Control Of Ribonucleic Acid Synthesis Research Articles

Formation of sugar phosphates in colicin K-treated Escherichia coli.

Colicin K greatly decreased the incorporation of 32P-labeled inorganic orthophosphate into nucleotides and nucleic acids, causing a concomitant increase in the formation of 32P-labeled sugar phosphates in sensitive cells of Escherichia coli. These sugar phosphates were formed in aerobically growing cells, as well as in cells under stringent control of ribonucleic acid synthesis. The main 32P-labeled product was identified as sedoheptulose 7-phosphate in two strains (B1 and K-12 MK-1) and fructose 1,6-diphosphate in one strain (K-12 CP78). The formation of sugar phosphates induced by colicin K was inhibited by carbonyl cyanide m-chlorophenylhydrazone. It was also not observed in N,N'-dicyclohexylcarbodiimide-treated cells or Mg2+-(Ca2+)-adenosine triphosphatase-less mutant (strain K-12 AN120) cells. Thus, the formation of sugar phosphates in colicin K-treated cells is dependent on the formation of adenosine 5'-triphosphate by oxidative phosphorylation.

Control of ribonucleic acid synthesis in eukaryotes. 2. The effect of protein synthesis on the activities of nuclear and total DNA-dependent RNA polymerase in yeast.

A thermosensitive conditional yeast mutant (ts-187) which suppresses protein synthesis at the nonpermissive temperature (36 degrees C) also suppresses RNA synthesis. The effect of temperature on the mutant is similar to the addition of cycloheximide--it inhibits the incorporation of labeled precursors into RNA in both whole cells and isolated nuclei. The effect of temperature is selective for the RNA polymerases bound to the nuclear template but not for the total RNA polymerases. Thus, the specific activities and total amounts of RNA polymerase species extracted and assayed with exogenous DNA template are similar in the ts-187 cultured at 23 degrees C and at 36 degrees C. On the contrary, the nuclear polymerases, i.e., RNA synthesis in isolated nuclei, are dramatically inhibited in cells cultured at 36 degrees C. When amino acid starved ts-187 cells are transferred to 36 degrees C, release from the inhibtion of RNA synthesis is observed. As with the addition of cycloheximide, this relaxation is observed in cells but not in isolated nuclei. The parental strain, A364A, which responds by stimulating instead of inhibiting protein synthesis when the temperature is increased to 36 degrees C, also exhibits an inhibition in the incorporation of labeled precursor into RNA as well as reducing RNA synthesis in isolated nuclei. However, these are transitory inhibitions and afterward there is reinitiation of both processes. Reinitiation of RNA synthesis in isolated nuclei is similar to the relaxed phenomenon and it is called "nuclear relaxation". This relaxation can only be obtained if protein synthesis is not inhibited; however, cellular relaxation occurs in the absence of protein synthesis. The repression of the nuclear RNA polymerase activities which starvation and inhibition of protein synthesis produce appears to be due to a restriction in the nuclear DNA template. This notion is supported by the fact that a net diminution of these nuclear enzyme activities is observed in spheroplasts cultured under starving conditions. Studies of the four main ribonucleotide pools indicate that stringency and inhibition of protein synthesis (ts-187 cultured at 36 degrees C) produce an increase in UTP and CTP pools. This is consistent with the concept that stringency and inhibition of protein synthesis affect the rate of utilization rather than the synthesis of these ribonucleotide residues. In the A364A and ts-187 yeast strains, the conversion of uracil but not of uridine into the UTP and CTP is inhibited when there is inhibition of the nuclear RNA polymerases. This indicates that the uracil phosphoribosyltransferase but not the uridine-cytidine kinase is allosterically inhibited by UTP and CTP in yeast. The feedback inhibition in the metabolic pathway of the base explains why relaxation cannot be detected when uracil instead of uridine is used as the labeled RNA precursor.

Control of ribonucleic acid synthesis in eukaryotes. 3. The effect of cycloheximide and edeine on rna synthesis in yeast.

