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Studies on the in vitro synthesis of ppGpp and pppGpp
80
Biochimica et Biophysica Acta, 395 ( 1 9 7 5 ) 8 0 - - 9 0 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
BBA 98316
STUDIES ON THE IN VITRO SYNTHESIS OF ppGpp AND pppGpp
LASZLO BERES and JEAN LUCAS-LENARD
Biochemistry and Biophysics Section of the Biological Sciences Group, University of Connecticut, Storrs, Conn. 06268 (U.S.A.) (Received November 4th, 1974)
Summary The effect of a number of inhibitors of protein synthesis on ppGpp and pppGpp synthesis in vitro has been examined. As expected from in vivo results, chloramphenicol is without effect on this reaction. Aurintricarboxylic acid and chlortetracycline, on the other hand rapidly and specifically inhibit ppGpp synthesis. Fusidic acid in the presence of saturating amounts of EF G also inhibits the reaction completely, suggesting that an e m p t y ribosomal acceptor site is necessary for this reaction. On the other hand, the 50-S subunit proteins L7 and L12 are not required for stringent factor activity. Ribosomes from Pseudomonas fluorescens can replace those from Escherichia coli in the complete system, while ribosomes from Ehrlich ascites cannot. A small but reproducible synthesis of ppGpp is observed when the ribosomal wash from E. coli is complemented with ribosomes from wheat germ cytoplasm.
Introduction In stringent strains of Escherichia coli reIA ÷ a decreased charging of tRNA results in a n u m b e r of metabolic changes in the cell, including an inhibition of stable RNA accumulation (for a review see ref. 1). The effect on RNA accumulation, termed the "stringent response", has been extensively studied for it suggests a possible coupling of the transcription of stable RNA and the translation of proteins. Cashel and Gallant [2] were first to observe the appearance of two unusual guanine nucleotides, during amino acid starvation of stringent cells which were later characterized as guanosine 5'
Abbreviations: TPCK, L-l-tosylamido-2-phenylethyl c h l o r o m e t h y l ketone; TLCK, L-l-tosylamido2-aminobutyl c h l o r o m e t h y l ketone; ATA, aurintricarboxylic acid ( a m m o n i u m salt); EF G, EF Tu, and EF Ts, bacterial polypeptide elongation factors.
81
(ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) [3,4,9]. It has been hypothesized that these guanosine tetra- and pentaphosphates mediate the various cellular responses to amino acid starvation, including the stringent response [5--7]. Thus, the recent demonstration by Haseltine et al. [4,8] that ppGpp and pppGpp can be synthesized from ATP and GTP in vitro in a ribosomal system under conditions in which protein synthesis is idling has generated considerable interest [9,10]. In a purified system the reaction requires mRNA, codon-specific tRNA, both ribosomal subunits and a protein factor found in the initial high-salt wash of ribosomes obtained from stringent cells [8,10]. Cochran and Byrne have recently reported the purification of the stringent factor to near homogeneity [11]. As an extension of our previous studies on the mechanism of peptide chain elongation in bacteria, we have examined some aspects of the in vitro synthesis of ppGpp and pppGpp. Here we report on the effect of various protein synthesis inhibitors, such as chloramphenicol, chlortetracycline, fusidic acid, puromycin and sparsomycin, TPCK and ATA, on ppGpp synthesis. In addition, our results on the role in ppGpp synthesis, of the ribosomal proteins L7 and L12 which are needed for elongation factor binding [16,20], are described. Finally the synthesis of ppGpp in some heterologous systems is discussed. Our experiments using the various protein synthesis inhibitors confirm and extend previous work on this subject [4,8,10--12]. While this work was in progress, Richter reported on the effect of the removal of proteins L7 and L12 on ppGpp synthesis [13]. The relevant data in this paper agrees with the results of our studies. Materials and Methods Growth o f cells and preparation o f cell extracts Escherichia coli cells strain CP 78 reIA ÷ (leu, thr-, his, arg, thi-) was kindly provided by Dr J. Speyer. This strain was grown either in our laboratory or by the New England Enzyme Center,' Boston, Mass. The growth conditions and media were as described by Haseltine et al. [4]. E. coil A-19 and Pseudomonas fluorescens cells were purchased from General Biochemicals. The cells were lysed by grinding frozen pellets with twice their weight of levigated alumina. After disruption, 1 volume of buffer containing 10 mM Tris • acetate, pH 7.8, 14 mM magnesium acetate, 60 mM potassium acetate and 1 mM dithiothreitol (Buffer B of Haseltine et al.) [4] was added and the alumina and cell debris were removed by two 30 minute centrifugations at 10 000 ×g. The preparation of sucrose-washed and high salt-washed ribosomes as well as the 0.5 M NH4 C1 ribosomal wash containing the stringent factor was carried out as described by Haseltine et al. [4]. The ribosomes and the ribosomal wash were stored in liquid nitrogen in small aliquots. The preparation of ribosomes from E. coli A-19 cells and from P. fluorescens was carried out as described previously [14]. Crude wheat germ ribosomes and high salt-washed ribosomes from Ehrlich ascites cells were a gift of Sergio Abreu. The crude ribosome preparation from wheat germ was pelleted through Buffer B containing 0.5 M NH4 C1. The pellet
82 was resuspended in the same buffer without NH4 C1 and stored in liquid nitrogen before use. The high salt.washed ascites ribosomes were suspended in buffer containing 0.25 M sucrose, i mM magnesium acetate, 10 mM KCI, 0.1 mM EDTA, 1 mM dithiothreitol, and 10 mM Tris • acetate, pH 7.0, and stored in liquid nitrogen. In vitro synthesis o f ppGpp and pppGpp The procedure for the cell-free synthesis o f ppGpp and p p p G p p was primarily that of Haseltine et al. [4] and Pedersen et al. [ 1 0 ] . Unless otherwise indicated assays for ppGpp and pppGpp synthesis were carried o u t at 32°C in 45 pl volume. The complete reaction mixture consisted of 39 mM Tris • acetate, pH 7.8; 19 mM magnesium acetate; 75 mM ammonium acetate; 20 mM potassium acetate; 4 mM dithiothreitol; 0.3 mg/ml polyuridylic acid; 4--6 A260 units/ml yeast t R N A e h e ; 1.3 mM ATP; 0.7 mM GTP; 0.7 • 10 -5 M ~4 Cor 32 P-labeled GTP; ribosomes and 0.5 M NH4 C1 ribosomal wash protein as indicated. The [U -14 C] GTP (512 Ci/mol) and [a -32 p] GTP (535 Ci/mol ) were obtained from Amersham Searle Inc. Polyuridylic acid and t R N A Phe were included in the assays because in our hands even with this crude system the initial rate of the reaction was stimulated 4--5-fold by the addition of these substances. This is in