq1pGp1’ is rather tedious, :111 attempt was made to w-we rihosomes for this purpose. This ran be accomplished in a variety of ways. The simplist is to recover the rihosomes by high speed centrifugation and store the resulting swpcnded pellets in liquid nitrogen. LIltcri~ati\-c~lg rihosomcs liar-c been placed in a dialysis bag and the reaction allowed to proceed by eschangc of suhstratcs and products through the dialysis incmbranc. Although the rates of the reaction arc substantially slower than normal, the removal of the rihosomes and their re-use for other twctions in “tcx-bag” fashion is greatly facilitated. Storage of rihosomw in the dialysis bag is 1)~ immersing the bag in buRcr containiug 50%’ glycerol and after equilibration placing at -20°C’. A third altcriiatiw is to run the reaction n-it11 small amounts of riboeomes l)y plaring tllc rihosomal susprnsion in :t tlialysis flow cell, with the otllcr side of the mcmbranc pcrfu& \\-ith fresh reactants at a rate consistent with nearly cluantitative ronvc,rsion of the substrates. Of the three mcthotls mentioned. tlicl first is routinely used as it is quickest. Preparation
of
isotopicall,r/
Irrbelwl
ppGpp
find
pppC;pp.
Suitable
s;Ilt)-
stitution of the suhstratrs with the appropriate radioactive nuc1cotide leads to the synthesis of radionctiw ~)ro~lrrctr labeled in sl’ccific popit,ions (6,7).
PKEPARATIOS
OF
l’I’l’c;]‘l’
ASD
l’pc;l,l’
107
BIO(‘liKMISTHY
Preparation and
57,
100-107
(1974)
of Guanosine
Tetraphosphate
(ppGpp)
Guanosine Pentaphosphate (pppGpp) from Escherichia co/i Ribosomes
co/i has thv c:tp:wity to syttthcsizc two unusual nucleotides as well as the ability to pwciscly regulate the ccllulnr concentrations of these cotnpouttd~ in respottw to phpkologically stressful conditions. A close correlation exista heta.~~cti the opcratiott of the stringent cellular response to amino acid st:trv:ttioti atitl the :tccutnul:ttion of tliesc conlpounds in stringent sttxittr lw:trittg tlw wl allele of the R?;h C’ontrol gent ; a tlistinctly clitictwit rc~llulnr reqxnse 1)y sitnihrly stnrl-cad ~1~ mutants is nssoci:~ted with the f:Glrtw to ncct!tnul:~W tlicse cornpounds (1.2). Howvvcr the :tcctttnul:ttiott of at Icast OII(’ ol tltcw cottt1~outtds is for sourrw uf erokcrl in both wl ant1 wl strains during tlryrivatioti carl,oti or nitrogen such that growth raks arc liinitccl by tii~ans other than simple miiiio acid stnrmtioti (3.4 1. (he of tlicw c~~rnpouticl~ , :L gu:m:rsinc t~,tr:~l)liosl)li:rtc, has heti isolated from acid extracts ol siznlAc fmncvttor culturc>s :tt1(1 chnrnctwizcd as gu:~nosinc 5’ tlipliospli:ttc~ 2’ or 3’ (li~ho.q~li:~to (lqK;pl) I (5 1. B ~ccond nuclrotidc. u~unlly hut not, inr-arinhly nssocint,cd with pp(:pp, diffrrs from pp(+pp only in tltnt it, posscssrs an ndditional l~liosplinte residue on tltc rihow 5’ position nn~l thus is guatiosinc I)c~tit~ty)liosl)li:tte (ppp( :pp 1 (5,6’1. \‘cry rwrntly, thr 2’ or 3’ I’yrol)liosl)tt:ttc, twiduc of pp(:pp was low lized to tlic 3’ rihsotnc~ position on tlic Im bpis of 3’-spwific phosplioEscherichia
100
102
MICHAEL
CBSHEL
of the reaction during their subsequent izc the nucleotide lwoducts purifirat,ion. (‘02~~7~ ch~oi~~wfog~nphz~. Column cl~ron~atogr:~l~l~ic isolnt,ion of the rcnction products was performed as before Ilsing I)EAE-Sephadcx, A-25 (Pharmacia 1 in 2.5 X 40 cm columns and eluting with linear I,iC’I gradients (0.1-0.5 NJ in 0.05 M Tris-(‘1 (pH 7.4) (5). C’hewicnI nssn~s. Determinations for ribosc. phosphate, and uv spectral propertics of the reaction products were carried out as describccl (5 1. Chemicals nil isotopes. The GDP, GTP, and ATP uwd in the reactions were purchnscd from t,he Sigma Chemical (‘ompan;v. Thwe subst,ratcs used for preparative purposes were I~sunlly not further purified unless contamination with 5’ linear tetrn1~l~osl~hates was present, as indicated from cl~ron~atograpl~p of 2.0 /moles of nucleoticle. The (:TP analog, p-7 methylrnpl (;TP was purcl~nst~d from Miles Laboratories. P;vruvic kinase was obtained in Iyophilized form from \T’orthington Biochcmicals and the sodium salt of I)lioaplloerlolI)~rnT-atc was uaetl as it.s suhtmte. Carrier-free H”Pi was purchased from TCN-Tracer Labs and [“H]GTP from Yew England Nuclear. Radioactive counting and autoradiographic methodologies have been described (5).
