Selective inhibition of tRNATyr transcription by guanosine 3'-diphosphate 5'-diphosphate.

Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) selectively reduces the synthesis of su+III tRNA from omega 80 psu+III DNA relative to the synthesis of omega 80 RNA in a system in vitro containing DNA and Escherichia coli RNA polymerase holoenzyme as the sole macromolecular components. The response of su+III tRNA synthesis to increasing salt and to temperature in the presence of ppGpp suggests that the nucleotide may reduce the affinity of the enzyme for su+III promoters. The Ki for the selective inhibition of tRNA synthesis by ppGpp is 4 muM in contrast to the value of 150 muM for the inhibition of rRNA synthesis.     

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.     

The effect of alcohols on guanosine 5'-diphosphate-3'-diphosphate metabolism in stringent and relaxed Escherichia coli

The effects of a series of alcohols on the stringent response system of Escherichia coli were studied. The alcohols used could be divided into two groups on the basis of the response of pppGpp and ppGpp to the growth downshift induced by the alcohols. The cells responded to the alcohols, methanol, ethanol, and propanol, as if they were being starved of amino acids. In the stringent strain CP78 these alcohols induced pppGpp and ppGpp accumulation and curtailed RNA synthesis, whereas in the relaxed strain CP79, both of these responses were absent. It was determined that this response was most likely due to an interference by these alcohols with the uptake of amino acids required by these strains. By contrast both stringent and relaxed cells elevated their level of ppGpp and decreased RNA accumulation when treated with butanol or pentanol. This response is similar to the effect of carbon source limitation. It was determined that the elevation of ppGpp in the stringent strain was primarily the result of increased ppGpp synthesis in response to these alcohols. In the relaxed strain the rise in ppGpp was dependent on a decrease in ppGpp degradation coupled with a moderate increase in ppGpp synthesis. This stimulation of ppGpp synthesis in relaxed cells, although small, suggests the existence of an enzyme distinct from stringent factor which is capable of synthesizing ppGpp. Data are presented which suggest that the activity of this enzyme is coupled to the potential for protein synthesis and energy availability of the cell, perhaps being regulated by the overall ratio of unchanged to amino-acylated tRNA.     

Guanosine 5'-diphosphate 3'-diphosphate levels, carbon source, and ribonucleic acid synthesis in a mutant strain of Escherichia coli

We have previously described a mutant strain of Escherichia coli (2S142) which shows a specific inhibition of stable RNA synthesis at 42 degrees C. The temperature-sensitive lesion mimics a carbon source downshift (diauxie lag). We therefore measured RNA synthesis and levels of ppGpp (guanosine 5'-diphosphate 3'-diphosphate) on a number of different carbon sources. There is a 6-fold variation in ppGpp levels at 42 degrees C, depending on the carbon source present. Much of the variation in ppGpp levels at 42 degrees C can be explained by variations in the decay rate of ppGpp at 42 degrees C. The rates of ribosomal RNA and total RNA synthesis also vary with the carbon source at 42 degrees C. Linear regression analysis shows only a moderately good correlation (correlation coefficient = 0.62, P = 0.0001) between the ppGpp level at 42 degrees C and the rate of rRNA synthesis at 42 degrees C. In fact, ppGpp levels are a slightly better predictor of the rate of total RNA synthesis (correlation coefficient = 0.69, P = 0.0001) at 42 degrees C. Other variables such as rate of carbon source uptake appear to have very little, if any, relationship to the rate of rRNA synthesis on the different carbon sources. Segmented linear regression analysis indicates that ppGpp levels and rates of RNA synthesis correlate best when the carbon sources are divided into two groups: 6- and 12-carbon sugars and other carbon sources. The rate of rRNA synthesis in 2S142 at 42 degrees C appears to be relatively insensitive to ppGpp levels with 6- and 12-carbon sugars as the carbon source. These data raise the possibility that carbon source may affect rRNA synthesis in a manner that is at least partially unrelated to ppGpp levels.     

Dependence of RelA-mediated (p)ppGpp formation on tRNA identity

The bacterial stringent response is a cellular response to amino acid limitations and is characterized by the accumulation of the alarmone polyphosphate guanosine ((p)ppGpp). A key molecular event leading to (p)ppGpp synthesis is the binding of a deacylated tRNA to the vacant A-Site of a ribosome. The resulting ribosomal complex is recognized by and activates RelA, the (p)ppGpp synthetase. Activated RelA catalyzes (p)ppGpp formation until the deacylated tRNA passively dissociates from the ribosomal A-Site. In this report, we have investigated a novel role for the identity of A-Site bound tRNA in RelA-mediated (p)ppGpp synthesis. A comparison in the stimulation of RelA activity was made using ribosome complexes with either a tightly or weakly binding deacylated tRNA occupying the A-Site. In vitro analysis reveals that ribosome complexes formed with tight binding tRNA(Val) stimulate RelA activity at lower concentrations than that required for ribosome complexes formed with the weaker binding tRNA(Phe). The data suggest that the recovery from the stringent response may be dependent on the identity of the amino acid that was initially limiting for the bacteria.     

Temperature dependence of RNA synthesis parameters in Escherichia coli

For Escherichia coli B/r growing in glucose minimal medium, the following parameters of RNA synthesis remained invariant between 20 and 40 degrees C: RNA polymerase concentration (RNA polymerase/mass), rRNA and tRNA concentration (RNA/mass), RNA polymerase activity (fraction of total RNA polymerase actively engaged in RNA chain elongation), and stable RNA synthesis relative to total RNA synthesis. The following parameters increased 3.4-fold over the same temperature range: rRNA chain elongation rate, guanosine tetraphosphate (ppGpp) concentration, and culture growth rate. Above 40 degrees C, the changes became more complex, and the growth rate began to decrease. The observation that most RNA synthesis parameters are temperature invariant despite the increase of ppGpp suggests that the mechanism of RNA synthesis control by ppGpp, assumed to involve an interaction of RNA polymerase wtih ppGpp, is itself temperature dependent such that, with increasing temperature, higher concentrations of ppGpp are required to affect the RNA polymerase.     