agreement with the report of Cochran and Byrne [11] indicating that in the presence of saturating amounts of stringent factor only a small fraction of high salt-washed ribosomes are active in ppGpp synthesis in the absence of exogenous m R N A and tRNA. The reactions were quenched b y the addition of 1 pl of 88% formic acid. After mixing and centrifugation, 5 ~1 aliquots of the supernatant were spotted on polyethyleneimine-cellulose thin-layer chromatography sheets (EM Laboratories, Inc., Elmsford, New York) and chromatographed using 1.5 M KH2 PO4, pH 3.4 [15] buffer as solvent. The guanine nucleotides, localized either b y ultraviolet scanning or autoradiography using Kodak No-Screen Medical X-ray Film, were cut o u t and counted in a Nuclear Chicago Isocap-300 liquid scintillation counter using either a toluene- or xylene-based scintillation solution. The ratio of the cpm found in the region of the chromatogram containing ppGpp and p p p G p p to the total cpm per sample spotted was used to calculate the % conversion of GTP to ppGpp and pppGpp. Because of the presence of EFG in the ribosomal wash, both guanosine tetra- and pentaphosphates are synthesized in the reaction. The protein synthesis inhibitors used in these experiments, chloramphenicol, chlortetracycline hydrochloride, puromycin hydrochloride, TPCK and TLCK were purchased from Sigma Chemicals, and fusidic acid and ATA, from Leo Pharmaceuticals and Aldrich Chemical Co., respectively. Preparation of L7 and L12 depleted ribosomes Purified ribosomes from either E. coli A-19 or CP 78 relA ÷were treated to remove the proteins L7 and L12 as described by Brot et al. [ 1 6 ] , except that the ribosomes were washed with the buffer containing 1 M NH4 C1 and 40% ethanol twice and then suspended in Buffer A (10 mM Tris • HC1, pH 7.4; 10 mM magnesium acetate; 1 mM dithiothreitol). These treated ribosomes and the NH4 C1/ethanol wash were stored in liquid nitrogen. Assays for polyphenyl-
83
alanine synthesis, as well as the preparation of components for the assays, were as previously reported [14] and protein determinations were carried out according to the method of Lowry et al. [ 1 7 ] . Results The effect o f antibiotics on ppGpp synthesis In vivo the protein synthesis inhibitor chloramphenicol reverses the stringent response under conditions of amino acid starvation or when certain aminoacyl-tRNA synthetase temperature-sensitive mutants are grown at the nonpermissive temperature. In vitro chloramphenicol up to a concentration of 0.7 mM (Table I), does not inhibit ppGpp synthesis, in agreement with a previous report [ 1 0 ] . The in vivo effect of chloramphenicol on the stringent response in temperature-sensitive aminoacyl-tRNA synthetase mutants has been explained by a "leakiness" in the mutant, which allows the accumulation of charged tRNA [ 1 8 ] . Chloramphenicol, for example, has no effect on stringent control when non-leaky temperature-sensitive synthetase mutants are studied [ 1 8 ] . Tetracycline also inhibits the stringent response in vivo [ 1 8 ] , but unlike chloramphenicol, it is also a potent inhibitor in vitro (Table I and Fig. 1). The drastic inhibitory effect of chlortetracycline on ppGpp synthesis suggested the possibility of using this system to study the mechanism of degradation of these nucleotides. In vivo such degradation must take place, since tetracycline induces a drastic and rapid reduction on the level of ppGpp if the antibiotic is added after the nucleotide has accumulated under starvation conditions [ 1 9 ] . In Fig. 1 it is shown that if chlortetracycline is added to the ribosomal system after a significant percentage of GTP has been converted to ppGpp, further increase in ppGpp formation is inhibited, but no decrease is evident in the concentration of this nucleotide as the incubation is continued. It appears then TABLE I T H E E F F E C T O F A N T I B I O T I C S ON p p G p p A N D p p p G p p S Y N T H E S I S IN V I T R O Details o f the e x p e r i m e n t a l p r o c e d u r e arc given in the t e x t . T h e c o m p l e t e s y s t e m c o n t a i n e d 5 ~ug o f 0.5 M NH4C1 w a s h p r o t e i n and 0 . 3 4 m g high s a l t - w a s h e d r i b o s o m e s w h i c h w e r e a d d e d last. T h e r e a c t i o n s w e r e quenched after 2 h incubation. System
Antibiotic
c p m in p p G p p and p p p G p p
Total cpm
Conversion (%)
Inhibition (%)
Complete -- Ribosomes - - Wash p r o t e i n
----
16 2 2 0 804 1291
18 4 6 7 19 4 8 6 19 3 3 6
88 4 7
Complete
chloramphenicol (0.7 m M ) chlortetracycline ( 1 . 1 raM) fusidic acid (1.1 raM) puromycin (1.1 raM) sparsomycin (0.4 raM)
16 6 5 7
19 0 3 9
87
1
383
18 4 6 5
2
98
11 4 5 5
18 7 4 4
61
31
14 6 4 4
18 511
79
10
15 0 4 6
17 7 4 0
85
3
84 I
I
I
- chl°rtetr°cY~/~~O
o
_
so
¢1
+ch 0¢tefrQcyclne (t:70 m n) 40
!,o Z
00 0
I 50
i tOO
i 150
200
INCUBATION TIME, MIN Fig. I . T h e e f f e c t o f c h l o r t e t r a c y c l i n e o n p p G p p s y n t h e s i s . T h e t i m e c o u r s e of p p G p p s y n t h e s i s was d e t e r m i n e d in t h e p r e s e n c e and a b s e n c e o f t h e a n t i b i o t i c using t h e p r o c e d u r e d e s c r i b e d in t h e t e x t . C h l o r t e t r a c y c U n e w a s a d d e d as i n d i c a t e d either at t h e start o f t h e r e a c t i o n ( e ) o r a f t e r 70 m i n (o) to a final c o n c e n t r a t i o n o f 0.7 m M . Each i n c u b a t i o n m i x t u r e c o n t a i n e d 15/~g o f 0 . 0 5 M N H 4 C I w a s h p r o t e i n and 0.34 m g high salt-washed ribosomes.
that unless chlortetracycline also inhibits some c o m p o n e n t of this system which may be involved in ppGpp degradation, the breakdown of these nucleotides does not take place on the ribosome under these conditions. Similar results were obtained when sucrose-washed ribosomes were used in the experiment described in Fig. 1 (not shown). Of a number of other antibiotics tested only fusidic acid was found to be inhibitory (Table I). Fusidic acid binds the EF G present in the ribosomal wash to the ribosomes at the acceptor (A) site and thus according to current models of ppGpp synthesis, would be expected to inhibit the reaction b y interfering with the interaction of t R N A at this site. If this is true, then it should be possible to completely block p p G p p synthesis by saturating the A site with EF G and fusidic acid. Indeed, if the experiment described in Table I is repeated in the presence of 2.0 mM fusidic acid and 6.6 pg of added EF G, the synthesis of ppGpp and p p p G p p is 99% inhibited (data not shown). The lack of effect of puromycin and sparsomycin in this system suggests that their interaction with ribosomes does not inhibit the binding of uncharged t R N A to the A site.
