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At least 19 recordsLinked to original sources
PubMed1974

Effects of guanosine tetraphosphate, guanosine pentaphosphate, and beta-gamma methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro.

The effects of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), both produced by E. coli, were measured on the activities of several genes in a cell-free system. Gene activity is measured as gene-directed synthesis of biochemically competent protein or transfer RNA. Both ppGpp and pppGpp stimulated the activities of the ara, lac, and trp operons and inhibited the arg operon. Production of transfer-RNA(Tyr) was unaffected by moderate levels of either ppGpp or pppGpp and only slightly inhibited at higher levels of ppGpp. Since the cell-free reaction mixtures hydrolyze pppGpp to ppGpp, we performed similar studies with a hydrolysis-resistant analog of pppGpp, the beta-gamma methylenyl derivative (pcppGpp). In general, pcppGpp shows the same inhibitory potency as pppGpp for the arg operon, but lacks the stimulatory effects on the ara, lac, and trp operons. This result suggests that the stimulation of these gene activities is specific for ppGpp.Under similar conditions, pppGpp and ppGpp show a slight inhibitory effect on the messenger-directed synthesis of beta-galactosidase and no effect on the messenger-directed synthesis of MS2 viral-coat protein. These observations, together with the fact that in the same system these nucleotides affect coupled transcription and translation, lead us to surmise that the activities of pppGpp and ppGpp are exerted at the level of RNA polymerase activity.

Acetamides↗
PubMed1980

Accumulation of guanosine tetraphosphate and guanosine pentaphosphate in Myxococcus xanthus during starvation and myxospore formation.

Cultures of Myxococcus xanthus develop multicellular fruiting bodies when starved for carbon and nitrogen sources on an agar surface. Under these conditions of severe starvation, cultures rapidly accumulated a compound identified as guanosine tetraphosphate by chromatographic migration of the compound and of its major acid and alkali breakdown products. The accumulation of guanosine tetraphosphate was reduced in the presence of tetracycline, indicating that it may be synthesized by mechanisms similar to those of Escherichia coli. The guanosine tetraphosphate level was also reduced in starved cultures of a mutant unable to fruit normally, although it has been determined whether the defect in guanosine tetraphosphate accumulation is responsible for the inability to fruit. Induction of spores by glycerol addition led to transient increases in both guanosine tetraphosphate and guanosine pentaphosphate at a stage following most cell shortening, but before spores had acquired full refractility.

Adenosine Triphosphate↗
PubMedDate not supplied

Inhibition of mammalian soluble guanylate cyclase activity by adenosine 5'-tetraphosphate, guanosine 5'-tetraphosphate and other nucleotides.

The effects of a variety of purine and pyrimidine nucleotides were tested for their capacity to inhibit mammalian soluble guanylate cyclase activity. Adenosine 5'-tetraphosphate (ATetP), ATP, ADP, AMP, guanosine 5'-tetraphosphate (GTetP) and GDP were found to inhibit soluble guanylate cyclase activity from rat lung and other mammalian tissues. The corresponding cytosine and thymine nucleotides showed little or no inhibitory activity, except for thymidine 5'-tetraphosphate, which inhibited glanylate cyclase activity but to a lesser extent than did the purine nucleoside tetraphosphates. ATetP and GTetP were found to be potent inhibitors of soluble guanylate cyclase activity from rat, guinea pig and mouse lung, rat heart and rat brain. Both purine nucleoside tetraphosphates were competitive inhibitors of the rat lung soluble enzyme. ATetP and GTetP had Ki values of 1 muM and 2.5 muM, respectively. The experimental data suggest that purine nucleoside tetraphosphates, and perhaps other purine nucleotides, may play a biologic role in modulating mammalian soluble guanylate cyclase activity.

Adenine Nucleotides↗
PubMed1980

Guanosine pentaphosphate and guanosine tetraphosphate accumulation and induction of Myxococcus xanthus fruiting body development.

Development of multicellular fruiting bodies of Myxococcus xanthus can be induced by limitation of any of a number of different classes of amino acids. Investigated were amino acids that wild-type strains of M. xanthus are unable to synthesize (isoleucine, leucine, and valine), can synthesize at a low rate (phenylalanine), or can normally synthesize at an adequate rate (tryptophan and serine). In general, gradual rather than abrupt starvation for an essential amino acid was required for the induction of fruiting. Perhaps gradual starvation in general minimizes antagonism between amino acids present in the medium, as was documented for valine starvation. The previously reported induction of fruiting by a high concentration of threonine was shown to be specifically reversed by lysine. Threonine addition may starve cells for lysine by feedback inhibition of aspartokinase activity. Starvation for carbon-energy sources or inorganic phosphate also induced fruiting. As in other bacteria, amino acid starvation of M. xanthus leads to increases in cellular guanosine polyphosphate, usually consisting of large increases in the amount of guanosine pentaphosphate with smaller increases in the level of guanosine tetraphosphate. Guanosine polyphosphate accumulation is thus shown to be correlated with nutritional conditions that induce fruiting, and therefore may serve as an intracellular signal to trigger cells to end vegetative growth and initiate fruiting body development.