Preparation of ribosomes. Frown cells are mixed wit,112 vol of buffer and thawd by blending at low speedswith a rheostated blcndor. RKasefree DNase Worthington) is added (5 ,,g/ml) and the cells broken by two passagesin n French Press or a Gaulin homogenizer Press with a maximal t’empernture of 15°C. The l.i,OOO~/,60 min supernatnnt from this lpsate is sedimented at 30,000 rlnn in a Spinco 30 rotor for 6 hr. The pellets are resuspended in standard buffer containing O.Sy&,Triton X-100 ancl sedimcntcd for 12 hr at 30,OOOrpm. The rcrulting Ivllct is again wsuspendedand similarly pellctcd. Usually in the second or third pc~llc~ting, t,he rihosomcs separate out into a clear lower layer and a superficial layer of brown m:rtjerinl; n-hen this occurs t’hr brown layer is wmorcd with a spatula and discarded. The ribosomnl lwllct is finally rcwspendcd in st’andard lnlffer (lacking Triton) and pelleted twice at 30,OsOO rlun for 6 hr. These .5X pclletcd ribosomcs arc colorless and are rcsuapenrle~lin standard buffer at an A,,;, of 600-1000 and constitute the low salt washed riboPomc>s. Ribsomal ppGpp synthetic activity semis best preservrd by fast freezing in liquid nitrogen. In this manner low salt, washed ribosomes have maintained the pp(;pp synthetic act’ivitics ohserrrd at tile time of
W and 3pP Activity
TABLE Remaining 0 Mill
0.0.5 \f:Tris-Cl (pH 0.4 M sodium formate (pH 3.4) 1 M formic arid 0.:; M KOTT
Sj
1 in pp(+pp
aftar
Hydrolgsis
30 Ylrin
60 klirl
3H cpm
32P rprn
VT cpm
3?P cpm
3IT cprn
S?P cpm
6000 6200
3120 41 no
5901) 6SOO
2800 :3050
6550 .57.50
3100 L’n4n
6000 7200
3090 3100
:%l60 141
lS70 6”
1370 :is
770 27
Acid and alkali lability uf ppGpp made in rilv and in ~,i/ro. A preparation of (“2P]ppGpp (1000 epm/nmolei from amino acid starved E. coli (.i I was mixed with [3H]ppGpp 11.500 cpm/nmule) synthesized from [“H]GTP in the irk zsi/,ro rilwsc,mal reart.ion together with lullabeled ~~p(;pp. After irlclthntions at 60°C under the cI,nditiorrs indicated, 3 ~1 aliqklots wntainiq 2.5 Iunoles ppGpp were c,hlomatogra.phetl itI I ..i M KH2P04 and t,he residrxtl 3zP and 3lT activities in ppGpp mrasured. Altholtgh Iuk shown, the vast majority of both “H ;IIK~ ?:I’ :ic+iviticx in the hydrolysis prothlrt cc unigratc~d u-ith ppGp.
IO4
MICHAEL
C4SHEL
FIN. 1. The effect, of pyruvic kinasc on t)ppGpp and ppGpp synthesis reaction mixtures contained the indicated amounts of fcu-“‘PIGTP (10,000 cpm/nmole) and twice these concentrations of unlnheltd ATP as well as 60 AZ,:,, units of low salt washed ribowmes. Each tancl shows tltc concrntmlions of I)hosphoc,noll,~rl1~:ltc~ ; when present, such mixtures also contained 36 units/ml pvruvic kinasr. after incttbat,ion (1 hr. 37~‘C) 5 ~1 aliquots were chromnloyrnI~llrt1 with 1.5 RI RH2P0, aud t11c acti\ritics of pppGpp (-0-j and ppGpp (-0-j mearurc~d. Prrincubation of mixtures lacking ribosomes with phospl~ocnolp~rl~v:~te and I,>-ruvic kinasc ,virlded even lrw ppGpp synt.hesized after ribosome addition.
