On the control of ribosomal protein biosynthesis in Escherichia coli. II. Studies during recovery from amino acid starvation.

The rate of synthesis of ribosomal proteins relative to that of total protein was measured at various times during recovery from arginine starvation in isogenic re+ and rel- strains of Escherichia coli K 12. Total ribosomal proteins are preferentially synthesized early during recovery. Higher rates of synthesis are obtained in the rel+ strain than in the rel- strain. Differential rates of synthesis of individual ribosomal proteins are observed at the various times studied. The rate of synthesis of individual proteins increases with time up to maximum values then the rates come down to values similar to those found in exponentially growing cells. The time of restart of synthesis of each protein has been estimated (1) by the time at which the maximum value is reached, and (2) by measuring the rate of synthesis at early time (3 min). Most ribosomal proteins behave similarlly in rel- and rel+ strains. Proteins have been listed from highly labelled (early proteins) to poorly labelled (late proteins). The significance of the order of restart is considered.     

Synthesis of beta, beta'-subunits of RNA-polymerase in E. coli cells starved for an essential amino acid]

The differential rate of RNA-polymerase beta,beta'-subunits synthesis in E. coli rel+ and rel- cells starved for an essential amino acid, i. e. leucine, is decreased. Inhibition of the rpo BC genes expression proceeds faster in the rel A strain. Guanosine tetraphosphate within the concentration range of 0,2--0,6 mM specifically inhibits lambda drifd18 DNA directed beta,beta'-subunits synthesis in vitro. rpo BC genes are believed to be subject to a weak "stringent control".     

On the control of ribosomal protein biosynthesis in Escherichia coli. I. Studies on ribosomal protein biosynthesis in amino acid-starved cells.

The rate of individual ribosomal protein synthesis relative to total protein synthesis has been determined in Escherichia coli rel+ and rel- cells, under valyltRNA deprivation. These strains have a temperature-sensitive valyl-tRNA synthetase. Starvation was obtained following transfer to the cells to non-permissive temperature. Ribosomal proteins were obtained by treatment of either total lysates of freeze-thawed lysozyme spheroplasts or ammonium sulphate precipitate of ribosomes, with acetic acid. Differential labelling of the ribosomal proteins was observed in both strains: proteins from the rel+ strain appear more labelled than those from the rel- strain, the rate of labelling of individual proteins being about the same in both strains. Moreover ribosomal proteins were found as stable during starvation as total protein. It is thus concluded that in starving cells individual ribosomal proteins are not synthesized at equal rates. This indicates that the synthesis of ribosomal proteins is not only under the control of the rel gene.     

The suppression of defective translation by ppGpp and its role in the stringent response.

Amino acid starvation is shown to decrease the fidelity of translation in E. coli. When proteins are analyzed by two-dimensional gel electrophoresis, missense errors are detected as an unusual heterogeneity in their isoelectric points, while premature termination of protein synthesis can be recognized by a decreased relative rate of synthesis of higher molecular weight proteins and by the the accumulation of a complex group of new small polypeptides. The types of translational errors observed are amino acid-specific. For example, starvation of a rel- strain for histidine produces severe isoelectric point heterogeneity with little evidence of premature termination, while starvation for leucine has little effect on the isoelectric points, but produces a drastic decrease in the average molecular weight of the newly synthesized protein. These differences suggest codon-specific errors in reading the genetic code. In these rel- cells, the effect of amino acid starvation on the rates of synthesis of complete individual proteins is both protein- and amino acid-specific. For example, ribosomal protein L7/12, which lacks histidine, is made at a higher level during histidine starvation than during isoleucine or leucine starvation. This suggests that in rel- cells, the modulation of gene expression caused by the lack of a particular amino acid is, at least in part, a function of the abundance of that amino acid in particular proteins-that is, the response of rel- cells to starvation is consistent with the theory that the inhibition of protein synthesis and the accompanying increase in error frequency both result from low levels of the correct substrate. In marked contrast, virtually no starvation-induced translational errors are detected in a rel+ strain, and the response is not amino acid-specific. Varoius data strongly imply that in this rel+ strain, essentially all the changes caused by starvation are due to the accumulation of ppGpp, which independently reduces protein synthesis, thereby suppressing all the direct effects of amino acid limitation seen in rel- strains (where ppGpp does not accumulate upon starvation). A model is presented which describes how ppGpp might suppress the direct effects of starvation and avoid the loss of translational fidelity. In addition, the direct and specific effects of ppGpp on gene expression are examined independently of amino acid starvation.     