Amino Acids↗
PubMed1983

Regulation of Escherichia coli aspartate transcarbamylase synthesis by guanosine tetraphosphate and pyrimidine ribonucleoside triphosphates.

The effects of guanosine tetraphosphate (ppGpp) and pyrimidine ribonucleoside triphosphates on Escherichia coli aspartate transcarbamylase (ATCase) synthesis were examined. To determine the effect of ppGpp, a stringent (relA+) and relaxed (relA) isogenic pair of E. coli K-12 strains was starved for isoleucine, and the residual rate of synthesis of this enzyme was measured. It was necessary to starve the strains for uracil before the isoleucine limitation to maintain similar, low levels of UTP, the putative pyrimidine effector of ATCase synthesis. The isoleucine starvation of the stringent strain caused an immediate 10-fold increase in the intracellular concentration of ppGpp, which was coincident with the cessation of the synthesis of the enzyme. The elevated level of ppGpp then decayed until it reached an intracellular concentration similar to that found in unstarved cells. Enzyme synthesis resumed at this time. In the relaxed strain, the intracellular concentration of ppGpp did not increase upon isoleucine starvation and synthesis of the enzyme was not repressed. These experiments strongly indicated that ppGpp acts as a negative effector of ATCase synthesis. The repression of ATCase synthesis by ppGpp was demonstrated directly by using a Salmonella typhimurium (relA) in vitro coupled transcription-translation system with a lambda specialized transducing phage carrying the E. coli K-12 operon encoding the subunits of this enzyme (pyrBI) as a source of DNA. This in vitro system was also used to measure the effects of UTP and CTP on ATCase synthesis. Increasing the concentration of UTP in the in vitro reaction mixture resulted in strong repression of this synthesis, whereas increasing the CTP concentration did not affect synthesis significantly. Possible mechanisms for the regulation of pyr gene expression, including attenuation control, are discussed.

Aspartate Carbamoyltransferase↗
PubMed1983

The elongation factor Tu from Escherichia coli, aminoacyl-tRNA, and guanosine tetraphosphate form a ternary complex which is bound by programmed ribosomes.

The interaction of the Escherichia coli elongation factor Tu guanosine tetraphosphate complex (EF-Tu ppGpp) with aminoacyl-tRNAs(aa-tRNA) was reinvestigated by gel filtration and hydrolysis protection experiments. These experiments show that EF-Tu X ppGpp like EF-Tu X GDP (Pingoud, A., Block, W., Wittinghofer, A., Wolf, H. & Fischer, E. (1982) J. Biol. Chem. 257, 11261-11267) forms a fairly stable complex with Phe-tRNAPhe, KAss being 0.6 X 10(5) M-1 at 25 degrees C. The binding of the EF-Tu X ppGpp X aa-tRNA complex to programmed ribosomes was investigated by a centrifugation technique. It is shown that this complex is bound codon-specific with KAss = 3 X 10(7) M-1 at 0 degrees C and that it stimulates peptidyl transfer. A numerical estimation of the intracellular concentration of EF-Tu X GTP X aa-tRNA and EF-Tu X ppGpp X aa-tRNA during normal growth and under the stringent response indicates that ppGpp accumulation does affect the EF-Tu X GTP X aa-tRNA concentration but does not lead to major depletion of this pool. Furthermore, due to the higher affinity of EF-Tu X GTP to aa-tRNA and of the ternary complex EF-Tu X GTP X aa-tRNA to the ribosome, EF-Tu X ppGpp X aa-tRNA binding to the ribosome is not significant. According to our measurements and calculations, therefore, a direct participation of EF-Tu in slowing down the rate of protein biosynthesis and improving its accuracy during amino acid starvation is not obvious.

Escherichia coli↗
PubMed1973

Identification of the synthesis of guanosine tetraphosphate (MS I) as insertion of a pyrophosphoryl group into the 3'-position in guanosine 5'-diphosphate.

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 (32)P in the beta- and gamma-positions as donor. The beta-label was found to produce a ppGp⃰p and the gamma-label a ppGpp⃰. Furthermore, [(3)H]GDP + [gamma-(32)P]ATP yielded ppGpp in a (3)H:(32)P ratio of 1:1. The results indicate a transfer of the terminal pyrophosphoryl group of ATP as a unit. The position of the transferred pyrophosphoryl was assayed for by preparation of pGp⃰ from ppGp⃰p with Zn(++)-activated inorganic pyrophosphatase from yeast. The pGp⃰ was then assayed with 3'-nucleotidase, which liberated practically all the labeled phosphate. This result indicate that the phosphate transfer from ATP to GDP yields guanosine 5'-diphosphate-3'-diphosphate.

Adenosine Triphosphate↗
PubMed1973

Regulation of phospholipid synthesis in Escherichia coli by guanosine tetraphosphate.

Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with (32)P-inorganic phosphate and (32)P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells.

Acetyltransferases↗
PubMed1983

Accumulation of guanosine tetraphosphate induced by polymixin and gramicidin in Escherichia coli.

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA+) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.

