The control of ribonucleic acid synthesis in bacteria. Fluctuations in messenger ribonucleic acid synthesis in cultures recovering from amino acid starvation

Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNA(fMet) for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RC(str) strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RC(rel) strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RC(str) and RC(rel) cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.     

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.     

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli

The biosynthesis and stability of various RNA fractions was studied in RC(str) and RC(rel) multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RC(str) mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA-RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60-70% of the labelled RNA formed in RC(str) mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RC(rel) strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RC(rel) strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RC(str) or RC(rel) strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RC(str) auxotrophs was unchanged from that found in normally growing cells. In contrast, RC(rel) strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.     

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.     

Ribonucleic Acid Synthesis and Glutamate Excretion in Escherichia coli

Cultures of Escherichia coli excreted glutamate into the medium when protein synthesis was blocked in RC(rel) strains or when it was blocked with chloramphenicol in either RC(str) or RC(rel) strains. Both of these conditions resulted in continued ribonucleic acid (RNA) synthesis in the absence of protein synthesis. Glutamate was also excreted by both RC(str) and RC(rel) strains when RNA synthesis was inhibited by uracil starvation or by treatment with actinomycin D. It is proposed that, in each of these cases, glutamate excretion resulted from an increase in the permeability of the cell membrane.     

The control of ribonucleic acid synthesis in bacteria. Polymerization rates for ribonucleic acids in amino acid-starved relaxed and stringent auxotrophs of Escherichia coli

Polymerization rates of newly formed chains of various RNA fractions were measured in Escherichia coli CP78 (RC(str)) and CP79 (RC(rel)) multiple amino acid auxotrophs, deprived of four amino acids essential for growth. Immediately after the onset of severe amino acid deprivation, in RC(str) strains the rate of labelling of RNA by exogenous nucleotide bases was greatly diminished. At first, the initiation of new RNA chains declined faster than the rate of polymerization in RC(str) organisms, but as starvation proceeded the rate of polymerization was eventually lowered to about 10% of that found during normal growth. In strain CP79 (RC(rel)), on the other hand, chain-polymerization rates were unaffected by amino acid withdrawal. Artificial depletion of the intracellular purine nucleotide pools in RC(str) or RC(rel) strains by trimethoprim, before the onset of amino acid deprivation, showed that in the RC(str), but not the RC(rel) strain, amino acid withdrawal gave rise to an inability of the cells to utilize exogenously supplied purine or pyrimidine bases for RNA synthesis. During a prolonged starvation, the observed 100-fold decrease in the total rate of incorporation of exogenous nucleotide bases into the RNA of RC(str) organisms was ascribed to a combination of a tenfold decrease in the overall rate of RNA chain polymerization, at least a fivefold decrease in the ability of the cells to utilize exogenous bases and a preferential inhibition of initiation of stable RNA chains. None of these changes occurred in the corresponding RC(rel) strain.     

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.     

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

Starvation for a required amino acid of normal or RC(str)Escherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RC(rel)E. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RC(str) phenotype but not in cells of RC(rel) phenotype. Inhibition of phage DNA synthesis in amino acid-starved RC(str) host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought.     

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. General effects on ribonucleic acid and protein synthesis

In trimethoprim-inhibited RC(str) strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RC(rel) strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RC(str) strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RC(rel) strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RC(str) strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RC(str) or RC(rel) bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.     

Translational control and the cytoskeleton in Physarum polycephalum

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.     

Isolation and properties of polyribosomes and fragments of the endoplasmic reticulum from rat liver

1. A centrifugation method for the fractionation of the postmitochondrial fraction from rat-liver homogenates is described. The technique, in which no detergent is used, may be used as a tool to discriminate between two classes of ribosomes. One class is firmly bound to membranes and the other consists either of free polysomes or of ribosomes attached by weaker forces to the membranes of the endoplasmic reticulum. 2. Electron-micrograph studies revealed that the polysomes were not contaminated with bound ribosomes or with membranous fragments. 3. The separated fractions were characterized by their RNA, protein, ribonuclease and phospholipid content. 4. The influence of starvation on the RNA and protein contents of the different fractions was investigated. 5. Labelling of the various centrifugal fractions in vivo revealed no difference in uptake of radioactive amino acid between the two classes of ribosomes. 6. Incorporation of radioactive leucine in vitro and the polyuridylic acid-directed phenylalanine incorporation were similar for both classes of ribosomes.     

Polysome activity in relation to growth and protein starvation in brains and hearts of cultured early chick embryos

In previous studies, brains but not hearts of intact early chick embryos were found to be sensitive to protein starvation. In this study, the in vitro protein synthetic activity of polysomes isolated from brains was found to be greater than those isolated from hearts. Starvation reduced the protein synthetic activity of polysomes in vitro but the extent of the reduction was approximately the same for both brains and hearts. A reduction in the amount of ribosomes as polysomes may have contributed to the lower synthetic activity of polysomes from tissues of starved embryos but not to the differences in synthetic activities between brains and hearts. In addition, neither the stability of isolated polysomes nor ribosome-associated ribonuclease activity appeared responsible for the differences observed in polysome synthetic activities. In direct relationship to the differential sensitivity of brains and hearts to starvation observed in the intact embryo, ribosomes isolated from brains of both growing and starved embryos were more readily degraded during in vitro incubation than those from hearts.     

