The sequence of ppGpp and pppGpp in the reaction scheme for magic spot synthesis

123

Biochimica et Biophysica Acta, 442 ( 1 9 7 6 ) 123--127 © Elsevier Scientific Publishing Company, A m s t e r d a m -- Printed in The Netherlands

BBA Report BBA 91433

THE SEQUENCE OF ppGpp AND pppGpp IN THE REACTION SCHEME FOR MAGIC SPOT SYNTHESIS

W I C H E R J. W E Y E R , H E R M A N MAX GRUBER

A. de BOER, J O H A N G. de B O E R and

Biochemisch Laboratorium, The University,Zernikelaan, Groningen (The Netherlands) (Received March 26th, 1976)

Summary The kinetics of ppGpp (guanosine 5'-diphosphate,3'-diphosphate) and pppGpp (guanosine 5'-triphosphate,3'-diphosphate) synthesis, at the onset of amino acid starvation, and of their decay after inhibiting synthesis, were analysed in Escherichia coli. The pentaphosphate, but not ppGpp, is the first product of the stringent response to amino acid starvation. The pentapho~ phate is rapidly converted with first order kinetics (tl/2 = 6 s) to ppGpp which is broken down less rapidly (tl/2 = 20 s).

In amino acid deficiency [ 1,2] the concentration of the regulatory nucleotides guanosine 5'-diphosphate,3'-diphosphate (ppGpp) and guanosine 5'-triphosphate,3'-diphosphate (pppGpp), originally termed Magic Spot I and II, respectively, is increased to a new steady state level which is maintained by continuous synthesis and breakdown [3,5]. Under conditions of reduced energy production the concentration of ppGpp increases, but not that of pppGpp [3--5]. The synthesis of these compounds in vitro [6,7], and also in vivo [8], has been shown to require ribosomes, uncharged tRNA, mRNA, the ribosome-bound stringent factor, ATP as pyrophosphate donor and GDP or GTP as pyrophosphate acceptor [9]. Although from the m o m e n t of discovery of the Magic Spots a high rate of degradation with first order constants of 1--3/min has been observed in vivo [2,10] no degradation of ppGpp in vitro, for instance in a cell-free system or permeable cells, has been effected (ref. 11 and unpublished results). Several authors [ 12--15] preferred a degradation scheme in which ppGpp is first phosphorylated to pppGpp which would then be further degraded in an unknown way. They based their conclusion on the 10--30-fold lowered rate of breakdown of ppGpp and the absence of pppGpp in so-called s p o T - mutants [12,13]. A similar p h e n o m e n o n can be phenotypically induced by interference with the cellular energy

124

production [4,5] and by levallorphan treatment [14,15]. This model implies a pyrophosphorylation of GDP to form ppGpp as the first product of the stringent reaction. However, some arguments can be put forward for a model in which GTP, instead of GDP, is pyrophosphorylated to form pppGpp as the first product of the stringent reaction. First, all ribosomal reactions require GTP, secondly, the intracellular level of GTP is 6--10 fold that of GDP and thirdly, the in vitro ribosome-stringent factor system can pyrophosphorylate GTP as well as GDP [16]. The former reaction results in the formation of pppGpp wnich in turn can be converted to ppGpp by the ribosome-elongation factor G (EF G) complex [16]. The conversion of ppGpp to pppGpp, an obligatory step in the current model has, to our knowledge, never been observec~ Each of both models can be represented by its reaction scheme. Both are based on a simple precursor-product relationship since the two nucleotides are metabolically related. (I)

ATP + GDP ~ ppGpp ~ pppGpp ~ Y

(II)