Adenosine Triphosphate↗
PubMed1982

Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate.

The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments.

Escherichia coli↗
PubMed1976

Free 3'-OH group of the terminal adenosine of the tRNA molecule is essential for the synthesis in vitro of guanosine tetraphosphate and pentaphosphate in a ribosomal system from Escherichia coli.

tRNAPhe from yeast modified at the 3'-terminal adenosine residue or missing part of its CpCpA end was investigated for its ability to participate in the synthesis of guanosine tetraphosphate and pentaphosphate using Escherichia coli ribosomes. Only tRNA containing complete CpCpA end and an intact ribose residue in the terminal adenosine was active in this process. The absence of the 2'-hydroxyl group of the terminal adenosine does not influence the reaction, but the 3'-hydroxyl group is essential and cannot be replaced by an amino group or a hydrogen atom. tRNA in which the 3'-terminal adenosine is replaced by formycin is active in the induction of guanosine tetraphosphate and pentaphosphate synthesis, but the reaction proceeds relatively slowly.

Adenosine↗
PubMed1997

Adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate: new substrates of the cytosolic exopolyphosphatase of the yeast Saccharomyces cerevisiae.

A cytosolic preparation of Saccharomyces cerevisiae is capable of hydrolyzing adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate with activities which are 1.5-2 times greater than that with polyP15. The apparent K(m) values for hydrolysis of adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate are 100 and 80 microM, respectively. A comparative study of inhibitors shows that these activities are inherent characteristics of these exopolyphosphatases.

Acid Anhydride Hydrolases↗
PubMed1973

Accumulation of guanosine tetraphosphate in T7 bacteriophage-infected Escherichia coli.

Accumulation of guanosine tetraphosphate (ppGpp) in Escherichia coli strain AS19 valS, carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and infected with bacteriophage T7, was studied. Valine starvation was achieved by culturing this strain at 42 C. Addition of rifampin to an uninfected culture at the nonpermissive temperature resulted in loss of accumulated ppGpp; however, cultures infected with phage T7, treated with rifampin, and then shifted to the nonpermissive temperature maintained the ability to accumulate ppGpp. Moreover, treatment of the T7-infected culture with rubidomycin, an antibiotic which inhibits transcription, did not reduce the amount of ppGpp accumulated following shift to the nonpermissive temperature. Measurements of the instantaneous rate of T7 transcription showed that it is not under stringent control of amino acids. ppGpp synthesized in T7-infected E. coli appears to be more stable than its counterpart in an uninfected culture. These results are interpreted to mean that ppGpp production is not directly dependent on transcription and arises instead from inhibition of another reaction, most likely some aspect of translation.

Adenine↗
PubMed1981

Accumulation of guanosine tetraphosphate (ppGpp) under nitrogen starvation in Anacystis nidulans, a cyanobacterium.

The effect of nitrate deprivation on cell growth and nucleotide level was studied in Anacystis nidulans. A 10-fold reduction in nitrate level resulted in a drastic slowdown of growth. Upon addition of nitrate to the starving cultures, after a lag period, the cells resumed growth. Nutritional shift-down induced a transitory expansion of the guanosine tetraphosphate (ppGpp) pool, preceded by a transitory increase in GTP and ATP concentrations. After having reached peak values, the concentration of ppGpp, GTP and ATP dropped to the respective base levels. The expansion of the ppGpp pool was found to be due to an increase in ppGpp synthesis, rather than to a decrease in ppGpp breakdown. After nutritional shift-up, no decrease in the ppGpp level was found. In starving cells, a decrease in free amino acids was observed to occur concomitantly with the expansion of the ppGpp pool. The level of free amino acids started to increase simultaneously with the contraction of the ppGpp pool.

Adenosine Triphosphate↗
PubMed1998

The differential effects of guanosine tetraphosphate on open complex formation at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1.

The effects of guanosine tetraphosphate (ppGpp) on inhibition of single-round in vitro transcription and on the kinetics of open complex formation were investigated at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1. The two promoters differ in their saturation characteristics and sensitivities to ppGpp. With a 10:1 molar ratio of RNA polymerase (RNAP) to DNA, saturation of transcription activity and weak inhibition (approximately 30%) are observed at rplJ, in contrast to the weak activity and strong inhibition (approximately 80%) at rpsA P1. In the absence of ppGpp, the two promoters show a threefold difference in the overall rate constants of association (ka) (6.5 x 10(7) M-1 s-1 at rplJ and 2.0 x 10(7) M-1 s-1 at rpsA P1), while the dissociation rate constants (kd) are similar (approximately 4.8 x 10(-5) s-1). The addition of ppGpp causes a twofold reduction in k2 (isomerisation constant) rplJ and a threefold decrease in KB (equilibrium constant of RNAP binding) at rpsA P1. There is a significant twofold increase in kd at rplJ, compared with smaller changes at rpsA P1 and at the non-stringent lacUV5 promoter. These results indicate that ppGpp affects the formation and stability of the open complex at the rplJ promoter, in contrast to the inhibition of RNAP binding to the rpsA P1 promoter.

Bacterial Proteins↗