The lack of requirement for proteins L7 and L12 in ppGpp synthesis Two proteins of the 50 S subunit have been demonstrated to be necessary for the optimal interaction between ribosomes and the peptide chain elongation factors EF Tu and EF G, as well as their associated GTPase activities [16,20]. In order to test whether these proteins, designated as L7 and L12 [21,22], are also involved in the activity of the ribosomes in synthesizing ppGpp and p p p G p p in the presence of the stringent factor, they were extracted from ribosomes by washing with buffer containing 1 M NH4C1 and 40% ethanol. The deficient ribosomes were then tested in the presence and absence of the NH4 C1/ethanol wash for their ability to participate in peptide chain elongation and in t h e synthesis of ppGpp. Tables II and III demonstrate that these
85 T A B L E II EFFECT OF NH4C1-ETHANOL WASH PROTEINS ON POLYPHENYLALANINE
SYNTHESIS
In a d d i t i o n t o t h e c o m p o n e n t s i n d i c a t e d a b o v e , e a c h a s s a y m i x t u r e c o n t a i n e d in 250/~1 v o l u m e , 6 . 6 p g a n d 4 . 3 Dg o f p a r t i a l l y ptk~ified E F G a n d E F T, r e s p e c t i v e l y ; 6 . 4 p m o l ( 8 0 0 c p m / p m o l ) o f p u r i f i e d y e a s t [ 14C ] p h e n y l a l a n y l - t R N A l ' n e [ 2 3 ] , 2 5 m M Tris • HCI, p H 7 . 4 ; 1 0 m M m a g n e s i u m a c e t a t e ; 1 6 0 m M a m m o n i u m acetate; 10 mM dithiothreitol; 25 #g/ml polyuridylic acid; 0.4 mM GTP. Incubations were at 37°C for 2 rain w i t h o u t G T P a n d f o r a n a d d i t i o n a l 1 0 r a i n a f t e r t h e a d d i t i o n o f G T P . A f t e r p r e c i p i t a t i o n w i t h c o l d 5% t r i c h l o r o a c e t i c a c i d t h e r e a c t i o n m i x t u r e s w e r e h e a t e d a t 9 0 ° C f o r 1 5 m i n , c h i l l e d , f i l t e r e d o n MKlipore, a n d t h e w e t filters w e r e c o u n t e d in B r a y ' s s o l u t i o n . Ethanol-washed ribosomes (mg)
NH4C1/ethanol wash (~ttl)
14C c p m
Incorporation (%)
-0.14 0.14 0.14
20 -10 20
66 94 835 1 201
1 2 16 23
treated ribosomes which are inactive in polyphenylalanine synthesis are able to participate in ppGpp synthesis in the absence of the wash containing L7 and L12. Furthermore, the results also indicate that re-addition of the NH4C1/ethanol wash to these systems greatly stimulates the peptide chain elongation reaction, while it has little stimulatory effect on the synthesis of ppGpp. EF G in saturating amounts was also included in the ppGpp synthesizing system in order to eliminate a possible artifact which could have arisen of the NH4C1/ ethanol wash contained this factor, since in this crude system EF G has been shown to stimulate the reaction [4]. Separate control experiments designed to determine if the stringent-factor-containing ribosomal wash contained L7 and L12, gave negative results (not shown). The effect o f TPCK and TLCK on ppGpp synthesis TPCK has been shown to inhibit the formation of the ternary complex consisting of EF Tu, phenylalanyl-tRNA and GTP by inactivating the aminoacyl-tRNA binding site on EF Tu [24]. The effect of TPCK is very specific, since the related compound TLCK has no such effect. In order to test whether T A B L E III EFFECT OF NH4C1-ETHANOL WASH PROTEINS ON ppGpp SYNTHESIS The assays w e r e c a r r i e d o u t as d e s c r i b e d in the t e x t , e x c e p t t h a t the f i n a l v o l u m e w a s 9 2 #1 a n d e a c h i n c u b a t i o n also c o n t a i n e d 6 . 6 ~ g o f p a r t i a l l y p u r i f i e d E. coli E F G. T h e c o m p l e t e s y s t e m c o n t a i n e d the indicated amount of ethanol washed ribosomes and 4.5 ~g of 0.5 M NH4CI wash protein. Ribosomes (mg)
NH4Cl/ethanol wash (/~1)
Incubation t i m e (h)
% Conversion of GTP to ppGpp and pppGpp
0.14 0.14 0.14 0.14 -0.14"
-20 -20 20 20
1 1 3 3 3 3
20 25 41 53 3 3
* C o m p l e t e s y s t e m w i t h 0 . 5 M N H 4 C I w a s h c o n t a i n i n g the stringent f a c t o r o m i t t e d .
86 T A B L E IV T H E E F F E C T O F P R E I N C U B A T I N G T H E S T R I N G E N T F A C T O R W I T H T P C K OR T L C K ON p p G p p SYNTHESIS B e f o r e a d d i t i o n t o the complete r e a c t i o n m i x t u r e d e s c r i b e d in t h e t e x t t h e 0.5 M NH4C1 w a s h p r o t e i n w a s i n c u b a t e d o v e r n i g h t a t 4 ° w i t h m e t h a n o l , or T P C K a n d T L C K dissolved in m e t h a n o l , as i n d i c a t e d . The p r o t e i n c o n c e n t r a t i o n d u r i n g p r e i n c u b a t i o n was 0 . 2 2 m g / m l of w h i c h 3.3 # g was a d d e d t o t h e r e a c t i o n m i x t u r e s . T h e c o m p l e t e r e a c t i o n m i x t u r e c o n t a i n e d the components d e s c r i b e d in the t e x t plus 0 . 4 4 m g r i b o s o m e s in a v o l u m e of 70 ~1, T h e final i n c u b a t i o n was at 30°C.
Preincubation conditions
Incubation time (h)
% Conversion of G T P to p p G p p and p p p G p p
% Inhibition
No additions
1 3
13 36
---
M e t h a n o l (2,5%)
1 3
11 31
15 14
M e t h a n o l (2.5%)+ TPCK(0.23 mM)
1 3
3 7
77 81
M e t h a n o l (2.5%)+ T L C K ( 0 . 2 5 raM)
1 3
6 17
54 53
such specificity also exists in the case of the factor involved in the synthesis of ppGpp, we examined the effect of these alkylating agents on this system. It appears (Table IV) that preincubating the ribosomal wash containing the stringent factor with TPCK nearly completely inhibits the reaction. On the other hand, a smaller but significant inhibition effect can be observed in the case of similar concentrations of TLCK. Control experiments demonstrated that in both cases the effect of the inhibitors is due to their action on the stringent factor because the addition of comparable amounts of TPCK and TLCK to the reaction mixtures at the start of the incubation was much less inhibitory (not shown).
The inhibition of ppGpp synthesis by ATA ATA has been shown to inhibit m R N A binding to ribosomes and thus inhibit protein synthesis initiation in vitro [ 2 5 , 2 6 ] , and also to decrease EF Ts activity, resulting in the inhibition of peptide chain elongation [27]. In addition, a recent report indicates that ATA may be a general inhibitor of nucleic acid binding proteins involved in protein and nucleic acid synthesis [ 28 ]. For these reasons, the effect of ATA on ppGpp synthesis in vitro was tested. Our results indicate that it also inhibits the in vitro synthesis of ppGpp (Table V). Under the assay conditions specified, 50% inhibition is observed at 5 • 10 -s M ATA. In addition, as illustrated in Fig. 2, the extent of the reaction at a specified ATA concentration depends on the amount of ribosomes added to the reaction under conditions in which the stringent factor is saturated with ribosomes.
ppGpp and pppGpp synthesis in heterologous systems Richter has shown that ribosomes from yeast, reticulocyte and calf brain cytoplasm cannot substitute for E. coli ribosomes in the synthesis of ppGpp, while yeast mitochondrial ribosomes have some activity when complemented
87 TABLE V T H E I N H I B I T I O N O F p p G p p S Y N T H E S I S BY A T A The experimental complete
in t h e t e x t , e x c e p t t h a t t h e i n c u b a t i o n s w e r e a t 37°C. T h e 6.2 # g of 0 . 5 M NH4C1 w a s h p r o t e i n a n d 0 . 3 4 m g h i g h s a l t - w a s h e d w h i c h w e r e a d d e d l a s t . T h e r e a c t i o n t i m e w a s 2 h. procedure
reaction
ribosomes
w a s as d e s c r i b e d
mixtures
contained
Final ATA
% Inhibition
(X 105M)
% Conversion of GTP to p p G p p and pppGpp
Complete --Ribosomes
---
90 4
---
--Stringent factor
--
9
--
System
concentration
Complete
1.3
84
7
2.6 5.2
81 45
10 50
6.7 10.7
22 4
76 93
with the bacterial stringent factor [ 1 3 ] . Sy et al. have recently reported that the E. cola stringent factor can also complement ribosomes obtained from Bacillus brevis and Chlamydomonas reinhardtii chloroplasts [ 2 9 ] . In this respect we have observed that ribosomes from P. fluorescens can efficiently substitute for E. cola ribosomes in the complete system containing the 0.5 M NH4C1 wash of E. cola ribosomes (Table VI). In addition, a much less efficient but reproducible reaction can be observed when the ribosomal wash from E. cola is complemented with ribosomes obtained from wheat germ. However, there is no detectable conversion of GTP to either ppGpp or pppGpp when E. cola ribosomes are replaced with ribosomes obtained from Ehrlich ascites cells. The bacterial ribosomes and the ribosomes obtained from the eukaryotic cells
8C l
o
LO
40
"-
"x "-
2O
"x ".