Effect of ppGpp on the accuracy of protein biosynthesis

The maintenance of accuracy in protein biosynthesis in amino acid-starved rel+ strains of Escherichia coli has been attributed to an effect of ppGpp on the accuracy of aa-tRNA selection by the ribosome. It has been determined that concentrations of ppGpp characteristic of those found in amino acid-starved cells have no effect on the rate of reaction of poly(U)-programmed ribosomes with either the cognate (Phe) or the near-cognate (Leu2) ternary complexes. Neither the rate of GTP hydrolysis, which signals selection of the ternary complex, nor the rate of peptide formation, which signals the acceptance of the aa-tRNA after proofreading, is affected by the nucleotide. The results indicate that the effect of ppGpp in maintaining the accuracy of protein biosynthesis in cells starved for an amino acid is not due to a direct effect on the rate constants for substrate selection by the ribosome.     

DNA synthesis in vitro in human fibroblast preparations

With several pairs of rel+ and rel- strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K+ and the rate of uptake of 42K+ were investigated. In the early phase of the experiments, the intracellular concentration of K+ was estimated by the conventional method in which the cell volume per A660 value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K+ concentration of rel+ cells was kept almost constant, while that of rel- cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A660 changed only slightly in CP78 (rel+) cells, while it increased markedly in CP79 (rel-) cells after the exposure to amino acid starvation. Reestimation of the K+ concentrations based on the estimated respective values of cell volumes per A660 revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel+ cells was arrested upon amino acid starvation whereas that in the rel- cells was not. The 42K+ uptake by the rel+ cells was depressed upon amino acid starvation, whereas that by the rel- cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K+ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A660 of the culture, was pointed out.     

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

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

Ribosome biosynthesis in Tetrahymena pyriformis. Regulation in response to nutritional changes

Ribosome contents of growing and 12-h-starved Tetrahymena pyriformis (strain B) were compared. These studies indicate that (a) starved cells contain 74% of the ribosomes found in growing cells, (b) growing cells devote 20% of their protein synthetic activity to ribosomal protein production, and (c) less than 3% of the protein synthesized in starved cells is ribosomal protein. Ribosome metabolism was also studied in starved cells which had been refed. For the first 1.5 h after refeeding, there is no change in ribosome number per cell. Between 1.5 and 2 h, there is an abrupt increase in rate of ribosome accumulation but little change in rate of cell division. By 3.5 h, the number of ribosomes per cell has increased to that found in growing cells. At this time, the culture begins to grow exponentially at a normal rate. During the first 2 h after refeeding, cells devote 30-40% of their protein synthetic activity to ribosomal protein production. We estimate that the rate of ribosomal protein synthesis per cell increases at least 80-fold during the first 1-1.5 h after refeeding, reaching the level found in exponentially growing cells. This occurs before any detectable change in ribosome number per cell. The transit time for the incorporation of these newly synthesized proteins into ribosomes is from 1 to 2 h during early refeeding, whereas in exponentially growing cells it is less than 30 min. The relationship between ribosomal protein synthesis and ribosome accumulation is discussed.     

Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli.

The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation.     

Turnover of chloroplast and cytoplasmic ribosomes during gametogenesis in Chlamydomonas reinhardi

A number of novel observations on ribosomal metabolism were made during gametic differentiation of Chlamydomonas reinhardi. Throughout the gametogenic process the amount of chloroplast and cytoplasmic ribosomes decreased steadily. The kinetics and extent of such decreases were different for each of the two ribosomal species. Comparable rRNA degradation accompanied this ribosome degradation. Concurrent with the substantial ribosome degradation was the synthesis of rRNA, ribosomal proteins and the assembly of new chloroplast and cytoplasmic ribosomes throughout gametogenesis. The newly synthesized chloroplast ribosomes exhibited distinctively faster turnover than their cytoplasmic counterpart. Cytoplasmic ribosomes, pulse-labeled in early gametogenic stages, retained label until differentiation was nearly complete even though a net decrease in the level of cytoplasmic ribosomes continued, indicating that the newly synthesized cytoplasmic ribosomes were preferentially retained during differentiation. Hence the regulation of ribosome metabolism during gametogenesis contrasts with the conservation of ribosomes obtained during vegetative growth of C. reinhardi and other organisms. This unique pattern of ribosome metabolism suggests that new ribosome synthesis is necessary during gametogenesis and that some specific structural or functional difference relating to the development stage of the life cycle might exist between degraded and newly synthesized ribosomes.     

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.     