The addition of cycloheximide to a thermosensitive conditional yeast mutant (ts-187) before and after transfer to the nonpermissive temperature (36 degrees C) for initiation of protein synthesis produces the uncoupling of the RNA and protein synthetic machineries. Since the drug can produce this relaxation in the presence and absence of protein synthesis, it is concluded that the coupling of protein and RNA synthesis, which a temperature shift produces, is not exclusively related to the inhibition of protein synthesis. Support for this assumption has been obtained using the parental (A364A) strain. Transferring this strain to 36 degrees C produces inhibition of RNA synthesis in the presence of stimulation of protein synthesis. Furthermore, cycloheximide and edeine prevent this inhibtion of RNA synthesis that temperature shift produces. It is, therefore, postulated that this inhibition of RNA synthesis results from the synthesis or activation of a factor(s) elicited by the increase in temperature whose function is to repress the transcriptional apparatus. Cycloheximide or edeine can prevent the function of this repressor-like factor by binding to the factor or by preventing its synthesis. The fact that inhibition of protein synthesis either by cycloheximide action or temperature shift in ts-187 produces inhibition of RNA synthesis in isolated nuclei indicates that, in addition to the aforementioned repressor, other factor(s) having a promoter function may exist. Since a slight inhibition of protein synthesis produces nuclear template restrictions, it is postulated that the promoter-like factor(s) is a polypeptide different from the RNA polymerase and, at least in yeast, has a high turnover.

Stringent control of ribonucleic acid synthesis in Bacillus subtilis treated with granaticin.

The antibiotic granaticin interferes in Bacillus subtilis with the charging process of tRNALeu causing both the arrest of protein synthesis and bacteriostasis [A. Ogilvie, K. Wiebauer & W. Kersten (1975) Biochem. J. 152, 511-515]. A concomitant inhibition of RNA synthesis is observed. This inhibition was studied with mutant strains of B. subtilis. 2. Granaticin inhibits protein and RNA synthesis in stringently controlled B. subtilis (rel+) to about the same extent. In a relaxed mutant strain (rel-) of B. subtilis, protein synthesis is also inhibited, but the accumulation of RNA continues after the addition of the drug. 3. Chloramphenicol, which is known to abolish the stringent control mechanism, added simultaneously with granaticin, allows the synthesis of RNA to proceed in the stringent strain. 4. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate in granaticin-treated stringently controlled B. subtilis but not in the rel- mutant. 5. It is concluded that the inhibition of RNA synthesis granaticin can adequately be explained as a stringent response caused by the interference by the drug with leucyl-tRNA synthetase.

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.

Effects of leucine starvation on control of ribonucleic acid synthesis in strains of Bacillus subtilis differing in deoxyribonucleic acid regulation.

When starved for leucine, strains of Bacillus subtilis do not complete chromosome replication to the terminus. The amount of deoxyribonucleic acid (DNA) made poststarvation is characteristic of the strain. In this study, four strains differing in their DNA response were examined for ribonucleic acid (RNA) regulation during leucine starvation. Each of the strains was judged to be stringent for RNA control based on the amount of RNA made poststarvation. Sucrose gradient profiles on RNA made with and without leucine starvation support this conclusion. Accumulation of guanosine tetraphosphate during leucine starvation showed no correlation with the amount of DNA synthesized. We concluded that modulation control of DNA synthesis during leucine starvation is independent of RNA control.

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.

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.

Factors affecting virulence of Shigella flexneri: strain with relaxed control of ribonucleic acid synthesis.

An Escherichia coli-Shigella flexneri hybrid strain which produces an abortive, nonfatal experimental dysentery has inherited relaxed control of ribonucleic acid synthesis from its E. coli parent. This property, coupled with a partial methionine requirement, has been used to explain its long lag phase in vitro, relative to its Shigella parent. It was suggested that such characteristics may contribute to the hybrid's poor survival in the gut resulting in the abortive infection.

The control of ribonucleic acid synthesis in bacteria. The synthesis and stbility of ribonucleic acid in rifampicin-inhibited cultures of Escherichia coli.

