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.     

Responses to multiple-nutrient starvation in marine Vibrio sp. strain CCUG 15956.

The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.     

Delayed-relaxed response explained by hyperactivation of RelE

Escherichia coli encodes two rel loci, both of which contribute to the control of synthesis of macromolecules during amino acid starvation. The product of relA (ppGpp synthetase I) is responsible for the synthesis of guanosine tetraphosphate, ppGpp, the signal molecule that exerts stringent control of stable RNA synthesis. The second rel locus, relBE, was identified by mutations in relB that confer a so-called 'delayed-relaxed response' characterized by continued RNA synthesis after a lag period of approximately 10 min after the onset of amino acid starvation. We show here that the delayed-relaxed response is a consequence of hyperactivation of RelE. As in wild-type cells, [ppGpp] increased sharply in relB101 relE cells after the onset of starvation, but returned rapidly to the prestarvation level. RelE is a global inhibitor of translation that is neutralized by RelB by direct protein-protein interaction. Lon protease activates RelE during amino acid starvation by degradation of RelB. We found that mutations in relB that conferred the delayed-relaxed phenotype destabilized RelB. Such mutations confer severe RelE-dependent inhibition of translation during amino acid starvation, indicating hyperactivation of RelE. Hyperactivation of RelE during amino acid starvation was shown directly by measurement of RelE-mediated cleavage of tmRNA. The RelE-mediated shutdown of translation terminated amino acid consumption and explains the rapid restoration of the ppGpp level observed in relB mutant cells. Restoration of the prestarvation level of ppGpp, in turn, allows for the resumption of stable RNA synthesis seen during the delayed-relaxed response.     

Noncoordinate control of RNA, lipopolysaccharide, and phospholipid syntheses during amino acid starvation in stringent and relaxed strains of Escherichia coli.

The syntheses of RNA, lipopolysaccharides, and phospholipids were measured simultaneously in stringent and relaxed cells of Escherichia coli during normal growth or starvation for amino acids. The synthesis of all these molecules was inhibited by amino acid starvation, but the reduction in synthesis was not coordinated.     

Mammalian cells do not have a stringent response

A key attribute of the stringent response of bacteria is the rapid inhibition of ribosomal RNA synthesis mediated by unusual nucleotides in response to uncharged tRNA. The question as to whether mammalian cells show a stringent response analogous to that of bacteria was critically tested by the effective rapid amino acid starvation of both normal and transformed cells. Rapid starvation giving a high proportion of uncharged tRNA for leucine was produced within 7 minutes of expression of a nonleaky ts leucyl tRNA synthetase mutation in transformed CHO cells (tsH1) and in its normal growth control revertant (L-73). To control for the effect of temperature alone, tsrevertants of tsH1 and L-73 were included in the study, and to control for effects due simply to the inhibition of protein synthesis, the translational elongation inhibitor cycloheximide was used. In addition, rapid starvation for histidine was effected by incubation of both the CHO cell lines and of freshly explanted normal Chinese hamster embryo fibroblasts in histidine-free medium containing high concentrations of histidinol. The rate of preribosomal RNA synthesis and the extent of its maturation to mature rRNA was measured using (³H-methyl) methionine as a donor of methyl groups during synthesis and methylation of pre-rRNA. There was no effect on pre-rRNA synthesis of the rapid generation of uncharged tRNA for 45 minutes for any of the cell types tested. A nonspecific inhibition of maturation of 18S rRNA and late (3 hour) inhibition of pre-rRNA synthesis was observed, but could be mimicked by the inhibition of protein synthesis to comparable levels with cycloheximide. Less severe amino acid starvation resulting in a more physiological inhibition of protein synthesis to 30% also had no specific effect on pre-rRNA synthesis and maturation. Intracellular nucleotide pools were also examined for the appearance of unusual nucleotides such as guanosine tetraphosphate or pentaphosphate and for changes in the levels of normal nucleotides after severe amino acid starvation. No such changes could be detected. We conclude that although mammalian cells may have some biochemical reactions which respond to uncharged tRNA, they do not possess a macromolecular control system analogous to the stringent response of bacteria.     

Studies on the turnover of endogenous choline-containing phospholipids of cultured neuroblastoma cells

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.     

