Proc. Nat. Acad. Sci. USA Vol. 71, No. 9, pp. 3470-3473, September 1974 Reversibility of the Pyrophosphoryl Transfer from ATP to GTP by Escherichia coli Stringent Factor (guanosine 5'-diphosphate-3'-diphosphate/guanosine 5'-triphosphate-3'-diphosphate/relaxed control/ribosomes) JOSE SY The Rockefeller University, New York, N.Y. 10021 Communicated by Fritz Lipmann, July 1, 1974 ABSTRACT The stringent factor-catalyzed, ribosome- was incubated for 60 min at 300 in a 1W0-1 mixture contain- dependent synthesis of guanosine polyplosphates is ing: 50 mM Tris OAc, pH 8.1; 4 mM dithiothreitol; 20 mM found to be reversible. The reverse reaction specifically requires 5'-AMP as the pyrophosphoryl acceptor, and Mg(OAc)2; 5 mM ATP; 10 ,ug of poly(A, U, G); 27 pgof tRNA; guanosine 5'-triphosphate-3'-diphospbate is preferentially 90',g of ethanol-washed ribosomes; and 2 p&g of fraction II utilized as the pyrophosphoryl donor. The primary prod- stringent factor. By minimizing GTPase activity with the ucts of the reaction are GTP and ANP. The reverse reaction use of high Mg++ concentration and ethanol-washed ribo- is strongly inhibited by the antibiotics thiostrepton and a ob- tetracycline, and b' A P and,1-y-pnethylene-adenosixie- somes with a minimal time of incubation, product is triphosphate, but not by ADP, GTP, and GIDP. The reverse tained that is more than 95% pppGpp. After incubation, 1 Al reaction occurs under conditions for nonribosomal syn- of 88% HCOOH was added and the resulting precipitate dis- thesis. The ove'rill reaction for stringent factor-catalyzed carded. The supernatant was neutralized with KOH and diluted guanosine polyphosphate formation may thus be formu- to 1 ml with 50 mM triethylamine-HCOs buffer, pH 7.7. The lated: '+ ppp5'A = (p)pp5'og'pp p5'A. (p)pp5'G + sample was then applied to a 1-ml DEAE-cellulose (Whatman The synthesis of guanosine 5'-tri-(or di-)phosphate-3'-di- 52) column that had been equilibrated with 50 mM triethyl- phosphate has been elucidated recently. This formation is amine-HCO3 buffer. The column was washed successively with catalyzed by a pyrophosphoryl transferase (stringent factor) the 'following concentrations of triethylamine-HCO3 buffer: that transfers the f3 and y phosphates of ATP as a unit to the 1.0 ml each of 50 mM, 100 mM, and 150 mM; 7 ml of 200 3'-OH group of GDP and GTP (1-3). The transferase requires mM; and 0.5 ml of 0.5 M. pp*Gpp was then completely a complex of ribosome, rn1NA and codon-specific uncharged eluted from the column with an additional 1 ml of 0.5 M tri- tRNA for activity as-described (4, 5). Under appropriate con- ethylamine-HCOs buffer. The buffer was removed by lyophi- ditions, however, transferase activity can be obtained'in the lization and pp Gpp was redissolved in a small amount of absence of the ribosomal complex by performing the assay in water. The overall yield of pppGpp was about 70% of input 20%/ methanol and at lower temperatures (3). GTP. Aliquots of the nucleotides were stored in liquid nitro- Since the 3'-phosphate and pyrophosphate linkages of gen for maximal stability. pppGpp was also prepared by the ppGpp and pppGpp should be of a high-energy nature (6), same method with [3-,32P]ATP'(28 Ci/mmol) as the pyro- reversibility of the transferase reaction was to be expected. phosphate donor (2). Large quantities of unlabeled' ppGpp I wish to report that the stringent factor does catalyze a and pppQpp were prepared by a scaled-up version of this pyrophosphoryl transfer reaction in reverse, and that pppGpp method, or by a continuous-flow method using Aminco mem- preferentially acts as the pyrophosphoryl donor. branes. MATERIALS Assay for the Reverse Reaction. The standard reaction mix- ture contained in 20 pl: 20 mM Tris OAc at pH 8.1, 0.1 mM Escherichia coli K-19 (stringent) ribosomes and fraction II dithiothreitol, 10 mM Mg(OAc)2, 1.5 X 10- M pp*pGpp (20 stringent factor were prepared as described (3). [a-32P]GTP Ci/mole), 10 mM 5'-AMP, 29 pg of ammonium chloride- and [F-132P]ATP were obtained from ICN. E. coli uncharged washed ribosomes, 1 pug of fraction II stringent factor, 2 pAg of tRNA was purchased from Schwarz/Mann and poly(A, U, G) poly(A, U, G), and 5 pig of tRNA. Incubations were at 300 from Miles Laboratories. for 90 min and the reactions were stopped by the addition of METHODS HCOOH. The resulting precipitates were centrifuged and 10 pl of the supernatant spotted on polyethyleneimine-cellulose Preparation of Various Guanosine Polyphosphates. I'H]- plates. The thin-layer sheets were desalted by immersion in ppGpp was prepared as described (2) with [3H]GDP (9.5 Ci/ absolute methanol (7) and developed with 1.5 M KH2PO4, pH mmol) as acceptor, and 5'_[at-2P]pp*Gpp was prepared