Regulation of mRNA Utilization and Degradation by Amino-acid Starvation

Amino-acid starvation of a stringent strain of E. coli relieves the polar effect on distal messenger RNA imposed by a nonsense mutation. There is no relief of polarity starvation of a relaxed strain. In fact, starvation of a relaxed (but not of a stringent) strain by itself causes an artificial polar effect on distal mRNA. These findings are consistent with a mechanism for polarity based on mRNA degradation.     

Different cyclic AMP requirements for induction of the arabinose and lactose operons of Escherichia coli

Valyl-tRNA deprivation causes a threefold reduction of the polysome content of stringent cells but not of relaxed cells. The polysomes of valyl-tRNA-deprived stringent and relaxed cells decay in the presence of rifampin at a rate very similar to that observed in growing cells.Polysome assembly and decay were studied in valyl-tRNA-deprived stringent and relaxed strains after first causing the pre-existing polysomes to be converted to monosomes by glucose starvation. The capacity for polysome assembly is normal in relaxed cells and is reduced by a factor of three in stringent cells. The polysomes which reassemble in glucose-starved cells also decay in the presence of rifampin at a rate similar to that of the polysomes of growing cells. The polysomes which assemble in relaxed cells are potentially functionally competent, as shown by their ability to incorporate amino acids in an in vitro proteinsynthesizing system. Valyl-tRNA deprivation causes an intense shift in the polysome size distribution in stringent cells, but only a moderate shift in relaxed cells. A model for the control of the polysome level in amino acid-starved cells, based on these observations, is presented here.     

Functional aspects of bacterial polysomes during limited protein synthesis

The effects of amino acid starvation on the metabolic behavior of polysomes and the size distribution of proteins have been studied in an otherwise isogenic pair of stringent (relA+) and relaxed (relA) strains of Escherichia coli. The stability of polysomes has been analyzed by using two different approaches. First, the process of their degradation has been followed after treating the cells with rifampicin, an inhibitor of the synthesis of all classes of RNA including messenger RNA. Secondly, the process of their assembly has been studied after their previous conversion to monosomes, as induced by glucose deprivation of cells. It is shown that, in either type of bacterial strain, polysomes are continually broken down and re-synthesized during amino acid starvation. However, such polysome turnover is then less rapid than in normally growing bacteria and, moreover, it seems amino acid specific since it occurs at a lower rate during arginine starvation than during histidine starvation, namely, in the relaxed strain. The molecular weight distribution of proteins has been determined after labeling of cells with radioactive methionine and separation of polypeptides by one-dimensional polyacrylamide gel electrophoresis. The average size of polypeptides synthesized in the stringent strain during starvation is quite similar to that measured during normal growth. By contrast, a significant shift towards smaller molecules is observed in the relaxed strain deprived of an essential amino acid. Here again, this reduction of the size of polypeptides seems amino acid specific since it is especially marked during arginine starvation. These results are discussed in terms of ribosomes translocation and premature peptide chain termination in connection with the accuracy of the translational process.     

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.     

Loss of translational control in yeast compromised for the major mRNA decay pathway

The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways. In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress. In the past, the paradigm of the eIF2alpha kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2alpha phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.     

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.     

Synthesis, no synthesis, or synthesis and degradation of ribosomal ribonucleic acid in various Escherichia coli strains starved for an amino acid

A standard stringent strain of Escherichia coli makes little or no ribosomal ribonucleic acid (RNA) during starvation for an essential amino acid, whereas the isogenic relaxed strain makes both ribosomal and messenger RNA. A third class of strains was found which continues to make ribosomal RNA during starvation, but the RNA made is apparently unstable. There is little accumulation of RNA in the third class of strains, and few complete newly formed chains of (3)H-ribosomal RNA are observed in sedimentation analyses, even after long labeling times.     

Replication and expression of plasmid pBR 322 during discontinuous culture of a stringent and relaxed Escherichia coli strain

The E. coli strains CP78 and CP79 carrying the plasmid pBR 322 display similar growth kinetics in discontinuous culture. During limitation of amino acids the stringent strain CP78 is able to synthesize guanosine-5'diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3' diphosphate, but the relaxed strain can not produce highly phosphorylated guanosine nucleotides. During the logarithmic phase of growth both strains contain similar amounts of plasmid DNA. During amino acid starvation plasmid DNA is amplified in the relaxed strain only, whereas in the stringent strain the plasmid content per cell remains constant. In stationary phase cells of CP78 a higher activity of plasmid-coded beta-lactamase than in CP79 cells was detected. Furthermore, remarkable differences between both strains were observed in the composition of proteins derived from the periplasmic fraction and separated by polyacrylamide gel electrophoresis. Our results might indicate a negative control of pBR 322 DNA replication by ppGpp during amino acid starvation.     