Stable Ribonucleic Acid Synthesis in Stringent (rel+) and Relaxed (rel−) Polyamine Auxotrophs of Escherichia coli K-12

The relationship of polyamines to stable ribonucleic acid (RNA) synthesis under conditions of amino acid withdrawal or chloramphenicol treatment was examined with the use of a closely related rel(+), rel(-) pair conditionally incapable of synthesizing putrescine. Under conditions of polyamine starvation, the cellular sperimidine level fell to one-third to one-half of the value observed in putrescine-supplemented cultures and putrescine became undetectable; cadaverine was synthesized by both strains, but the relaxed strain, MA 252, accumulated less cadaverine per cell than its stringent twin, MA 254. Upon amino acid withdrawal, the stringent strain remained stringent whether starved of or supplemented with polyamines. Similarly, the relaxed strain was capable of making RNA either with or without polyamine starvation. On the addition of chloramphenicol or upon amino acid withdrawal in the relaxed strain, supplementation with spermidine had no effect on the initial rate of RNA synthesis, although RNA accumulation was greater in the presence of added spermidine. Spermidine added at the conclusion of RNA synthesis prompted additional synthesis, although preincubation with spermidine again had no effect on the initial rate. All forms of stable RNA species were made with polyamine supplementation. The present data appear to rule out the possibility that polyamines are primary causative agents in stimulating RNA synthesis, but rather suggest an indirect or secondary role for spermidine in which the polyamines "stimulate" stable RNA synthesis probably by relieving RNA product inhibition of RNA synthesis.     

Polysome stability in relaxed and stringent strain of Escherichia coli during amino acid starvation

The influence of amino acid starvation on polysome content was examined in relaxed and stringent strains of Escherichia coli which were isogenic for the RC locus. No difference was observed between the polysome profiles obtained from two different sets of stringent and relaxed strains starved for the same amino acid. In both relaxed and stringent strains, starvation for amino acids other than methionine resulted in only a slight breakdown of polysomes with a concomitant increase of 70S ribosomes. However, starvation for methionine in both RC stringent and relaxed strains of E. coli resulted in a more extensive degradation of polysomes and accumulation of 70S ribosomes. The 70S ribosomes obtained as a result of methionine starvation were more sensitive to degradation to 50 and 30S subunits in 10(-3)m Mg(2+) than 70S monomers obtained either by degradation of polysomes with ribonuclease or by starvation of cells for amino acids other than methionine. The 70S ribosomes from methionine starvation were similar (sensitivity to 10(-3)m Mg(2+)) to 70S ribosomes obtained from cells in which initiation of protein synthesis had been prevented by trimethoprim, an inhibitor of formylation. Since N-formyl-methionyl-transfer ribonucleic acid is required for initiation, the 70S ribosomes obtained in both methionine-starved and trimethoprim-treated cells must result from association of 50 and 30S subunits for reasons other than reinitiation. These results suggest that the level of ribonucleic acid synthesis does not influence the distribution of ribosomes in the polysome profile and vice versa.     

Ribonucleic Acid Regulation in Amino Acid-Limited Cultures of Escherichia coli Grown in a Chemostat

The regulation of ribonucleic acid (RNA) synthesis was examined in cultures of bacteria whose growth was limited in the chemostat by the supply of a required amino acid. Strains possessing the relaxed (relA) mutation accumulated excess RNA (relative to protein) at low growth rates when growth was limited by arginine, histidine, or cysteine but not when limited by methionine. In contrast, stringent (relA(+)) strains maintained a constant RNA/protein ratio with decreasing growth rate regardless of the amino acid used to limit growth. The presence of excess RNA in relaxed strains was accompanied by an absence of increase in RNA production upon addition of chloramphenicol, a lag upon shift-up in growth by addition of excess of the limiting amino acid, and a decreased rate of production of beta-galactosidase upon induction. Analysis of the RNA accumulated in relaxed strains indicated it was present as transfer RNA as well as 50S and 30S ribosomal subunits. Microscope examination of the relaxed strains during histidine-, arginine-, or cysteine-limited growth in the chemostat showed them to be 10 to 20 times longer in size than the stringent strains. Also, cell density was reduced to one-tenth when the increased size was observed. An analysis of the amount of ppGpp present in all slow-growing amino acid-limited cultures (relaxed and stringent) demonstrated that only basal levels of ppGpp were made. These data are consistent with the hypothesis that when growth is limited in the chemostat by an initiation event in protein synthesis, i.e., limited methionine, RNA regulation occurs in relaxed as well as stringent strains. Also, when other amino acids are limiting in concentration during translation, errors occur in relaxed strains, resulting in misread proteins.     