Unusual effects of 5a,6-anhydrotetracycline and other tetracyclines. Inhibition of guanosine 5'-diphosphate 3'-diphosphate metabolism, RNA accumulation and other growth-related processes in Escherichia coli.

5a,6-Anhydrotetracycline was discovered to be unique among several tetracycline derivatives tested in its ability to inhibit RNA accumulation in vivo at low concentration (20 microgram/ml and less). In addition, in vivo protein, DNA, and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthesis were completely inhibited by 20 microgram/ml 5a,6-anhydrotetracycline. ppGpp decay in a spoT strain was inhibited by 20 microgram/ml 5a,6-anhydrotef RNA synthesis by a 5a,6-anhydrotetracycline may be due, in part, to reduced UTP and CTP synthesis. The effects of tetracyclines on in vitro ppGpp synthesis by crude stringent factor in the absence of ribosomes were investigated. It was determined that of six tetracyclines tested, four strongly inhibited the reaction (oxytetracycline, chlorotetracycline, dedimethylaminotetracycline, and tetracycline) whereas 5a,6-anhydrotetracycline gave a moderate inhibition and alpha-6-deoxyoxytetracycline resulted in only a slight reduction in ppGpp synthesis. It is proposed that tetracyclines interfere with factors involved in ppGpp metabolism and function.     

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.     

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.     

Toxic effects of high levels of ppGpp in Escherichia coli are relieved by rpoB mutations.

A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action. To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter. The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp. In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases. At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates. After continued incubation, these colonies form blue sectors of faster growing mutant cells. Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase. One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis. The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid. The presence of this plasmid conferred increased resistance to overproduction of ppGpp. These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.     

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.     

The stringent response to unacylated tRNA,energy- and temperature-downshift in Bacillus stearothermophilus

The response of the thermophile Bacillus stearothermophilus to inhibition of tRNA acylation, energy starvation and temperature downshift was characterized. We found that B. stearothermophilus, like other prokaryotic organisms, reacts with the so-called stringent response, which includes the accumulation of the unusual nucleotides guanosine 3′,5′ bis (dipphosphate) [ppGpp] and guanosine 3′-diphosphate, 5′-triphosphate [pppGpp] and concomitantly the reduction of RNA synthesis and growth rate. The amount of (p)ppGpp formed depended on the cause of the stringent response: when tRNA acylation was inhibited (p)ppGpp synthesis was much higher than after energy starvation or temperature downshift whereas RNA synthesis was totally blocked in each case.     

Anomalous synthesis of ppGpp in growing cells.

In E. coli cells, accumulation of ppGpp is normally triggered by conditions that restrict the aminoacylation of tRNA or interfere with carbon/energy source metabolism; in both cases, the nucleotide's accumulation is associated with control of stable RNA synthesis and is generally believed to bring it about. We have found an anomalous situation wherein vigorously growing cells accumulate a high level of ppGpp and there is no restriction of stable RNA synthesis. This occurs when wild-type cells are shifted up from an abnormally low growth temperature to one in the optimal range (35 degrees C-40 degrees C). The effect is partly, but not entirely, dependent upon the presence of a functional relA gene product. These results appear to call into question the simpler interpretations of the role of ppGpp in the control of stable RNA synthesis.     

Discrimination between purine and pyrimidine base at the 3' terminus of the tRNA molecule by the stringent factor system from Escherichia coli.

Crude stringent factor, prepared from a mutant strain with low levels of tRNA nucleotidyl transferase, synthesizes little or no (p)ppGpp in the presence of tRNA^Phe-CpC; addition of yeast tRNA nucleotidyl transferase, however, fully restores (p)ppGpp formation, indicating that the complete CCA terminus of the tRNA molecule is a prerequisite in the (p)ppGpp synthesizing reaction. When the terminal purine is replaced by a pyrimidine base as in the case of tRNA^Phe-CpCpC; or when the latter is extended by addition of AMP yielding tRNA^Phe-CpCpCpA, both modified tRNAs are low in stimulating the (p)ppGpp synthesizing reaction. Hence activation of the stringent factor by tRNA requires (i) the terminal purine base and (ii) the precise fitting of the CCA terminus to the acceptor site of the ribosome.     

Magic Spot Metabolism in an Escherichia coli Mutant Temperature Sensitive in Elongation Factor Ts

A temperature-sensitive mutant of Escherichia coli HAK88 which has been shown to have a lesion in elongation factor Ts (EFTs) was studied with repsect to its metabolism of guanosine 5′-diphosphate, 2′(3′)-diphosphate (ppGpp) and the associated failure of ribosomal ribonucleic acid (rRNA) accumulation at the nonpermissive temperature. Results reported here show that (i) when EFTs is nonfunctional, a full complement of charged transfer RNA (tRNA) cannot prevent accumulation of ppGpp (magic spot) and the stringent failure of rRNA accumulation; (ii) chloramphenicol prevents magic spot (MS) formation and the stringent response not by increasing the percentage of charged tRNA, but possibly by somehow interfering directly with the synthesis of MS; and (iii) tetracycline can lead to MS disappearance without resumption of RNA synthesis. Thus, the absence of MS and the presence of a functional RNA polymerase and charged tRNA are not sufficient to support rRNA accumulation in vivo. An additional element in the regulatory system is suggested.     

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.