§
"x -
>
00 ~ 0
I 0,01
0,02
i
I
0103
0.04
x i "s 0 ~
~
xc~
O, ~
007
FINAL ATA CONCENTRATION, mM
Fig. 2. T h e d e p e n d e n c e o f t h e i n h i b i t i o n
synthesis by A T A on ribosome concentration. The in t h e t e x t , e x c e p t t h a t t h e i n c u b a t i o n s w e r e at 3 7 ° C . T h e c o m p l e t e r e a c t i o n m i x t u r e s c o n t a i n e d 10.7 #g o f 0 . 5 M N H 4CI w a s h p r o t e i n , t h e i n d i c a t e d c o n c e n t r a t i o n o f A T A a n d e i t h e r 0 . 1 4 m g ( l ) , 0 . 2 7 m g (o) o r 0 . 3 4 m g (A) high s a l t - w a s h e d r i b o s o m e s . T h e r e a c t i o n t i m e was 1 h. experimental
procedure
was as described
of ppGpp
88 were active in the polyuridylic acid-dependent synthesis of polyphenylalanine when complemented respectively with bacterial and eukaryotic supernatant factors (not shown). Discussion Kaplan et al. [18] have recently demonstrated that chloramphenicol has no effect on ppGpp synthesis in vivo in a strain of E. coli (5F2 relA ÷) in which reaccumulation of charged tRNA does not take place at the nonpermissive temperature. In the same strain tetracycline was found to completely inhibit the reaction. The respective antibiotics mimic their in vivo effect in cell-free systems isolated from CP 78 reIA ÷. On the other hand the inhibitory effect of fusific acid in vitro was not found in the case of the intact cells. This antibiotic probably inhibits ppGpp synthesis in vitro by its ability to bind EF G present in the salt wash and GTP to the ribosomal acceptor site [30], thus preventing the interaction of uncharged tRNA and possibly the stringent factor with the ribosomes. Our finding that saturating the ribosomes with EF G, GTP and fusidic acid results in complete inhibition of the reaction, as well as the inhibitory effect of chlortetracycline supportsthis view. Tetracycline is known to inhibit the interaction of both aminoacyl-tRNA and uncharged tRNA with ribosomes [31,32]. The lack of inhibition in the case of chloramphenicol and sparsomycin suggests that the peptidyltransferase site on the ribosome is not involved in ppGpp synthesis. In view of the specific effects of the various antibiotics on this reaction, it is surprising that a lack of requirement for the ribosomal proteins L7 and L12 is ppGpp synthesis is observed. These proteins are involved in the activity of the elongation factors which interact with the acceptor site, namely EF Tu and EF G [16,20], although the requirement for L7 and L12 can be minimized under certain conditions [33]. These observations also indicate that ribosomes functionally inactivated with respect to protein synthesis retain the ability of synthesize ppGpp. Unlike the rapid and specific inhibition of EF Tu by TPCK [24], the stringent factor is inactivated by this alkylating reagent only after prolonged incubation. In addition, as has been pointed out by Jonak and Clark [12], complete inhibition is only observed at very low stringent factor concentrations, even though TPCK is in great excess in these experiments. These observations, along with our finding that TLCK also has a significant inhibitory effect in this system, suggest that these compounds inhibit the reaction by nonspecific alkylation of reactive groups on the stringent factor. On the other hand, ATA is a rapid and specific inhibitor of ppGpp synthesis, and this inhibition is dependent on ribosome concentration. It is likely that this inhibition is due to the previously reported ability of ATA to inhibit the binding of mRNA, including polyuridylic acid, to ribosomes [25] since ppGpp synthesis is dependent on messenger RNA [10]. It now appears that the stringent factor obtained from E. coli ribosomes can complement purified ribosomes from other bacteria, such as B. brevis [29], and P. fluorescens (Table VI), as well as ribosomes isolated from cell organelles such as chloroplasts [29] and mitochondria [13]. On the other hand, ribo-
89 T A B L E VI ppGpp AND pppGpp SYNTHESIS IN HETEROLOGOUS
SYSTEMS
Reaction mixtures were incubated at 37°C for 2 h. Where indicated, 10.5/~g of 0.5 M NH4CI wash protein o b t a i n e d f r o m E. coli r i b o s o m e s w a s i n c l u d e d in t h e r e a c t i o n . This stringent f a c t o r c o n t a i n i n g ribosomal w a s h p r e p a r a t i o n w a s r e c e n t r i f u g e d a t 1 3 5 0 0 0 X g f o r 4 h before use in o r d e r t o r e m o v e c o n t a m i n a t i n g E. coli ribosomes. Source of ribosomes (mg)
0.5 M NH4CI wash protein
% Conversion of GTP to ppGpp and pppGpp
None
+
1
E. coli ( 0 . 1 4 ) E. coli ( 0 . 0 7 )
-+
2 84
P. f l u o r e s c e n s ( 0 . 1 4 ) P. f l u o r e s c e n s ( 0 . 0 7 )
-+
2 87
Wheat g e r m ( 0 . 1 7 ) Wheat g e r m ( 0 . 1 7 )
-÷
2 7
Ehriich ascites (0.08) Ehrlich ascites ( 0 . 0 8 )
-+
1 1
somes obtained from the cytoplasm of eukaryotic cells such as yeast, brain [ 1 3 ] , C. reinhardtii [29] and Ehrlich ascites tumor cells (Table VI) are completely inactive in this system. The slight activity obtained in the case of the wheat germ ribosomes appears to be an exception which w e are investigating in more detail in order to eliminate a number of possible artifacts.
Acknowledgements This investigation was supported by a Public Health Service Research Career Development Award No. 6-K4-GM-9523 from the Institute of General Medical Sciences to J. L.-L., a Postdoctoral Research Fellowship Award No. 5 FO2 CA53660-02 from the National Cancer Institute to L.B., and by PHS Research Grant No. ROI CA 1 2 1 4 4 from the National Cancer Institute. We are grateful to John Micthell for helpful discussions and assistance with the initial experiments of this study.
References 1 L u c a s - L e n a r d , J . a n d Beres, L. ( 1 9 7 4 ) in T h e E n z y m e s , X ( B o y e r , P . D . , e d . ) , 3 r d E d n . p p . 5 3 - - 8 6 , A c a d e m i c Press, N e w Y o r k 2 Cashel, M. a n d G a l l a n t , J . ( 1 9 6 9 ) N a t u r e 2 2 1 , 8 3 8 - - 8 4 1 3 Cashel, M. and Kalbacher, B. ( 1 9 7 0 ) J. Biol. Chem. 2 4 5 , 2 3 0 9 - - 2 3 1 8 4 H a s e l t i n e , W . A . , B l o c k , R . , G i l b e r t , W. a n d W e b e r , K . ( 1 9 7 2 ) N a t u r e 2 3 8 , 3 8 1 - - 3 8 4 5 Cashel, M. ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 3 1 3 3 - - 3 1 4 1 6 G a l l a n t , J . , Irr, J . a n d Cashel, M. ( 1 9 7 1 ) J. BioL C h e m . 2 4 6 , 5 8 1 2 - - 5 8 1 6 7 H o c h s t a d t - O z c r , J . a n d Cashel, M. ( 1 9 7 2 ) J . Biol. C h e m . 2 4 7 , 7 0 6 7 ~ 7 0 7 2 8 H a s e l t i n e , W . A . a n d B l o c k , R . ( 1 9 7 3 ) P r o c . N a t l . A c a d . Sci. U . S . 7 0 , 1 5 6 4 - - 1 5 6 8 9 S y , J . a n d L i p m a n n , F. ( 1 9 7 2 ) P r o e . N a t l . A c a d . Sci. U.S. 7 0 , 3 0 6 - - 3 0 9 1 0 P e d c r s e n , F . S . , L u r i d , E. a n d K j e l d g a a r d , W . O . ( 1 9 7 3 ) N a t . N e w BioL 2 4 3 , 1 3 - - 1 5 11 C o c h r a n , J . W . a n d B y r n e , R . W . ( 1 9 7 4 ) J. Biol. Chem. 2 4 9 , 3 5 3 - - 3 6 0 12 Jonak, J. and Clark, B.F.C. (1973) FEBS Lett. 34, 81--84 1 3 Richter, D. ( 1 9 7 3 ) F E B S L e t t . 3 4 , 2 9 1 - - 2 9 4