Characterization of hibernating ribosomes in mammalian cells

Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.Key words: ribosome, translation, stress, starvation, polysome     

Characterization of hibernating ribosomes in mammalian cells

Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.     

Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes

The kinetics of labeled histone mRNA entry into polysomes was studied in nuclease-treated reticulocyte lysates. Added mRNA rapidly bound 1 or 2 ribosomes. However, the formation of full size polysomes required at least 16 min. The amount of mRNA bound to ribosomes reached a maximum (73%) within 2 min after mRNA addition and then declined slowly for the remainder of the experiment. Two initiation inhibitors, aurintricarboxylic acid and 7-methylguanosine 5'-triphosphate, were found to affect polysome size and the fraction of mRNA in polysomes in an opposite manner. These results suggest that initiation and reinitiation events may be intrinsically different. The relatively long time period required for the formation of large polysomes can be explained by large polysomes having higher initiation and/or reinitiation rates or slower elongation rates. These possibilities are not mutually exclusive. The results suggest that there exist several levels of control which can regulate polysome size and the fraction of mRNA in polysomes.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

Polysome formation and incorporation of new ribosomes into polysomes during germination of the embryonic axis of maize

Isolated axes of Zea mays L. cvs CiV₂ and CUZCO were imbibed for different periods of time, and free polysomes were extracted and analysed by centrifugation in a sucrose gradient. The amount of rRNA per axis was determined at different moments of germination. Polysome reassembly was practically completed by 8 h and 54% of the preformed ribosomes were found in the polysome fraction. An increase in the proportion of large polysomes was also observed during this period of germination. During the following period, the polysome content and the distribution of the various classes of polysomes remained unchanged.
The time of appearance of newly synthesized ribosomes into the polysomes was investigated using axes germinated in the presence of [³H]-uridine. Centrifugal analysis of EDTA-dissociated polysomes and gel electrophoretic analysis of polysomal RNA showed that new ribosomes appeared into polysomes a few hours after completion of the initial polysome assembly. When released into the cytoplasm, the new ribosomes were preferentially incorporated into polysomes rather than stored as free ribosomes.     

Starvation-induced alterations of ribosomal protein phosphorylation in Bombyx mori L. Evidence for different phosphorylation kinetics in free and membrane-bound ribosomes

In the posterior silk gland of Bombyx mori, ribosomal protein S1, homologous to S6 in mammals, is partially phosphorylated in a normally fed animal. Before the first meal of the fifth larval instar, S1 is completely dephosphorylated. Likewise, starvation induces rapid dephosphorylation of the protein in both free and membrane-bound ribosomes. Upon refeeding after 48 h of starvation, S1 becomes phosphorylated again, first on membrane-bound ribosomes, then on free ribosomes, with a lag time of about 3 h. Following 48 h of refeeding, the most highly phosphorylated form of S1 predominates in both populations of ribosomes. These variations in phosphorylation are correlated with the level of protein synthesis in the posterior silk gland, 70% of the ribosomes occurring in polysomes upon feeding and only 30% upon starvation [Prudhomme, J.-C. & Couble, P. (1979) Biochimie (Paris) 61, 215-227]. After in vivo 32P labelling, the phosphopeptides of S1 from free and membrane-bound ribosomes were found to be identical and phosphoserine (only) was found in each S1. These results suggest the involvement of S1 phosphorylation in the regulation of protein synthesis at the translational level and the existence of at least two different pathways controlling this phosphorylation: one for the free ribosomes, the other for the membrane-bound ribosomes.     

DISTRIBUTION OF POLYSOMES IN MOUSE BRAIN TISSUE

Abstract— Polysomes were isolated from several different fractions of mouse brain tissue. After homogenization, the extract was centrifuged to yield a post-mitochondrial supernatant fraction and a pellet fraction. Sucrose gradient analysis of the material in the post-mitochondrial supernatant fraction indicated that 80 per cent of the ribosomes were present in polysomes and that little, if any, of the pellet fraction was present. Sucrose gradient analysis of the solution obtained after washing the pellet showed that very little polysomal material was present. The remaining pellet fraction was resuspended in a detergent mixture of deoxycholate-Tween 40. Sucrose gradient analysis of the resulting detergent-soluble solution indicated that large amounts of ribosomal material, in which 60–70 per cent of the ribosomes were associated in polysomes, were present.
In brain tissue from young animals, 20 per cent of the polysomes were found in the post-mitochondrial supernatant fraction whereas 80 per cent of the polysomes were released from the pellet fraction by detergent treatment. In contrast, in brain tissue from adult animals, 40 per cent of the polysomes were found in the post-mitochondrial supernatant fraction, whereas 60 per cent of the polysomes were released from the pellet fraction by detergent treatment.