Post-transcriptional regulation of ribosome accumulation during myoblast differentiation

The synthesis, accumulation and stability of rRNA were examined in embryonic quail myoblasts differentiating in cell culture. Quail myoblasts initially divide rapidly in culture, and accumulate 28S and 18S rRNA and ribosomes at a rate which maintains a constant ribosome content during cell division. After these myoblasts fuse, cell division ceases and ribosomes accumulate in fibers, but at a reduced rate which is only one fourth that in dividing myoblasts. Measurements of rRNA stability by 3H-methyl-methionine pulse-chase analysis show that 28S and 18S rRNA formed by fibers turn over with half-lives of 45 hr, and rRNA formed by myoblasts remains stable until fusion and then also turns over in fibers. Turnover of rRNA in fibers accounts for only half the reduction in ribosome accumulation following myoblast fusion. Measurements of the incorporation of 3H-adenosine into rRNA and ATP pools show that the rates of synthesis of rRNA precursor do not decrease after myoblast fuse, but half the rRNA molecules synthesized by fibers are degraded during processing. Degradation of rRNA during processing reduces the rate of formation of 28S and 18S rRNA, and together with rRNA turnover quantitatively accounts for the reduced rate of ribosome accumulation in fibers.     

An analysis of the ribosomal ribonucleic acids of Escherichia coli by hybridization techniques

From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0.35+/-0.02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA-RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0.45% of the DNA would hybridize total rRNA and (b) when so little RNA was added to unit weight of DNA that the DNA sites were not saturated, only 70-75% of the input RNA would form hybrids. The reasons for the discrepancy between the results obtained by the two alternative analytical approaches were discussed. (3) For either 16s or 23s rRNA, hybridization analysis indicated that two principal weight fractions of rRNA may exist, hybridizing to two distinct groups of DNA sites. However, these groups seem to be incompletely divided between the 16s and 23s fractions. Analysis suggested that (a) 85% of the 16s rRNA was hybridized to about half the DNA that specifically binds rRNA (0.23% of the total DNA). (b) 70% of the 23s rRNA hybridized to a further 0.23% of the DNA and (c) the minor fraction (15%) of 16s rRNA may be competitive with the major fraction (70%) of 23s rRNA. Conversely, the minor fraction (30%) of the 23s rRNA may compete with the major fraction (85%) of 16s rRNA. Models were proposed to explain the apparent lack of segregation of distinct RNA species in the two subfractions of rRNA. (4) If protein synthesis and ribosome maturation were inhibited in cells of an RC(rel) mutant, E. coli W 1665, by depriving them of an amino acid (methionine) essential for growth, the inhibition had no discernible effect on the relative rates of synthesis of rRNA species. The rRNA that accumulates in RC(rel) strains of E. coli after amino acid deprivation is apparently identical in its content of RNA species with that of the pre-existing mature RNA in the ribosomes. On the other hand, the messenger RNA is stabilized, and accumulates as about 15% of the RNA formed after withdrawal of the amino acid.     

The connection between ribophagy, autophagy and ribosomal RNA decay

Ribosomes are essential components of all cells. A large body of knowledge has been accumulated regarding ribosome synthesis and assembly; however, the pathways of normal ribosome turnover, especially rRNA decay, are not known. Some information on ribosome recycling derives from studies on starved yeast cells that use a specialized type of autophagy, called ribophagy, to differentially target ribosomes for degradation. We found that Arabidopsis RNS2, a conserved ribonuclease of the RNase T2 family, is necessary for normal decay of rRNA. Mutants lacking RNS2 activity have longer-lived rRNA, accumulate RNA in the vacuole and show constitutive macroautophagy. Thus, it is clear that normal rRNA decay is necessary to maintain cellular homeostasis. These phenotypes and the subcellular localization of RNS2 in the endoplasmic reticulum and the vacuole suggest that RNS2 participates in a ribophagy-like mechanism that targets ribosomes for recycling under normal growth conditions.     

The connection between ribophagy,autophagy and ribosomal RNA decay

Ribosomes are essential components of all cells. A large body of knowledge has been accumulated regarding ribosome synthesis and assembly; however, the pathways of normal ribosome turnover, especially rRNA decay, are not known. Some information on ribosome recycling derives from studies on starved yeast cells that use a specialized type of autophagy, called ribophagy, to differentially target ribosomes for degradation. We found that Arabidopsis RNS2, a conserved ribonuclease of the RNase T2 family, is necessary for normal decay of rRNA. Mutants lacking RNS2 activity have longer-lived rRNA, accumulate RNA in the vacuole and show constitutive macroautophagy. Thus, it is clear that normal rRNA decay is necessary to maintain cellular homeostasis. These phenotypes and the subcellular localization of RNS2 in the endoplasmic reticulum and the vacuole suggest that RNS2 participates in a ribophagy-like mechanism that targets ribosomes for recycling under normal growth conditions.     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.     

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.