Characterization of the guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-diphosphate degradation reaction catalyzed by a specific pyrophosphorylase from Escherichia coli.

Guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) are specifically degraded by a manganese-dependent pyrophosphorylase present in spoT+ but not in spoT- strains of Escherichia coli, indicating that the enzyme is the spoT gene product. The enzyme catalyzes the release of pyrophosphate from the 3' position of ppGpp or pppGpp, yielding ppG and pppG, respectively; pppGpp could not be detected as an intermediate in the decay reaction. Degradation of (p)ppGpp is optimal in the presence of 200 to 300 mM potassium or sodium acetate, at a pH of 7.5 to 8 and a temperature of 37 degrees C.     

The guanosine 5'-diphosphate D-mannose: guanosine 5'-diphosphate L-galactose epimerase of Chlorella pyrenoidosa. Chemical synthesis of guanosine 5'-diphosphate L-galactose and further studies of the enzyme and the reaction it catalyzes.

An enzyme from extracts of the green alga Chlorella pyrenoidosa that catalyzes the reversible epimerization of guanosine 5′-diphosphate d-mannose to guanosine 5′-diphosphate l-galactose was further purified. The substrate guanosine 5′-diphosphate l-galactose was made chemically by the morpholidate procedure. An improved method was developed for the synthesis of an intermediate in that process, β-l-galactopyranosyl phosphate, via an orthoester of l-galactose. Various characteristics of the enzyme and the reaction it catalyzes were studied. A new method using gas-liquid chromatography was introduced for following the course of the reaction with unlabeled substrates.     

Correlation between RNA synthesis and basal level guanosine 5'-diphosphate 3'-diphosphate in relaxed mutants of Escherichia coli

Through the use of a new nucleotide extraction procedure, we had previously shown that relaxed mutants of Escherichia coli exhibit a unique response to amino acid starvation (Lagosky, P. A., and Chang, F. N. (1980) J. Bacteriol. 144, 499-508). The basal level amounts of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA and phenotypically relaxed relA+ rplK (relC) strains were shown to decrease at the onset of amino acid limitation and to remain severely depressed throughout the course of the starvation. Upon resupplementation of amino acid-starved relaxed mutants, the production of ppGpp resumes and results in the temporary overaccumulation of this nucleotide beyond its original basal level amount. We now show that the basal level ppGpp content of relaxed bacteria, as well as its subsequent fluctuations in response to amino acid starvation, is inversely correlated with the initial rates of RNA synthesis in these strains. The ability of ppGpp to control the rate of protein synthesis in relA mutants was also examined. It was observed that ppGpp had no apparent direct effect on the initial rates of protein synthesis in relA mutants. The constant inverse correlation which exists between ppGpp content in relA mutants, and their rates of RNa synthesis provide evidence which indicates that basal level ppGpp synthesis has definite physiological significance. It also suggests that the synthesis of basal level ppGpp might be an absolute requirement needed for normal bacterial growth.     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.     

Role of peptide chain elongation factor G in guanosine 5'-diphosphate 3'-diphosphate synthesis.

In a wild-type strain (relA+) of Escherichia coli, starvation of amino acid led to an immediate cessation of the synthesis of stable ribonucleic acids, together with the accumulation of an unusual nucleotide, guanosine 5'-diphosphate 3'-diphosphate, commonly known as ppGpp. This compound also accumulated during heat shock. When temperature-sensitive protein synthesis elongation factor G (EF-G) was introduced into E. coli NF859, a relA+ strain, the synthesis of ppGpp was reduced to approximately one-half that of wild-type EF-G+ cells at a nonpermissive temperature of 40 degrees C. Furthermore, fusidic acid, an inhibitor of protein synthesis which specifically inactivates EF-G, prevented any accumulation of ppGpp during the heat shock. We suggest that a functional EF-G protein is necessary for ppGpp accumulation under temperature shift conditions, possibly by mediating changes in the function of another protein, the relA gene product. However, EF-G is probably not required for the synthesis of ppGpp during the stringent response, since its inactivation did not prevent ppGpp accumulation during amino acid starvation.     

Synthesis of guanosine 5'-diphosphate, 3'-diphosphate in spot mutants of Escherichia coli.

The synthesis of ppGpp in spoT- mutants of Escherichia coli has been invesitgated. In these mutants the first-order rate constant for ppGpp breakdown is low, and pppGpp is barely detectable. It is shown that the rate of pppGpp, and hence ppGpp, synthesis is strongly reduced compared with that observed in spot+ strains. The low rate of magic spot synthesis satisfactorily explains the low levels of pppGpp in spoT- mutants. The pentaphosphate very probably is the precursor of ppGpp as it is in wild-type, i.e. spoT+, strains.     

Degradation of guanosine 3'-diphosphate 5'-diphosphate in vitro by the spoT gene product of Escherichia coli.

Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) is rapidly degraded to guanosine 5'-diphosphate (ppG) and probably pyrophosphate by an enzyme present in the ribosomal fraction prepared from spoT+ strains of Escherichia coli. The ppGpp-degrading enzyme was released from the ribosomes during dissociation at low ionic strength. Ribosomes are not essential for degradation of ppGpp, and decay of ppGpp is strictly dependent on manganese ions. The reaction is sensitive to inhibition by tetracycline, which can be reversed by MnCl2, indicating that the inhibitory effect is due to the previously described chelating properties of the antibiotic. When the ppGpp-degrading enzyme was complemented with adenosine 5'-triphosphate (pppA) and a nucleoside diphosphate kinase, decay of ppGpp was accelerated yielding pppG and ppG as major products. In the absence of pppA we have been unable to detect the ppGpp-degrading enzyme in various spoT- mutant strains indicating that this enzyme is the spoT gene product.     

Nitrogenase synthesis in Klebsiella pneumoniae: enhanced nif expression without accumulation of guanosine 5'-diphosphate 3'-diphosphate

Derepression of nitrogen fixation (nif) genes in Klebsiella pneumoniae following transfer from NH+4-sufficiency to N-free medium was preceded by rapid expansion of the guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool. When derepressed in N-free medium supplemented with glutamine (600 micrograms ml-1), expression from the nifH and nifL promoters, determined as beta-galactosidase activity in nif::lac merodiploid strains, was stimulated 7-fold and nitrogenase activity 26-fold; ppGpp did not accumulate, remaining at the levels found in NH+4-repressed populations. The relaxed mutant K. pneumoniae relA40, which accumulates only very low levels of ppGpp, showed partial derepression of nitrogenase activity in the presence of glutamine, thus ppGpp is unlikely to be an effector of nif expression. ATP and GTP levels were elevated under conditions where nif expression was enhanced, consistent with previous data suggesting that maintenance of ATP levels is a prerequisite for the expression of nif genes in K. pneumoniae.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Role of the spoT gene product and manganese ion in the metabolism of guanosine 5'-diphosphate 3'-diphosphate in Escherichia coli.

Addition of divalent ion chelating agents picolinic acid, 1,10-phenanthroline, or quinoline-2-carboxylic acid to wild type, relA, or relX, but not spoT strains of Escherichia coli increases the levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Poorly chelating analogs of these agents and a larger and more highly charged chelating agent, ethylene glycol bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid are ineffective. Mn2+ reverses the increase in ppGpp. The increase in ppGpp in wild type cells can be explained by an inhibition of degradation. In spoT cells the response is more complex; ppGpp does not increase although degradation is completely inhibited. The lack of increase in spoT cells suggests a role for spoT in synthesis of ppGpp in addition to its known role in degradation. Growth of both spoT+ and spoT cells is inhibited following chelator addition. This suggests that growth inhibition is through a mechanism not directly involving ppGpp. The results of this study provide evidence in intact cells for a role for Mn2+ and the spoT gene product in ppGpp degradation, and provide further evidence for an involvement of spoT and possibly divalent ions in ppGpp synthesis.     

Direct photoaffinity labeling of cysteine 211 or a nearby amino acid residue of beta-tubulin by guanosine 5'-diphosphate bound in the exchangeable site

Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.     

Specific binding of guanosine 5'-diphosphate with the NS protein of vesicular stomatitis virus

A soluble protein fraction containing L, NS, G and M proteins of vesicular stomatitis virus was prepared by treatment of Triton-disrupted virions with 0.8M NaCl. Incubation of the soluble fraction with beta-32P GDP followed by analysis of the proteins by polyacrylamide gel electrophoresis showed specific labeling of the NS protein. The NS-GDP complex was sensitive to phosphatase treatment, suggesting non-covalent binding. No binding of GDP to NS protein was detected when the soluble fraction was pre-heated at 100 degrees C for 1 min. or Mg++ was omitted from the incubation mixture. The binding was inhibited by ATP consistent with competition for a common nucleotide binding site.     

Activation of transcription by guanosine 5'-diphosphate,3'-diphosphate, transfer ribonucleic acid, and novel protein from Escherichia coli.

A protein factor TFms) that is required for ppGpp to stimulate RNA synthesis has been purified from an eluate of crude ribosomes. TFms also has the capacity to stimulate RNA synthesis without ppGpp present. Under standard conditions the action TFms and ppGpp requires uncharged tRNA. TFms and ppGpp act at inhibition to promote the formation of rifampicin-resistant or polytrI)-resistant preinitiation complexes. In the presence of rifampicin or poly(rI), tRNA is no longer required. With lambdah80dlacPs DNA as template, ppGpp together with TFms stimulated gal RNA synthesis to a much greater extent than total RNA synthesis. The stimulation of both lac and gel RNA synthesis was increased in the presence of cyclic AMP receptor and cyclic AMP.