by 3.4 (8). The resulting chromatograms were radioautographed the following method. [a-32P]GTP (1.72 Ci/mmol, 0.2 mM) overnight and spots corresponding to the various guanosine derivatives were cut out and counted. The % of GTP + GDP formed represents the amount of input radioactivity con- Abbreviations: ppGpp, guanosine 5'-diphosphate-3'-diphosphate; verted into GDP + GTP. pppGpp, guanosine 5'-triphosphate-3'-diphosphate; an asterisk RESULTS over a 'p', e.g., p, denotes a 82P-label in that position; AMPP- CP, ,--y-methylene-adenosine triphosphate; GMPPCP, f--y- In vitro synthesis of guanosine polyphosphates requires, for methylene-guanosine triphosphate; tRNA, transfer RNA. fast reaction, the activation of stringent factor by the ribo- 3470 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Reversibility of Guanosine Polyphosphate Synthesis 3471 some-mRNA-tRNA complex (3). The requirements for the reverse reaction are shown in Table 1 and are identical to those for the forward synthetic reaction (1, 4, 5), i.e., ribo- somes, stringent factor, poly(A, U, G) as synthetic message, and uncharged tRNA are essential, in addition to 5'-AMP as the pyrophosphoryl acceptor. Using pp*Gpp, the major radioactive products after a 25-min incubation are GTP and GDP (Fig. 1). After longer incubation (90 min) or when a lower initial pppGpp concen- tration is used, the final products usually are only GDP and ADP. This result is probably due to the presence of both GTPase and ATPase in our partially purified stringent factor preparation. With pppGpp, the major radioactive product is ATP, with some activity also in ADP, Pi, and PPi (Fig. 1); fi*5 - ADP after longer incubation, only ADP and Pi can be detected. All radioactive nucleotide products were further identified GDP -- by cochromatography with nucleotide standards in two ATP dimensional thin-layer chromatography on polyethylene- _" imine-cellulose: (1) 1st dimension, 3.3 M ammonium formate + 4.2% boric acid brought to pH 7 with NH40H, and 2nd GTP -_ dimension, 0.75 M KH2PO4, pH 3.4; (2) 1st 1.5 M dimension, - PPj LiCl, and 2nd dimension, 0.75 M KH2PO4, pH 3.4. Using double-labeled pppGpp, I have found that a stoichiometric amount of both guanosine and adenosine nucleotides is formed ppGpp ppGpp pppGpp - pppGpp in the reverse reaction (data not shown). -- Origin Table 2 shows that 5'-AMP is specific as the pyrophos- (a) (b) phate acceptor. Neither adenosine, ADP, ATP, guanosine, GMP, GDP, nor GTP has any acceptor activity. In the FIG. 1. Identification of nucleotide products. The reverse re- absence of 5'-AMP, there is a slow hydrolytic activity of action was assayed as described in Methods. An incubation mix- pppGpp by the stringent factor-ribosome complex. Under the ture in 20,4 contained: 50 mM Tris - OAc, pH 8. 1; 0.2 mM dithio- assay conditions, the reverse reaction as determined by GDP threitol; 15 mM Mg(OAc)2; 10 mM 5'-AMP; 29 ,ug of washed formation occurs at a linear rate for at least 50 min. Concen- ribosomes; 1 ug of fraction II stringent factor; 2 pug of poly- U, 5 pg was trations of 10-15 mM Mg++ were optimal for the reverse (A, G); and of tRNA. Incubation at 300 for 25 min. reaction. In contrast to the synthetic reaction (4, 9), Mg++ pppGpp (0.22 mM, 12,000 cpm) was used in (a); pppG~p (0.22 mM, 30,000 cpm) was used in (b). ppGpp, ATP, GTP, concentrations higher than 15 mM were found to be inhibi- ADP, and GDP were used as markers in chromatography. The reverse tory for the reaction. chromatograms were developed in 0.75 M KH2PO4, pH 3.4, and Both the synthetic and the reverse reactions were found then radioautographed. not to be dependent on the presence of the 50S ribosomal pro- teins L7 and L12. This is shown in Table 3 where ribosomes washed with ethanol-ammonium chloride were equally active analogous to the forward reaction in its equal sensitivity to in both reactions regardless of the presence or absence of the low concentrations of thiostrepton and tetracycline (Table 4). washed proteins L7 and L12. The EF G-dependent GTPase, Under the assay conditions, the formation of GDP from however, was strictly dependent on the presence of L and pppGpp is linear with the concentration of 5'-AMP up to 5 L12, as has been reported (10). The reverse reaction is also mM (Fig. 2). The presence of 10 mM 5'-AMP in the synthetic TABLE 1. Requirement for the reverse reaction TABLE 2. Specificity of the acceptor molecule % GDP % GDP formed Nucleotide formed Complete 24.6 None 2 -5'-AMP 1.3 5'-AMP 20 -ribosomes 1.7 ADP 1 -stringent factor 0.2 ATP 0 -poly(A, U, G) 7.3 GMP 1 -poly(A, U, G), tRNA 3.2 GDP 0 -ribosomes, stringent factor 0.3 GTP 0 No addition 0.3 Adenosine 2 Guanosine 2 The assays are described in Meds.