Synthesis of ppGpp by mouse embryonic ribosomes

The unusual nucleotide guanosine tetraphosphate (ppGpp) is synthesized in vitro by ribosomes isolated from mouse embryos, but is not formed by ribosomes isolated from adults. Analysis of specific organs shows a developmental change in detectable ppGpp-forming ability. Eleven day embryonic liver ribosomes synthesize ppGpp, but this ability is essentially lost by 14 day embryonic liver and adult liver. Eleven day embryonic brain ribosomes also synthesize ppGpp at a level comparable to that observed using E. coli ribosomes. The synthesis of ppGpp appears to be associated with the early stages of differentiation, when cell proliferation is rapid and specialized protein synthesis is low or absent. The potential role of ppGpp as an effector molecule or regulator in the eucaryotes is discussed.     

Eukaryotic ribosomal proteins stimulate Escherichia coli stringent factor to synthesize guanosine 5''-diphosphate, 3''-diphosphate (ppGpp) and guanosine 5''-triphosphate, 3''-diphosphate (ppGpp).

Summary When supplemented with Escherichia coli stringgent factor, 80S ribosomes from various sources failed to support guanosine tetra- and pentaphosphate ((p)ppGpp) synthesis. In contrast, ribosomal proteins from 80S, 60S or 40S particles (mouse embryos, rabbit reticulocytes) crossreacted with the E. coli stringent factor. Significant stimulation of (p)ppGpp synthesis was achieved proteins/ml. These observations may provide additional criteria to detect homologies between eukaryotic and prokaryotic ribosomal proteins.     

Dissection of the mechanism for the stringent factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.     

Synthesis of pppGpp by ribosomes from an Escherichia coli spoT mutant and the metabolic relationship between pppGpp and ppGpp.

Both ribosomes and a cell-free extract (S-30) prepared from an Escherichia coli spoT mutant catalyzed the synthesis of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) as efficiently as did ribosomes and S-30 from a spoT+ strain. In both cases, the level of pppGpp reached its maximum before ppGpp maximally accumulated. pppGpp added to the ribosome system was rapidly converted to ppGpp. These results indicate that the spoT+ gene product may not have a direct role in the synthesis of pppGpp and that pppGpp is a precursor of ppGpp.     

Feedback control of ribosome function in Escherichia coli

We have previously proposed that the rate of ribosome function during balanced growth in E. coli, expressed as the rate of peptide chain elongation, is adjusted by a feedback mechanism: whenever that rate is submaximal (i.e. below 22 amino acid residues polymerized per active ribosome at 37 degrees C), the feedback signal ppGpp is generated by an activation of the ppGpp synthetase expressed from the spoT gene. The accumulation of ppGpp reduces the synthesis of additional ribosomes and thereby reduces the consumption of amino acids which, in turn, allows the remaining ribosomes to function at a higher rate. Here we have described with supporting evidence the proposed feedback loop in greater detail and provided a mathematical analysis which predicts that the SpoT ppGpp synthetase activity should be highest when the ribosomes function at their half-maximal rate. This prediction is consistent with reported observations and is independent of the particular (unknown) mechanism by which the rate of translation controls the ppGpp synthetase activity of SpoT.     

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.     

Altered specificity of synthesis of guanosine tetraphosphate (ppGpp) and pentaphosphate (ppGpp) by salt-washed ribosomes

Salt-washed ribosomes from $\text{Escherichia coli}$, plus stringent protein, form more ppGpp than pppGpp from GTP at all times, but unwashed ribosomes are shown to synthesize primarily pppGpp as the initial product.     

Template-independent synthesis of guanosine tetra- and pentaphosphates on ribosomes

It is shown that Escherichia coli ribosomes carrying poly(Lys)-tRNA can form (p)ppGpp in the presence of stringent factor in the absence of the poly(A) template. Template-independent synthesis of (p)ppGpp is suppressed by tetracycline and partially decreases if deacylated tRNA is omitted.     

Methylgroups of ribosomal protein L11 are not related to the synthesis of ppGpp

Summary Ribosomes carrying either a normally methylated or an undermethylated L11, respectively, were tested with respect to the stringency reaction in the presence of crude stringent factor. Systems with either kind of ribosomes synthesize ppGpp with the same efficiency. The ppGpp synthesis in presence of ribosomes with undermethylated L11 depends also on stringent factor, mRNA and deacylated tRNA whereas aminoacyl-tRNA and peptidyl-analogous tRNA show no effect. Thus, the absence of methylation in L11 does not influence the stringency reaction.     

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.     

The interaction of guanosine 5'-diphosphate, 2' (3')-diphosphate with the bacterial elongation factor Tu

The unusual nucleotide guanosine tetraphosphate, ppGpp, which appears following amino acid starvation in “stringent” strains of bacteria binds to the elongation factor EFTu with a dissociation constant of about 8 × 10^?9m. ppGpp binds competitively with GDP and GTP, and EFTs catalyzes the exchange reaction of ppGpp with EFTu · GDP. ppGpp binds to EFTu about 50 times more tightly than does GTP, and, in the absence of elongation factor EFTs, it will effectively inhibit the formation of the ternary complex Phe-tRNA · EFTu · GTP. However, in the presence of EFTs there is rapid equilibration between EFTu · GTP and EFTu · ppGpp which allows EFTu to be rapidly and extensively incorporated into the stable ternary complex. A preliminary estimate of the constant for the dissociation of Phe-tRNA from the ternary complex is 10^?810^?9m. ppGpp inhibits the enzymatic binding of Phe-tRNA to ribosomes; however, EFTs reverses this inhibition. ppGpp moderately inhibits phenylalanine polymerization even in the presence of EFTs. This inhibition probably involves an interaction of ppGpp with elongation factor G, the translocation factor. It appears that in the intact cell ppGpp would not be an effective inhibitor of EFTu, and that little EFTu · ppGpp can exist in the cell.     