3H-thymidine incorporation following isoleucine limitation in stringent and relaxed controlled strains of Escherichia coli

Stringent and relaxed strains of E. coli subjected to isoleucine starvation were examined by follow-wing the incorporation of 3H-thymidine into chromosomal DNA. After valine treatment to trigger an isoleucine deprivation (p)ppGpp is synthesized in the stringent strain only. Remarkable differences in the morphology of the amino acid starved cells of the stringent and relaxed strains can be observed. Upon isoleucine limitation 3H-thymidine incorporation into DNA is reduced in both strains, but this inhibition is remarkably delayed in the relaxed strain. Our result show that the reduction of chromosomal DNA synthesis during amino acid limitation occurs also without ppGpp, but in the presence of ppGpp this process is accelerated.     

Erroneous synthesis of ribosomal proteins in amino acid starved E. coli

The effect of amino acid starvation on the accuracy of translation of ribosomal proteins was analyzed in a stringent (relA+)/relaxed (relA) pair of E. coli strains. The degree of misreading was estimated from the amount of cysteine erroneously incorporated into individual proteins during arginine starvation of bacteria. Illegitimate incorporation of cysteine was found to occur to a significant extent in several proteins from both the small and the large subunits of ribosomes, in either type of strain.     

The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

Guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a ‘rare’ actinomycete

With the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) and guanosine 5′‐triphosphate 3′‐diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)‐encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild‐type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic cluster‐specific dpgA mRNA were stabilized during the idiophase in the wild‐type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse‐labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined.     

Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same

We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli ams(ts) strain (originally defined by a prolonged bulk mRNA half-life) and in the me(ts) strain (originally defined by aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8 kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the ams(ts) and rne(ts) strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Ams(ts) and the Rne(ts) phenotype with regard to the ompA cleavages. In addition we showed that the ams(ts) strain suppresses 9S RNA processing to 5S RNA to the same extent as the rne(ts) strain, and that the rne(ts0 strain has a prolonged bulk mRNA half-life, as was reported for the ams(ts) strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K.     

Isolation of relaxed-control mutants of Escherichia coli K-12 which are sensitive to glucose starvation

Mutants of $\text{Escherichia coli}$ K-12 which are sensitive to glucose starvation were isolated by an enrichment procedure using thymine starvation to select for nongrowing cells. Eleven independent isolates were obtained by this method. The mutants are also sensitive to glycerol starvation and to a lesser extent to nitrogen or amino acid starvation. The mutants are more sensitive than the parental strain to inhibitors of protein synthesis but not inhibitors of RNA or DNA synthesis. [³H]-leucine incorporation experiments indicate that protein synthesis is blocked in the mutants during recovery from glucose starvation or chloramphenicol inhibition. Incorporation of [³H]uridine in amino acid-starved cells demonstrates that the mutants are partially relaxed for control of RNA synthesis. Physiological and genetic experiments indicate that these mutants are different from previously isolated relaxed-control mutants.     

Control of Protein Synthesis in Escherichia coli: Analysis of an Energy Source Shift-Down

The energy source shift-down described in the preceding paper (Molin et al., J. Bacteriol. 131: 7-17, 1977) was used to study the effects of shift-down on protein synthesis. The overall rate of protein synthesis was reduced immediately, and to the same extent, in stringent and relaxed strains. The primary effect of the shift was a slowing down of the polypeptide chain growth rate, a finding not previously reported. In stringent strains the normal, preshift rate was reestablished within 2 to 3 min, whereas in relaxed strains the chain growth rate remained low for about 20 min before slowly returning to the normal value, which was reestablished some 50 to 60 min after the shift. Throughout this transition, the stability of messenger ribonucleic acid (mRNA) remained unchanged in both strains. We interpret these findings as evidence of the more rapid reduction of the mRNA pool in the stringent strain after shift-down: we believe that very soon after the shift, the stringent strain reduces its pool of mRNA and with it the number of ribosomes engaged in protein synthesis. In this manner the number of active ribosomes is adjusted to the availability of energy and carbon. The relaxed strain cannot rapidly reduce its mRNA pool, which thus remains large enough to engage a near-preshift number of ribosomes during a prolonged period; as a consequence its ribosomes must work at a reduced rate. The possibility that ppGpp is involved in the control of mRNA production is discussed. After shift-down, the initial part of beta-galactosidase (the auto-alpha fragment) was produced at a higher rate than complete beta-galactosidase in the relaxed strain, as expected when translation is impeded.