90 14 Beres, L. and Lucas-Lenard, J. (1973) Arch. Biochem. Biophys. 154, 555--562 15 Cashel, M., Lazzarini, R.A. and Kalbacher, B. (1969) J. Chromatogr. 40, 103--109 16 Brot, N., Yamasaki, E., Redfield, B. and Weissbaeh, H. (1972) Arch. Biochem. Biophys. 148, 148-155 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 18 Kaplan, S., Atherly, A.G. and Barrett, A. (1973) Proc. Natl. Acad. Sci. U.S. 70, 689--692 19 Boquet, P.L., Devynck, M.-A., Monnier, C. and Fromageot, P. (1973) Eur. J. Biochem. 40, 31--42 20 Hamel, E., Koka, M. and N a k a m o t o , T. (1972) J. Biol. Chem. 2 4 7 , 8 0 5 - - 8 1 4 21 M611er, W., Groene, A., Terhorst, C. and Amons, R. (1972) Eur. J. Biochem. 25, 5--12 22 Kaltschmidt, E., Dzionara, M. and Wittman, H.G. (1970) Mol. Gen. Genet. 109, 292--297 23 Beres, L. and Lucas-Lenard, J. (1973) Biochemistry 12, 3998--4002 24 Richman, N. and Bodley, J.W. (1973) J. Biol. Chem. 248, 381--383 25 Grollman, A.P. and Stewart, M.L. (1968) Proc. Natl. Acad. Sci. U.S. 6 1 , 7 1 9 - - 7 2 5 26 Wilhelm, J.M. and Haselkorn, R. (1970) Proc. Natl. Acad. Sci. U.S. 65, 388--394 27 Weissbach, H. and Brot, N. (1970) Biochem. Biophys. Res. Commun. 39, 1194--1198 28 Blumenthal, T. and Landers, T.A. (1973) Biochem. Biophys. Res. C ommun. 55, 680---688 29 Sy, J., Chua, N.-H., Ogawa, Y. and Lipmann, F. (1974) Bioehem. Biophys. Res. Commun. 56, 611--616 30 Cabrer, B.~ V~zquez, D. and Modoeleil, J. (1972) Proc. Natl. Acad. Sci. U.S. 69, 733--736 31 Hierowski, M. (1965) Proc. Natl. Acad. Sci. U.S. 53, 594--599 32 Levin, J.G. (1970) J. Biol. Chem. 245, 3195--3202 33 Hamel, E. and N a k a m o t o , T. (1972) J. Biol. Chem. 247, 6810---6817
Biochimica et Biophysica Acta, 395 ( 1 9 7 5 ) 8 0 - - 9 0 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
BBA 98316
STUDIES ON THE IN VITRO SYNTHESIS OF ppGpp AND pppGpp
LASZLO BERES and JEAN LUCAS-LENARD
Biochemistry and Biophysics Section of the Biological Sciences Group, University of Connecticut, Storrs, Conn. 06268 (U.S.A.) (Received November 4th, 1974)
Summary The effect of a number of inhibitors of protein synthesis on ppGpp and pppGpp synthesis in vitro has been examined. As expected from in vivo results, chloramphenicol is without effect on this reaction. Aurintricarboxylic acid and chlortetracycline, on the other hand rapidly and specifically inhibit ppGpp synthesis. Fusidic acid in the presence of saturating amounts of EF G also inhibits the reaction completely, suggesting that an e m p t y ribosomal acceptor site is necessary for this reaction. On the other hand, the 50-S subunit proteins L7 and L12 are not required for stringent factor activity. Ribosomes from Pseudomonas fluorescens can replace those from Escherichia coli in the complete system, while ribosomes from Ehrlich ascites cannot. A small but reproducible synthesis of ppGpp is observed when the ribosomal wash from E. coli is complemented with ribosomes from wheat germ cytoplasm.
Introduction In stringent strains of Escherichia coli reIA ÷ a decreased charging of tRNA results in a n u m b e r of metabolic changes in the cell, including an inhibition of stable RNA accumulation (for a review see ref. 1). The effect on RNA accumulation, termed the "stringent response", has been extensively studied for it suggests a possible coupling of the transcription of stable RNA and the translation of proteins. Cashel and Gallant [2] were first to observe the appearance of two unusual guanine nucleotides, during amino acid starvation of stringent cells which were later characterized as guanosine 5'
Abbreviations: TPCK, L-l-tosylamido-2-phenylethyl c h l o r o m e t h y l ketone; TLCK, L-l-tosylamido2-aminobutyl c h l o r o m e t h y l ketone; ATA, aurintricarboxylic acid ( a m m o n i u m salt); EF G, EF Tu, and EF Ts, bacterial polypeptide elongation factors.
81
(ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) [3,4,9]. It has been hypothesized that these guanosine tetra- and pentaphosphates mediate the various cellular responses to amino acid starvation, including the stringent response [5--7]. Thus, the recent demonstration by Haseltine et al. [4,8] that ppGpp and pppGpp can be synthesized from ATP and GTP in vitro in a ribosomal system under conditions in which protein synthesis is idling has generated considerable interest [9,10]. In a purified system the reaction requires mRNA, codon-specific tRNA, both ribosomal subunits and a protein factor found in the initial high-salt wash of ribosomes obtained from stringent cells [8,10]. Cochran and Byrne have recently reported the purification of the stringent factor to near homogeneity [11]. As an extension of our previous studies on the mechanism of peptide chain elongation in bacteria, we have examined some aspects of the in vitro synthesis of ppGpp and pppGpp. Here we report on the effect of various protein synthesis inhibitors, such as chloramphenicol, chlortetracycline, fusidic acid, puromycin and sparsomycin, TPCK and ATA, on ppGpp synthesis. In addition, our results on the role in ppGpp synthesis, of the ribosomal proteins L7 and L12 which are needed for elongation factor binding [16,20], are described. Finally the synthesis of ppGpp in some heterologous systems is discussed. Our experiments using the various protein synthesis inhibitors confirm and extend previous work on this subject [4,8,10--12]. While this work was in progress, Richter reported on the effect of the removal of proteins L7 and L12 on ppGpp synthesis [13]. The relevant data in this paper agrees with the results of our studies. Materials and Methods Growth o f cells and preparation o f cell extracts Escherichia coli cells strain CP 78 reIA ÷ (leu, thr-, his, arg, thi-) was kindly provided by Dr J. Speyer. This strain was grown either in our laboratory or by the New England Enzyme Center,' Boston, Mass. The growth conditions and media were as described by Haseltine et al. [4]. E. coil A-19 and Pseudomonas fluorescens cells were purchased from General Biochemicals. The cells were lysed by grinding frozen pellets with twice their weight of levigated alumina. After disruption, 1 volume of buffer containing 10 mM Tris • acetate, pH 7.8, 14 mM magnesium acetate, 60 mM potassium acetate and 1 mM dithiothreitol (Buffer B of Haseltine et al.) [4] was added and the alumina and cell debris were removed by two 30 minute centrifugations at 10 000 ×g. The preparation of sucrose-washed and high salt-washed ribosomes as well as the 0.5 M NH4 C1 ribosomal wash containing the stringent factor was carried out as described by Haseltine et al. [4]. The ribosomes and the ribosomal wash were stored in liquid nitrogen in small aliquots. The preparation of ribosomes from E. coli A-19 cells and from P. fluorescens was carried out as described previously [14]. Crude wheat germ ribosomes and high salt-washed ribosomes from Ehrlich ascites cells were a gift of Sergio Abreu. The crude ribosome preparation from wheat germ was pelleted through Buffer B containing 0.5 M NH4 C1. The pellet