Mouse embryos fail to synthesize detectable quantities of guanosine 5'-diphosphate 3'-diphosphate.

The unusual highly phosphorylated nucleotide, guanosine 5′-diphosphate 3′-diphosphate, has been implicated in the control of development of the mouse (Irr, J. D., et al. (1974) Cell3, 249). We have been unable, however, to detect guanosine 5′-diphosphate 3′-diphosphate synthesis either in preimplantation and postimplantation mouse embryos cultured in the presence of [³²P]orthophosphate or in assays using ribosomes isolated from 10- to 13-day mouse embryos. Three unidentified phosphorous-containing compounds were detected in blastocyst stage mouse embryos.     

Stringent response of Bacillus stearothermophilus: evidence for the existence of two distinct guanosine 3',5'-polyphosphate synthetases.

Bacillus stearothermophilus reacted to pseudomonic acid-induced inhibition of isoleucine-transfer ribonucleic acid (RNA) acylation and to energy downshift caused by alpha-methylglucoside addition with accumulation of guanosine 3',5'-polyphosphates [(p)ppGpp] and restriction of RNA synthesis. In vitro studies indicated that (p)ppGpp was synthesized by two different enzymes. One enzyme, (p)ppGpp synthetase I, was present in the ribosomal fraction, required the addition of a ribosome-messenger RNA-transfer RNA complex for activation, and was inhibited by tetracycline and thiostrepton. It is suggested that (p)ppGpp synthetase I is comparable to the relA gene product from Escherichia coli and is responsible for (p)ppGpp accumulation during amino acid starvation. The other enzyme, (p)ppGpp synthetase II, was found in the high-speed supernatant fraction (S100). It functioned independently of ribosomes, transfer RNA, and messenger RNA and was not inhibited by the above-mentioned antibiotics. (p)ppGpp synthetase II is thought to be responsible for (p)ppGpp accumulation during carbon source downshift. The two enzymes differ in their Km values for adenosine triphosphate (ATP):2mM ATP for synthetase I and 0.05 mM ATP for synthetase II. They also have different molecular weights: apparent Mr of 86,000 (+/- 5,000) for synthetase I and 74,000 (+/- 5,000) for synthetase II.     

G-protein control of the ribosome-associated stress response protein SpoT

The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.     

The murine gene, Traube, is essential for the growth of preimplantation embryos

Little is known about the genetic control of preimplantation development. We have isolated, characterized, and mutated a previously undescribed mouse gene, Traube (Trb), essential for preimplantation development. Similar protein coding sequences are found in rats, humans, and yeast. The TRB protein contained two amino-terminal acidic domains, a leucine zipper, and three putative nuclear localization signals. The Trb gene was expressed at low levels ubiquitously early in development and became restricted to the liver and the central nervous system from E11.5 onward. Myc-tagged TRB protein was localized to the nucleus, and in a large proportion of the cells to the nucleoli. The Trb mutant embryos halted in development at the compacted morula stage at E2.5. At E3.5 they started to decompact and a day later they disintegrated and died. The observed defect was cell autonomous, as mutant cells failed to participate in the formation of chimeric embryos. The Trb mutant embryos showed a 50% reduction of the total cell number. The mutant embryos exhibited a paucity of ribosomes, polyribosomes, and rough endoplasmic reticulum. This paucity of ribosomes together with the localization of TRB to the nucleoli, the site of ribosome synthesis, suggests that TRB is involved in the synthesis of ribosomes.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes (rplK and rpsL) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants.     

Physiological characterization of Escherichia coli rpoB mutants with abnormal control of ribosome synthesis.

We have previously reported the isolation of Escherichia coli rpoB mutants in which the control of ribosome synthesis by the nucleotide effector guanosine tetraphosphate (ppGpp) is altered, owing to a 20-fold increased sensitivity of the mutant RNA polymerases to ppGpp. In these mutants, the level of ppGpp during exponential growth is decreased about 10-fold, relative to that of rpoB+ wild-type strains, such that a near normal partitioning of RNA polymerase occurs with respect to stable RNA (rRNA and tRNA) gene activity. Here, the physiological effects of two different rpoB alleles in a relA+ and relA background were analyzed in greater detail by comparison with their isogenic rpoB+ wild-type parents. For a given growth medium, the rpoB mutations were found to affect four parameters which resulted in a reduction of growth rate. The results reinforce a previous conclusion that a key element in control of the bacterial growth rate is a mutual relationship between control of ribosome synthesis by ppGpp and control of relA-independent ppGpp metabolism by the concentration and function of ribosomes.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes ( rplK and rpsL ) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants. Received: 12 May 1997 / Accepted: 22 July 1997