rate of the reaction (left panel). Higher levels of GTP t,and hTP) inhibit reaction rates, although detailed analyses of the extent of the reaction at severely inhihikd ratw have not been performed. The presencc of a tn-o-fold excess of ATP over C~+TPis suflicicnt for a ctuantitative conversion of the C+TP or C;DP substrate into product. As has been shown elscwherc (161, elongation factor G (EF C;I may not he a requirement for the reaction as initially proposed. Hoacwr the proportion of ppGpp and pppC+p produced by the reaction tloes depend upon the EF-G activity i 16). M’ith the low $alt washed ribosomes described here the ratios of pppC:pp to [)pGp~? are about I : 1 (Fig. 1, left panel). Ribosomes wnshcd fewer times in ion- salt mnkc prerlominatel~ ppCrpp (see 6,7). Attempts to phosphorylatc preparations of ppC:pl) with pyruvic kinasc (36 IT/ml ) in the absence of ribosonws with phosphoenolpvruvate concentrations from 2 to 10 mM were unsuccessful. Accordingly the preparative feasibility of utilizing pyrur-ate kinase to maintain high levels of both ATP and C$TP subskates has been explored. As indicated in Fig. 1, the presence of pyruvic kinnsc together with phos~~l~oenol pyruvate greatly augments the amount. of pppGpp produced at the cxpcnse of ppGpp. This study also indicates that optimal pliosplioenolpyruvatr conrent~rations rnngc from 2 to 5 ni>r. Preparntinn of ppGpp. Ribosomnl preparations woe first titr:~te(t in
106
MICHAEL
CASHEL
somes in the presence of l,21oe1’21ocnolpq’rLlvate and pyruvic kinase. A variable amount of ppGpp depending upon the length of incubation is recovered as well. The ribore and spectral analysis of pppGpp pwparations is identical with ppGpp while the phosphate content ranges from 4.9 to 5 moles ~~liosphate/rnolc ribosc. l-EPrepa.ra fion of pcppCpp. As demonetrutcd in analytical action mixtures, /3-y mcthylcnyl analog of (:TP can serve as a pyrophosphatc acceptor in the riboeomal reaction I61. Thus. in order to prepare the correspondin g guanosine pcntnphoaphatc analog (pcpp(;pp) , pcppC$ was substitutccl for GTY in the reaction at 1 mzx with ATP at 2 trill. The subsequent isolation of pcppGpp was carried out as abow; yield 74p.. The product was subjected to hydrolysis in the prcscnce of cndonuclease-free phosphomonoestcr:~s~,from E. coli (\\‘orthington alkaline phosphataseI (5 I which produced an ultrariolct fluorescing q)ot comigrating with pcpp(:, as cspccted hp the relative resistance of the met,hylcnyl bond to hydrolysis. AS reported elsewhere, prpp(;pp and pp(:pp aw relatively stable in crude $30 extracts capable of coupled transcription ant1 translation while pppGpp is unstable ( 17). So far, conditions have not been found which allow pyro1~lios1~liatr analog donation to pp( :pp as might hc cslwted with pcp~)A or pn~)pA. Re-use ribosomes for prepnrcrfille ~rencfions. Since the preparation of ribosomes for the synthesis of ppC+p and lq1pGp1’ is rather tedious, :111 attempt was made to w-we rihosomes for this purpose. This ran be accomplished in a variety of ways. The simplist is to recover the rihosomes by high speed centrifugation and store the resulting swpcnded pellets in liquid nitrogen. LIltcri~ati\-c~lg rihosomcs liar-c been placed in a dialysis bag and the reaction allowed to proceed by eschangc of suhstratcs and products through the dialysis incmbranc. Although the rates of the reaction arc substantially slower than normal, the removal of the rihosomes and their re-use for other twctions in “tcx-bag” fashion is greatly facilitated. Storage of rihosomw in the dialysis bag is 1)~ immersing the bag in buRcr containiug 50%’ glycerol and after equilibration placing at -20°C’. A third altcriiatiw is to run the reaction n-it11 small amounts of riboeomes l)y plaring tllc rihosomal susprnsion in :t tlialysis flow cell, with the otllcr side of the mcmbranc pcrfu& \\-ith fresh reactants at a rate consistent with nearly cluantitative ronvc,rsion of the substrates. Of the three mcthotls mentioned. tlicl first is routinely used as it is quickest. Preparation
of
isotopicall,r/
Irrbelwl
ppGpp
find
pppC;pp.
Suitable
s;Ilt)-
stitution of the suhstratrs with the appropriate radioactive nuc1cotide leads to the synthesis of radionctiw ~)ro~lrrctr labeled in sl’ccific popit,ions (6,7).
PKEPARATIOS
OF
l’I’l’c;]‘l’
ASD
l’pc;l,l’
107