Identification of the Synthesis of Guanosine Tetraphosphate (MS I) as Insertion of a Pyrophosphoryl Group into the 3′-Position in Guanosine 5′-Diphosphate

Abstract

The phosphate transfer system of Haseltine et al., consisting of a ribosomal wash obtained from a stringent strain of Escherichia coli, washed ribosomes, GTP, and ATP, was used to prepare large quantities of guanosine tetra-and pentaphosphates, the magic spot compounds MS I and MS II of Cashel and Gallant. In our hands, the Haseltine et al. system yielded predominantly guanosine tetraphosphate, ppGpp. This system was used exclusively in the described experiments, with ATP labeled with 32p in the A-and ey-positions as donor. The #-label was found to produce a ppGpp and the y-label a ppGp*. Furthermore the stringency effect, the translocation with which this laboratory has been concerned for some time (4). In the present report we have exploited the availability of a relatively easy method (2) of obtaining sizeable quantities of the "magic spot" guanosine polyphosphates for analysis of the mechanism of the transfer reaction. By obtaining transfer products from ATP marked at various positions, it has been possible to show: (i) that synthesis from ATP is due to pyrophosphoryl transfer, and (ii) that this transfer yields the 3'-pyrophosphoryl derivative of guanosine 5'-polyphosphates. MATERIALS The stringent strain used for preparation of the enzyme system was E. coli K-19 (strepr, thr-, leu-, B1-, galt, ac-, Fe); it was kindly supplied by Dr. Norton Zinder and checked for stringency by Dr. Peter Model. The organism was grown in bulk at the Oak Ridge National Laboratory, with the most valuable cooperation of Dr. Novelli's group in the Biology Division. There it was processed through to ribosomes, from which the enzyme system was prepared in this laboratory. The NH4Cl-washed ribosomes and 0.5 M NH4Cl ribosomal wash were prepared as described (2).Yeast inorganic pyrophosphatase (840 units/mg) and 3'-nucleotidase from Rye Grass (14 units/mg) were purchased from Sigma. Carrier-free 82p, [a-_2P] Since the aim of the present study was to explore the transfer mechanism of phosphate from ATP to GTP and/or GDP, we set up the system as described (2) and followed the course of reaction using GTP and ATP as the reactants. As shown in Fig. 1, the presently used system includes ribosomes and EF-G, and the GTP is rapidly converted to GDP. The figure shows that, in the beginning, with mostly GTP present, the reaction starts with relatively small amounts of guanosine 306

Guanosine 5′-Diphosphate, 3′-Diphosphate: Assignment of Structure by 13 C Nuclear Magnetic Resonance Spectroscopy

“…There is a clear indication from these spectra that the pyrophosphate residues are indeed attached to the 5' and 3' ribose carbons and that there are no indications of unusual spectral shifts in the guanosine moiety of ppGpp. Thus, these determinations with the intact compound clearly confirm the assignments made by Sy and Lipmann using evidence from enzyme specificity with guanosine 5'-phosphate, 3'-phosphate, the hydrolytic product of ppGpp (23).…”

“…While this work was in progress, Sy and Lipmann reported localization of the pyrophosphate residues to the ribose 5' and 3' positions based on the hydrolytic activities of rye grass 3' phosphomonoesterase towards guanosine 5'-phosphate, 3'-phosphate, which in turn was produced by hydrolysis of the ,3-phosphates of ppGpp with zinc-activated pyrophosphatase from yeast (23 The degree of hydrolysis of the compound was examined by PEI-cellulose thin-layer chromatography by the method of Cashel (6), which separates guanine nucleotides on the basis of their phosphate content. Chromatography of samples containing from 3.5-175 nmol of guanosine tetraphosphate revealed no contaminants despite the fact that as little as 1-2 nmol of nucleotide can be detected by UV absorption.…”

Relationship of the first step in protein synthesis to ppGpp: formation of A(5')ppp(5')Gpp.

“…The discovery by Cashel and Gallant (9) of the two guanine nucleotides, ppGpp and pppGpp, responsible for the stringent response of prokaryotic cells upon amino-acid starvation or a nutritional shift-down in growth medium, was followed by active research into their mechanism of formation (10,11), turnover (12,13), and mode of action (14). The accumulation of ppGpp was shown to be due to the recognition of a codon and binding of an uncharged tRNA to the acceptor site on the ribosomes (10,15).…”

Reversibility of the Pyrophosphoryl Transfer from ATP to GTP by Escherichia coli Stringent Factor

“…The guanosine nucleotides, GMPCPP (f3-y-methylene-guanosine triphos- The reverse reaction was assayed as described in Methods. Guanosine polyphosphate synthesis was assayed as described (2). GTPase was assayed by a chromatographic method: 50 ,l of reaction mixture containing 40 mM Tris.OAc, pH 8.0, 1 mM dithiothreitol, 10 mM Mg(OAc)2, 2 mM (a-32P]GTP (32 Ci/ mole), 45 jig of ethanol-ammonium-chloride-washed ribosomes, 3 jg of washed proteins L7 and L12, and 0.05 jug of EF G (elongation factor G) as indicated were incubated at 300 for 15 min.…”

“…The synthesis of guanosine 5'-tri-(or di-)phosphate-3'-diphosphate has been elucidated recently. This formation is catalyzed by a pyrophosphoryl transferase (stringent factor) that transfers the f3 and y phosphates of ATP as a unit to the 3'-OH group of GDP and GTP (1)(2)(3). The transferase requires a complex of ribosome, rn1NA and codon-specific uncharged tRNA for activity as-described (4,5).…”