Continued Expression of the Ribonucleic Acid Control Gene During Inhibition of Escherichia coli Ribonucleic Acid and Protein Synthesis

The effect of the ribonucleic acid (RNA) control (RC) gene on the biosynthesis of viral RNA has been examined in an RC(str) and an RC(rel) host infected with R17 RNA bacteriophage under conditions in which host RNA and protein synthesis were inhibited by the addition of rifampicin. Methionine and isoleucine starvation depressed viral RNA biosynthesis in an RC(str) host but not in an RC(rel) host. However, histidine starvation had little effect on viral RNA and protein synthesis in both RC(str) and RC(rel) cells, although it had a marked effect on host protein and RNA synthesis in an RC(str) host. Chloramphenicol relieved the effect of amino acid starvation on viral RNA synthesis in an RC(str) host. It is concluded that stringent control of viral RNA biosynthesis does not require the continued biosynthesis of the RC gene product (RNA or protein) and that a preformed RC gene product can regulate the biosynthesis of the exogenous RNA. It is suggested that the amino acid dependence of viral RNA biosynthesis is due to its obligatory coupling with the translation of the viral coat protein which lacks histidine. It may be inferred that the amino acid requirement of bacterial RNA is due to its coupling with the translation of a host-specific protein (other than the RC gene product) which requires a full complement of amino acids. Since chloramphenicol is known to permit ribosome movement in the absence of protein synthesis, it is suggested that ribosome movement along the nascent RNA chain is a sufficient condition for the continuation of RNA synthesis.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

Effect of amino acid starvation on glucose transport in two archaeal organisms

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.     

Relaxed mutants of Escherichia coli RNA polymerase

When Escherichia coli cells are treated with either polymixin or gramicidin at concentrations that block protein and RNA synthesis, they accumulate a significant amount of guanosine tetraphosphate ppGpp. Such accumulation occurs in stringent (relA+) as well as in relaxed (relA) strains and no guanosine pentaphosphate pppGpp is then detected within the cells. These observations suggest that polypeptide antibiotics elicit ppGpp formation through a mechanism different from the stringent control system triggered by amino acid starvation of bacteria. Experiments based on tetracycline action indicate, moreover, that the accumulation of ppGpp under polymixin or gramicidin treatment is connected with a strong restriction of the degradation rate of this nucleotide.     

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.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.     

Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli.

Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid.     

Stringent and growth control of rRNA synthesis in Escherichia coli are both mediated by ppGpp

Weak stringent or relaxed responses were induced in Escherichia coli (relA+), using mild amino acid starvation or treatment with chloramphenicol at low concentrations, respectively, such that the growth rate was barely reduced. In this manner, the intracellular concentration of the nucleotide guanosine tetraphosphate, ppGpp, could be varied in any desired range between 0 and 1000 pmol of ppGpp per OD460 unit of culture mass. At the same time, the rate of synthesis of stable RNA (rs; rRNA and tRNA) was measured, relative to the total instantaneous rate of RNA synthesis (rt). The correlation between the cytoplasmic concentration of ppGpp and stable RNA gene activity (rs/rt) was the same as that observed previously with relA+ and relA strains growing exponentially at different rates in different media. This suggests that the distinction between growth control and stringent control of stable RNA synthesis is arbitrary, and that both kinds of control reflect the same ppGpp-dependent phenomenon. By increasing the stable RNA gene dosage, using high copy number plasmids carrying an rrn gene, we have tested the idea that ppGpp partitions the bacterial RNA polymerase into two forms with different probabilities to initiate at stable RNA and mRNA promoters. The relaxed response was not significantly altered, but the extent of the stringent response was reduced by the presence of extra rrn genes. The results agree with quantitative predictions derived from the RNA polymerase partitioning hypothesis.