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Fig.1. Kinetics of p p G p p and p p p G p p b r e a k d o w n after chloramphenicol addition. E. co|i W 1 (|eu, arg, his, thr, pro, thi, spoT +, R C +) was exponentially g r o w n in a Tris/glucose low-phosphate m e d i u m [2] with appropriate supplements at 37 ° C. T h e culture was labeled for one generation time with about 1 0 0 ~Ci/ml 32Pi before induction of isoleucine starvation by addition of valine (800 ~Ug/ml). 30 m i n after the onset of a m i n o acid starvation, chloramphenicol (100 ~g/ml) w a s added. T h e intracelhlar concentration of the nucleotides p p O p p (o--o) and p p p G p p (o--~) was determined as d,,scribed (De Boer et al., submitted for publication). T h e arrows indicate the m o m e n t of chloramphenicol addition. T h e dotted lines represent the simulated degradation curves of p p G p p and p p P G p p based o n S c h e m e I (right panel) and based on S c h e m e II (left panel). T h e simulation was started 7 s after chloramPhenicol addition. In the right and left panels the s a m e experimental points axe used. N o t e that in the left panel the experimental points closely fit the simulated degradation curve. T h e equations used to calculate all the simulation curves were for S c h e m e I: d(MSl)/dt =--k I ( M S I ) +

v I andd(MSIl)/dt=k

I ( M S I)--k 2 ( M S II)

for S c h e m e If: d ( M S II)/dt =--k 2 ( M S If)+ v 2 a n d d ( M S I ) / d t = k 2

( M S II)--k] ( M S I )

(hl = first order degradation rate constant of M S I; k~ = first order degradation rate constant of M S If; vj = rate of M S I synthesis; v: = rate of M S II synthesis). In the equations the experimentally determ i n e d values for k I and k 2 of 3.2 m i n -I and 6.4 m i n -~ , respectively, were used and the experimentally determined values for the initial (steady-state) concentrations of M S I and M S II of 2 8 5 pmol/E450 and 114 pmol/E4s 0, respectively. In the simulation curves in this figure, v I = 0 and % = 0 by the effect of chloramphenic oL

125 The degradation of ppGpp proved to be energy requiring [5,13,15]. In the first scheme this energy-requiring step is supposed to be the phosphorylation of ppGpp to pppGpp, in the second scheme this requirement must involve the conversion of ppGpp in an u n k n o w n way and system. The present study was undertaken to determine the sequence of the tetra- and pentaphosphate in the turnover of Magic Spots. We worked at this problem by measuring the kinetics of breakdown of both nucleotides in a wildtype strain of E. coll. Accumulation of both components to a steady state concentration was induced by the addition of valine which induces a partial isoleucine starvation. By addition of chloramphenicol, Magic Spot synthesis was blocked and degradation ensued. The rapid declining concentration of both nucleotides was determined at very short intervals (1.8 s). Fig. 1 shows that the pentaphosphate pppGpp decreases according to a clear first-order degradation curve, after a lag time of 7 s. This lag time is probably necessary for the full action of chloramphenicol. However, the tetraphosphate ppGpp follows these kinetics only later and after the pentaphosphate concentration has dropped to low levels. In this way an apparent lag time of more than 7 s is observed. These data are in full agreement with Scheme II, b u t cannot be reconciled with Scheme I. The same results are obtained when isoleucine starvation is relieved by addition of this amino acid. Simulated curves using the final degradation constants and the initial steady state Magic Spot concentrations illustrate this point. In Cashel's paper (ref. 2, Fig. 5) describing the discovery of the unusual nucleotides an apparent longer lag time for Magic Spot I (MS I) than for Magic Spot II (MS II) after chloramphenicol treatment is also observed. If the tetraphosphate is formed via the pentaphosphate according to Scheme II this could be reflected in the shape of the accumulation curves of both components immediately after amino acid deficiency has started Magic Spot synthesis. Fig. 2 shows that this prediction is borne out. Again, as the simulated curves show (Fig. 3), only Scheme II agrees with the data. It is emphasized that with Scheme I and the measured degradation rate constants of 3.2 and 6.4 rain -1 of MS I and MS II, respectively, the concentration of both nucleotides can never become equal. The experimental curve clearly shows that this ratio is obtained. Thus, Scheme I must be rejected while Scheme II can explain our data without further assumptions. It must be emphasized that our data do not exclude the possibility of a minor pathway to ppGpp by pyrophosphorylation of GDP. This contribution can be estimated using the equation k 2 / k l = [ p p G p p ] / [ p p p G p p ] which is valid for Scheme II as well as Scheme 1 under steady state conditions. From Fig. 2 it can be seen that the quotient of the degradation constants is almost equal to the quotient of the nucleotide levels. So the contribution to the ppGpp concentration by independent synthesis will be within the experimental error, at most 10--20%. An apparent problem is formed by the spoT- strains in which no guanosine pentaphosphate can be detected. We have evidence, however, that besides the reduced rate of breakdown a strongly reduced rate of Magic Spot synthesis in such a strain occurs (unpublished). If the conversion of MS II into MS I proceeds normally, no MS II can be expected to accumulate in spoT- strains' according to Scheme II.