82 was resuspended in the same buffer without NH4 C1 and stored in liquid nitrogen before use. The high salt.washed ascites ribosomes were suspended in buffer containing 0.25 M sucrose, i mM magnesium acetate, 10 mM KCI, 0.1 mM EDTA, 1 mM dithiothreitol, and 10 mM Tris • acetate, pH 7.0, and stored in liquid nitrogen. In vitro synthesis o f ppGpp and pppGpp The procedure for the cell-free synthesis o f ppGpp and p p p G p p was primarily that of Haseltine et al. [4] and Pedersen et al. [ 1 0 ] . Unless otherwise indicated assays for ppGpp and pppGpp synthesis were carried o u t at 32°C in 45 pl volume. The complete reaction mixture consisted of 39 mM Tris • acetate, pH 7.8; 19 mM magnesium acetate; 75 mM ammonium acetate; 20 mM potassium acetate; 4 mM dithiothreitol; 0.3 mg/ml polyuridylic acid; 4--6 A260 units/ml yeast t R N A e h e ; 1.3 mM ATP; 0.7 mM GTP; 0.7 • 10 -5 M ~4 Cor 32 P-labeled GTP; ribosomes and 0.5 M NH4 C1 ribosomal wash protein as indicated. The [U -14 C] GTP (512 Ci/mol) and [a -32 p] GTP (535 Ci/mol ) were obtained from Amersham Searle Inc. Polyuridylic acid and t R N A Phe were included in the assays because in our hands even with this crude system the initial rate of the reaction was stimulated 4--5-fold by the addition of these substances. This is in agreement with the report of Cochran and Byrne [11] indicating that in the presence of saturating amounts of stringent factor only a small fraction of high salt-washed ribosomes are active in ppGpp synthesis in the absence of exogenous m R N A and tRNA. The reactions were quenched b y the addition of 1 pl of 88% formic acid. After mixing and centrifugation, 5 ~1 aliquots of the supernatant were spotted on polyethyleneimine-cellulose thin-layer chromatography sheets (EM Laboratories, Inc., Elmsford, New York) and chromatographed using 1.5 M KH2 PO4, pH 3.4 [15] buffer as solvent. The guanine nucleotides, localized either b y ultraviolet scanning or autoradiography using Kodak No-Screen Medical X-ray Film, were cut o u t and counted in a Nuclear Chicago Isocap-300 liquid scintillation counter using either a toluene- or xylene-based scintillation solution. The ratio of the cpm found in the region of the chromatogram containing ppGpp and p p p G p p to the total cpm per sample spotted was used to calculate the % conversion of GTP to ppGpp and pppGpp. Because of the presence of EFG in the ribosomal wash, both guanosine tetra- and pentaphosphates are synthesized in the reaction. The protein synthesis inhibitors used in these experiments, chloramphenicol, chlortetracycline hydrochloride, puromycin hydrochloride, TPCK and TLCK were purchased from Sigma Chemicals, and fusidic acid and ATA, from Leo Pharmaceuticals and Aldrich Chemical Co., respectively. Preparation of L7 and L12 depleted ribosomes Purified ribosomes from either E. coli A-19 or CP 78 relA ÷were treated to remove the proteins L7 and L12 as described by Brot et al. [ 1 6 ] , except that the ribosomes were washed with the buffer containing 1 M NH4 C1 and 40% ethanol twice and then suspended in Buffer A (10 mM Tris • HC1, pH 7.4; 10 mM magnesium acetate; 1 mM dithiothreitol). These treated ribosomes and the NH4 C1/ethanol wash were stored in liquid nitrogen. Assays for polyphenyl-
83
alanine synthesis, as well as the preparation of components for the assays, were as previously reported [14] and protein determinations were carried out according to the method of Lowry et al. [ 1 7 ] . Results The effect o f antibiotics on ppGpp synthesis In vivo the protein synthesis inhibitor chloramphenicol reverses the stringent response under conditions of amino acid starvation or when certain aminoacyl-tRNA synthetase temperature-sensitive mutants are grown at the nonpermissive temperature. In vitro chloramphenicol up to a concentration of 0.7 mM (Table I), does not inhibit ppGpp synthesis, in agreement with a previous report [ 1 0 ] . The in vivo effect of chloramphenicol on the stringent response in temperature-sensitive aminoacyl-tRNA synthetase mutants has been explained by a "leakiness" in the mutant, which allows the accumulation of charged tRNA [ 1 8 ] . Chloramphenicol, for example, has no effect on stringent control when non-leaky temperature-sensitive synthetase mutants are studied [ 1 8 ] . Tetracycline also inhibits the stringent response in vivo [ 1 8 ] , but unlike chloramphenicol, it is also a potent inhibitor in vitro (Table I and Fig. 1). The drastic inhibitory effect of chlortetracycline on ppGpp synthesis suggested the possibility of using this system to study the mechanism of degradation of these nucleotides. In vivo such degradation must take place, since tetracycline induces a drastic and rapid reduction on the level of ppGpp if the antibiotic is added after the nucleotide has accumulated under starvation conditions [ 1 9 ] . In Fig. 1 it is shown that if chlortetracycline is added to the ribosomal system after a significant percentage of GTP has been converted to ppGpp, further increase in ppGpp formation is inhibited, but no decrease is evident in the concentration of this nucleotide as the incubation is continued. It appears then TABLE I T H E E F F E C T O F A N T I B I O T I C S ON p p G p p A N D p p p G p p S Y N T H E S I S IN V I T R O Details o f the e x p e r i m e n t a l p r o c e d u r e arc given in the t e x t . T h e c o m p l e t e s y s t e m c o n t a i n e d 5 ~ug o f 0.5 M NH4C1 w a s h p r o t e i n and 0 . 3 4 m g high s a l t - w a s h e d r i b o s o m e s w h i c h w e r e a d d e d last. T h e r e a c t i o n s w e r e quenched after 2 h incubation. System
Antibiotic
c p m in p p G p p and p p p G p p
Total cpm
Conversion (%)
Inhibition (%)
Complete -- Ribosomes - - Wash p r o t e i n
----
16 2 2 0 804 1291
18 4 6 7 19 4 8 6 19 3 3 6
88 4 7
Complete
chloramphenicol (0.7 m M ) chlortetracycline ( 1 . 1 raM) fusidic acid (1.1 raM) puromycin (1.1 raM) sparsomycin (0.4 raM)
16 6 5 7
19 0 3 9
87
1
383
18 4 6 5
2
98
11 4 5 5
18 7 4 4
61
31
14 6 4 4
18 511
79
10
15 0 4 6
17 7 4 0
85
3
84 I
I
I
- chl°rtetr°cY~/~~O
o
_
so
¢1
+ch 0¢tefrQcyclne (t:70 m n) 40
!,o Z
00 0
I 50
i tOO
i 150
200
INCUBATION TIME, MIN Fig. I . T h e e f f e c t o f c h l o r t e t r a c y c l i n e o n p p G p p s y n t h e s i s . T h e t i m e c o u r s e of p p G p p s y n t h e s i s was d e t e r m i n e d in t h e p r e s e n c e and a b s e n c e o f t h e a n t i b i o t i c using t h e p r o c e d u r e d e s c r i b e d in t h e t e x t . C h l o r t e t r a c y c U n e w a s a d d e d as i n d i c a t e d either at t h e start o f t h e r e a c t i o n ( e ) o r a f t e r 70 m i n (o) to a final c o n c e n t r a t i o n o f 0.7 m M . Each i n c u b a t i o n m i x t u r e c o n t a i n e d 15/~g o f 0 . 0 5 M N H 4 C I w a s h p r o t e i n and 0.34 m g high salt-washed ribosomes.
that unless chlortetracycline also inhibits some c o m p o n e n t of this system which may be involved in ppGpp degradation, the breakdown of these nucleotides does not take place on the ribosome under these conditions. Similar results were obtained when sucrose-washed ribosomes were used in the experiment described in Fig. 1 (not shown). Of a number of other antibiotics tested only fusidic acid was found to be inhibitory (Table I). Fusidic acid binds the EF G present in the ribosomal wash to the ribosomes at the acceptor (A) site and thus according to current models of ppGpp synthesis, would be expected to inhibit the reaction b y interfering with the interaction of t R N A at this site. If this is true, then it should be possible to completely block p p G p p synthesis by saturating the A site with EF G and fusidic acid. Indeed, if the experiment described in Table I is repeated in the presence of 2.0 mM fusidic acid and 6.6 pg of added EF G, the synthesis of ppGpp and p p p G p p is 99% inhibited (data not shown). The lack of effect of puromycin and sparsomycin in this system suggests that their interaction with ribosomes does not inhibit the binding of uncharged t R N A to the A site.
The lack of requirement for proteins L7 and L12 in ppGpp synthesis Two proteins of the 50 S subunit have been demonstrated to be necessary for the optimal interaction between ribosomes and the peptide chain elongation factors EF Tu and EF G, as well as their associated GTPase activities [16,20]. In order to test whether these proteins, designated as L7 and L12 [21,22], are also involved in the activity of the ribosomes in synthesizing ppGpp and p p p G p p in the presence of the stringent factor, they were extracted from ribosomes by washing with buffer containing 1 M NH4C1 and 40% ethanol. The deficient ribosomes were then tested in the presence and absence of the NH4 C1/ethanol wash for their ability to participate in peptide chain elongation and in t h e synthesis of ppGpp. Tables II and III demonstrate that these
85 T A B L E II EFFECT OF NH4C1-ETHANOL WASH PROTEINS ON POLYPHENYLALANINE
SYNTHESIS
In a d d i t i o n t o t h e c o m p o n e n t s i n d i c a t e d a b o v e , e a c h a s s a y m i x t u r e c o n t a i n e d in 250/~1 v o l u m e , 6 . 6 p g a n d 4 . 3 Dg o f p a r t i a l l y ptk~ified E F G a n d E F T, r e s p e c t i v e l y ; 6 . 4 p m o l ( 8 0 0 c p m / p m o l ) o f p u r i f i e d y e a s t [ 14C ] p h e n y l a l a n y l - t R N A l ' n e [ 2 3 ] , 2 5 m M Tris • HCI, p H 7 . 4 ; 1 0 m M m a g n e s i u m a c e t a t e ; 1 6 0 m M a m m o n i u m acetate; 10 mM dithiothreitol; 25 #g/ml polyuridylic acid; 0.4 mM GTP. Incubations were at 37°C for 2 rain w i t h o u t G T P a n d f o r a n a d d i t i o n a l 1 0 r a i n a f t e r t h e a d d i t i o n o f G T P . A f t e r p r e c i p i t a t i o n w i t h c o l d 5% t r i c h l o r o a c e t i c a c i d t h e r e a c t i o n m i x t u r e s w e r e h e a t e d a t 9 0 ° C f o r 1 5 m i n , c h i l l e d , f i l t e r e d o n MKlipore, a n d t h e w e t filters w e r e c o u n t e d in B r a y ' s s o l u t i o n . Ethanol-washed ribosomes (mg)
NH4C1/ethanol wash (~ttl)
14C c p m
Incorporation (%)
-0.14 0.14 0.14
20 -10 20
66 94 835 1 201
1 2 16 23
treated ribosomes which are inactive in polyphenylalanine synthesis are able to participate in ppGpp synthesis in the absence of the wash containing L7 and L12. Furthermore, the results also indicate that re-addition of the NH4C1/ethanol wash to these systems greatly stimulates the peptide chain elongation reaction, while it has little stimulatory effect on the synthesis of ppGpp. EF G in saturating amounts was also included in the ppGpp synthesizing system in order to eliminate a possible artifact which could have arisen of the NH4C1/ ethanol wash contained this factor, since in this crude system EF G has been shown to stimulate the reaction [4]. Separate control experiments designed to determine if the stringent-factor-containing ribosomal wash contained L7 and L12, gave negative results (not shown). The effect o f TPCK and TLCK on ppGpp synthesis TPCK has been shown to inhibit the formation of the ternary complex consisting of EF Tu, phenylalanyl-tRNA and GTP by inactivating the aminoacyl-tRNA binding site on EF Tu [24]. The effect of TPCK is very specific, since the related compound TLCK has no such effect. In order to test whether T A B L E III EFFECT OF NH4C1-ETHANOL WASH PROTEINS ON ppGpp SYNTHESIS The assays w e r e c a r r i e d o u t as d e s c r i b e d in the t e x t , e x c e p t t h a t the f i n a l v o l u m e w a s 9 2 #1 a n d e a c h i n c u b a t i o n also c o n t a i n e d 6 . 6 ~ g o f p a r t i a l l y p u r i f i e d E. coli E F G. T h e c o m p l e t e s y s t e m c o n t a i n e d the indicated amount of ethanol washed ribosomes and 4.5 ~g of 0.5 M NH4CI wash protein. Ribosomes (mg)
NH4Cl/ethanol wash (/~1)
Incubation t i m e (h)
% Conversion of GTP to ppGpp and pppGpp
0.14 0.14 0.14 0.14 -0.14"
-20 -20 20 20
1 1 3 3 3 3
20 25 41 53 3 3
* C o m p l e t e s y s t e m w i t h 0 . 5 M N H 4 C I w a s h c o n t a i n i n g the stringent f a c t o r o m i t t e d .
86 T A B L E IV T H E E F F E C T O F P R E I N C U B A T I N G T H E S T R I N G E N T F A C T O R W I T H T P C K OR T L C K ON p p G p p SYNTHESIS B e f o r e a d d i t i o n t o the complete r e a c t i o n m i x t u r e d e s c r i b e d in t h e t e x t t h e 0.5 M NH4C1 w a s h p r o t e i n w a s i n c u b a t e d o v e r n i g h t a t 4 ° w i t h m e t h a n o l , or T P C K a n d T L C K dissolved in m e t h a n o l , as i n d i c a t e d . The p r o t e i n c o n c e n t r a t i o n d u r i n g p r e i n c u b a t i o n was 0 . 2 2 m g / m l of w h i c h 3.3 # g was a d d e d t o t h e r e a c t i o n m i x t u r e s . T h e c o m p l e t e r e a c t i o n m i x t u r e c o n t a i n e d the components d e s c r i b e d in the t e x t plus 0 . 4 4 m g r i b o s o m e s in a v o l u m e of 70 ~1, T h e final i n c u b a t i o n was at 30°C.
Preincubation conditions
Incubation time (h)
% Conversion of G T P to p p G p p and p p p G p p
% Inhibition
No additions
1 3
13 36
---
M e t h a n o l (2,5%)
1 3
11 31
15 14
M e t h a n o l (2.5%)+ TPCK(0.23 mM)
1 3
3 7
77 81
M e t h a n o l (2.5%)+ T L C K ( 0 . 2 5 raM)
1 3
6 17
54 53
such specificity also exists in the case of the factor involved in the synthesis of ppGpp, we examined the effect of these alkylating agents on this system. It appears (Table IV) that preincubating the ribosomal wash containing the stringent factor with TPCK nearly completely inhibits the reaction. On the other hand, a smaller but significant inhibition effect can be observed in the case of similar concentrations of TLCK. Control experiments demonstrated that in both cases the effect of the inhibitors is due to their action on the stringent factor because the addition of comparable amounts of TPCK and TLCK to the reaction mixtures at the start of the incubation was much less inhibitory (not shown).