126

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MSII~MS[

MS I---MS II

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Fig.3. S i m u l a t e d a c c u m u l a t i o n c u r v e s o f p p G p p a n d p p p G p p a f t e r a m i n o a c i d s t a r v a t i o n . Basal level o f ppGpp (@--o) a n d p p p G p p ( o - - o ) . T h e d o t t e d lines r e p r e s e n t t h e s i m u l a t i o n c u r v e s f o r p p G p p a n d pppGpp a c c u m u l a t i o n b a s e d o n S c h e m e I ( r i g h t p a n e l ) a n d b a s e d o n S c h e m e I I (left p a n e l ) . T h e e q u a t i o n s u s e d a r e d e s c r i b e d in t h e l e g e n d o f Fig. 1. F o r k I a n d k 2 v a l u e s o f 3.2 m i n - I a n d 6.4 r a i n - I , r e s p e c t i v e l y , w e r e u s e d ( t a k e n f r o m Fig. 1). T h e s e v a l u e s h a v e b e e n s h o w n in o t h e r e x p e r i m e n t s t o b e c o n s t a n t b e f o r e a n d at l e a s t up to I h a f t e r a m i n o a c i d s t a s v a t i o n . F o r t h e i n i t i a l ( b a s a l ) c o n c e n t r a t i o n s o f MS I a n d MS II t h e e x p e r i m e n t a l l y d e t e r m i n e d v a l u e s o f 2 0 p m o l / E 4 s 0 a n d 7 . 5 p m o l / E 4 5 0 w e r e u s e d ( t a k e n f r o m Fig. 2). v I a n d % w e r e c a l c u l a t e d f r o m t h e e x p e r i m e n t a l a c c u m u l a t i o n c u r v e s ( F i g . 2) u s i n g t h e e q u a t i o n s d ( M S I ) / d t = - - k I (MS I) + v I a n d d ( M S I I ) / d t = - - k 2 (MS I I ) + v 2 , r e s p e c t i v e l y , w h e r e d ( M S I ) / d t a n d d ( M S I I ) / d t is t h e s l o p e o f t h e a c c u m u l a t i o n c u r v e .

127

We conclude that Scheme II is the most probable sequence. The pentaphosphate then would be the immediate product of the stringent reaction and the precursor of the tetraphosphate. We propose that this reaction is catalyzed by EF G. The ppGpp degradation reaction which requires energy is probably therefore not its phosphorylation to pppGpp. Its nature still remains a mystery, We thank Jenny Brands for her excellent and dedicated technical assistance, Apple van Ooyen and Geert AB for helpful discussions and critically reading the manuscript. We a r e i n d e b t e d t o Dr. J. G a l l a n t f o r p r o v i d i n g u s w i t h t h e b a c t e r i a l s t r a i n u s e d in t h i s s t u d y . The present investigations were carried out under the auspices of the Netherlands Foundation of Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).

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