The inhibition of ppGpp synthesis by ATA ATA has been shown to inhibit m R N A binding to ribosomes and thus inhibit protein synthesis initiation in vitro [ 2 5 , 2 6 ] , and also to decrease EF Ts activity, resulting in the inhibition of peptide chain elongation [27]. In addition, a recent report indicates that ATA may be a general inhibitor of nucleic acid binding proteins involved in protein and nucleic acid synthesis [ 28 ]. For these reasons, the effect of ATA on ppGpp synthesis in vitro was tested. Our results indicate that it also inhibits the in vitro synthesis of ppGpp (Table V). Under the assay conditions specified, 50% inhibition is observed at 5 • 10 -s M ATA. In addition, as illustrated in Fig. 2, the extent of the reaction at a specified ATA concentration depends on the amount of ribosomes added to the reaction under conditions in which the stringent factor is saturated with ribosomes.
ppGpp and pppGpp synthesis in heterologous systems Richter has shown that ribosomes from yeast, reticulocyte and calf brain cytoplasm cannot substitute for E. coli ribosomes in the synthesis of ppGpp, while yeast mitochondrial ribosomes have some activity when complemented
87 TABLE V T H E I N H I B I T I O N O F p p G p p S Y N T H E S I S BY A T A The experimental complete
in t h e t e x t , e x c e p t t h a t t h e i n c u b a t i o n s w e r e a t 37°C. T h e 6.2 # g of 0 . 5 M NH4C1 w a s h p r o t e i n a n d 0 . 3 4 m g h i g h s a l t - w a s h e d w h i c h w e r e a d d e d l a s t . T h e r e a c t i o n t i m e w a s 2 h. procedure
reaction
ribosomes
w a s as d e s c r i b e d
mixtures
contained
Final ATA
% Inhibition
(X 105M)
% Conversion of GTP to p p G p p and pppGpp
Complete --Ribosomes
---
90 4
---
--Stringent factor
--
9
--
System
concentration
Complete
1.3
84
7
2.6 5.2
81 45
10 50
6.7 10.7
22 4
76 93
with the bacterial stringent factor [ 1 3 ] . Sy et al. have recently reported that the E. cola stringent factor can also complement ribosomes obtained from Bacillus brevis and Chlamydomonas reinhardtii chloroplasts [ 2 9 ] . In this respect we have observed that ribosomes from P. fluorescens can efficiently substitute for E. cola ribosomes in the complete system containing the 0.5 M NH4C1 wash of E. cola ribosomes (Table VI). In addition, a much less efficient but reproducible reaction can be observed when the ribosomal wash from E. cola is complemented with ribosomes obtained from wheat germ. However, there is no detectable conversion of GTP to either ppGpp or pppGpp when E. cola ribosomes are replaced with ribosomes obtained from Ehrlich ascites cells. The bacterial ribosomes and the ribosomes obtained from the eukaryotic cells
8C l
o
LO
40
"-
"x "-
2O
"x ".
§
"x -
>
00 ~ 0
I 0,01
0,02
i
I
0103
0.04
x i "s 0 ~
~
xc~
O, ~
007
FINAL ATA CONCENTRATION, mM
Fig. 2. T h e d e p e n d e n c e o f t h e i n h i b i t i o n
synthesis by A T A on ribosome concentration. The in t h e t e x t , e x c e p t t h a t t h e i n c u b a t i o n s w e r e at 3 7 ° C . T h e c o m p l e t e r e a c t i o n m i x t u r e s c o n t a i n e d 10.7 #g o f 0 . 5 M N H 4CI w a s h p r o t e i n , t h e i n d i c a t e d c o n c e n t r a t i o n o f A T A a n d e i t h e r 0 . 1 4 m g ( l ) , 0 . 2 7 m g (o) o r 0 . 3 4 m g (A) high s a l t - w a s h e d r i b o s o m e s . T h e r e a c t i o n t i m e was 1 h. experimental
procedure
was as described
of ppGpp
88 were active in the polyuridylic acid-dependent synthesis of polyphenylalanine when complemented respectively with bacterial and eukaryotic supernatant factors (not shown). Discussion Kaplan et al. [18] have recently demonstrated that chloramphenicol has no effect on ppGpp synthesis in vivo in a strain of E. coli (5F2 relA ÷) in which reaccumulation of charged tRNA does not take place at the nonpermissive temperature. In the same strain tetracycline was found to completely inhibit the reaction. The respective antibiotics mimic their in vivo effect in cell-free systems isolated from CP 78 reIA ÷. On the other hand the inhibitory effect of fusific acid in vitro was not found in the case of the intact cells. This antibiotic probably inhibits ppGpp synthesis in vitro by its ability to bind EF G present in the salt wash and GTP to the ribosomal acceptor site [30], thus preventing the interaction of uncharged tRNA and possibly the stringent factor with the ribosomes. Our finding that saturating the ribosomes with EF G, GTP and fusidic acid results in complete inhibition of the reaction, as well as the inhibitory effect of chlortetracycline supportsthis view. Tetracycline is known to inhibit the interaction of both aminoacyl-tRNA and uncharged tRNA with ribosomes [31,32]. The lack of inhibition in the case of chloramphenicol and sparsomycin suggests that the peptidyltransferase site on the ribosome is not involved in ppGpp synthesis. In view of the specific effects of the various antibiotics on this reaction, it is surprising that a lack of requirement for the ribosomal proteins L7 and L12 is ppGpp synthesis is observed. These proteins are involved in the activity of the elongation factors which interact with the acceptor site, namely EF Tu and EF G [16,20], although the requirement for L7 and L12 can be minimized under certain conditions [33]. These observations also indicate that ribosomes functionally inactivated with respect to protein synthesis retain the ability of synthesize ppGpp. Unlike the rapid and specific inhibition of EF Tu by TPCK [24], the stringent factor is inactivated by this alkylating reagent only after prolonged incubation. In addition, as has been pointed out by Jonak and Clark [12], complete inhibition is only observed at very low stringent factor concentrations, even though TPCK is in great excess in these experiments. These observations, along with our finding that TLCK also has a significant inhibitory effect in this system, suggest that these compounds inhibit the reaction by nonspecific alkylation of reactive groups on the stringent factor. On the other hand, ATA is a rapid and specific inhibitor of ppGpp synthesis, and this inhibition is dependent on ribosome concentration. It is likely that this inhibition is due to the previously reported ability of ATA to inhibit the binding of mRNA, including polyuridylic acid, to ribosomes [25] since ppGpp synthesis is dependent on messenger RNA [10]. It now appears that the stringent factor obtained from E. coli ribosomes can complement purified ribosomes from other bacteria, such as B. brevis [29], and P. fluorescens (Table VI), as well as ribosomes isolated from cell organelles such as chloroplasts [29] and mitochondria [13]. On the other hand, ribo-
89 T A B L E VI ppGpp AND pppGpp SYNTHESIS IN HETEROLOGOUS
SYSTEMS
Reaction mixtures were incubated at 37°C for 2 h. Where indicated, 10.5/~g of 0.5 M NH4CI wash protein o b t a i n e d f r o m E. coli r i b o s o m e s w a s i n c l u d e d in t h e r e a c t i o n . This stringent f a c t o r c o n t a i n i n g ribosomal w a s h p r e p a r a t i o n w a s r e c e n t r i f u g e d a t 1 3 5 0 0 0 X g f o r 4 h before use in o r d e r t o r e m o v e c o n t a m i n a t i n g E. coli ribosomes. Source of ribosomes (mg)
0.5 M NH4CI wash protein
% Conversion of GTP to ppGpp and pppGpp
None
+
1
E. coli ( 0 . 1 4 ) E. coli ( 0 . 0 7 )
-+
2 84
P. f l u o r e s c e n s ( 0 . 1 4 ) P. f l u o r e s c e n s ( 0 . 0 7 )
-+
2 87
Wheat g e r m ( 0 . 1 7 ) Wheat g e r m ( 0 . 1 7 )
-÷
2 7
Ehriich ascites (0.08) Ehrlich ascites ( 0 . 0 8 )
-+
1 1
somes obtained from the cytoplasm of eukaryotic cells such as yeast, brain [ 1 3 ] , C. reinhardtii [29] and Ehrlich ascites tumor cells (Table VI) are completely inactive in this system. The slight activity obtained in the case of the wheat germ ribosomes appears to be an exception which w e are investigating in more detail in order to eliminate a number of possible artifacts.
Acknowledgements This investigation was supported by a Public Health Service Research Career Development Award No. 6-K4-GM-9523 from the Institute of General Medical Sciences to J. L.-L., a Postdoctoral Research Fellowship Award No. 5 FO2 CA53660-02 from the National Cancer Institute to L.B., and by PHS Research Grant No. ROI CA 1 2 1 4 4 from the National Cancer Institute. We are grateful to John Micthell for helpful discussions and assistance with the initial experiments of this study.
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