A study was made of the kinetics of labelling of the stable ribonucleic acids (rRNA+tRNA) and the unstable mRNA fraction in cultures of Escherichia coli M.R.E.600, inhibited by the addition of 0.1g of rifampicin/l. Labelling was carried out by adding either [2-(14)C]- or [5-(3)H]-uracil as an exogenous precursor of the cellular nucleic acids. From studies using DNA RNA hybridization, the kinetics of the synthesis and degradation of mRNA was followed in the inhibited cultures. Although a considerable proportion of the mRNA labelled in the presence of rifampicin decayed to non-hybridizable products, about 25% was stabilized beyond the point where protein synthesis had finally ceased. It therefore seems unwise to extrapolate the results of studies on mRNA stability in rifampicin-inhibited cultures to the situation existing in the rate of steady growth, where there appears to be little, if any, stable messenger. The kinetics of labelling of RNA in inhibited cultures indicated that the clapsed time from the addition of rifampicin to the point at which radioactivity no longer enters the total cellular ribonucleic acids is a measure of the time required to polymerize a molecule of rRNA. At 37 degrees C, in culture grown in broth, glucose-salts or lactate salts media, exogenous [2-(14)C]uracil entered rifampicin-inhibited cells and was incorporated into RNA for 2 3min after the antibiotic was added. Taking this time as that required to polymerize a complete chain of 23S rRNA, the polymerization rate of this fraction in the three media was 25, 22 and 19 nucleotides added/s to the growing chains. Similar experiments in cultures previously inhibited by 0.2g of chloramphenicol/l showed virtually identical behaviour. This confirmed the work of Midgley & Gray (1971), who, by a different approach, showed that the polymerization rate of rRNA in steadily growing and chloramphenicol-inhibited cultures of E. coli at 37 degrees C was essentially constant at about 22 nucleotides added/s. It was thus confirmed that the rate of polymerization of at least the rRNA fraction in E. coli is virtually unaffected by the nature of the growth medium and therefore by bacterial growth rate.

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in chloramphenicol-inhibited cultures of Escherichia coli.

The rate of polymerization of ribosomal ribonucleic acid chains was estimated for steadily growing cultures of Escherichia coli M.R.E.600, from the kinetics of incorporation of exogenous [5-(3)H]uracil into completed 23S rRNA molecules. The analytical method of Avery & Midgley (1971) was used. Measurements were made at 37 degrees C, in the presence or the absence of chloramphenicol, in each of three media; enriched broth, glucose-salts or sodium lactate-salts. The rate of chain elongation of 23S rRNA was virtually constant in all media at 37 degrees C, as 24+/-4 nucleotides added/s. Accelerations in the rate of biosynthesis of rRNA by chloramphenicol in growth-limiting media are due primarily to an increase in the rate of initiation of new RNA chains, up to the rates existing in cultures growing rapidly in broth. Thus, in poorer media, only a small fraction of the available DNA-dependent RNA polymerase molecules are active at any given instant, since the chain-initiation rate is limiting in these conditions. In cultures growing rapidly in enriched broth, antibiotic inhibition caused a rise of some 12% in the rate of incorporation of exogenous uracil into total RNA. This small acceleration was due entirely to the partial stabilization of the mRNA fraction, which accumulated as 14% of the RNA formed after the addition of chloramphenicol. In cultures growing more slowly in glucose-salts or lactate-salts media, chloramphenicol caused an immediate acceleration of two- to three-fold in the overall rate of RNA synthesis. Studies by DNA-RNA hybridization showed that the synthesis of mRNA was accelerated in harmony with the other affected species. However, just over half the mRNA formed after the addition of chloramphenicol quickly decayed to acid-soluble products, whereas the remainder was more stable and accumulated in the cells. The mRNA fraction constituted about 6% of the total cellular RNA after 3h inhibition. A model was suggested to explain the partial stabilization and accumulation of the mRNA fraction and the acceleration in the rate of synthesis of mRNA when chloramphenicol was added to cultures in growth-limiting media.

The Control of Ribonucleic Acid Synthesis during Amino Acid Deprivation in Escherichia coli

Abstract The rate of RNA synthesis in a stringent strain of Escherichia coli was measured before and during amino acid deprivation. Upon amino acid deprivation the rate of RNA synthesis rapidly fell to 30% of the control rate and did not significantly change from that value upon prolonged starvation. The composition of RNA synthesized during amino acid deprivation was investigated by hybridization-competition, methyl labeling with 14C-methyl-labeled methionine, and polyacrylamide-agarose gel electrophoresis. Of the RNA pulse labeled during amino acid deprivation, ribosomal and transfer species comprise about 12% of the total, as opposed to about 40% in the unstarved control. Consequently, although some ribosomal and transfer RNAs are synthesized during amino acid starvation, the RNA is considerably enriched in messenger RNA, and the major effect of amino acid deprivation is the preferential curtailment of stable RNA synthesis. Our results also indicate that the rate of messenger RNA synthesis is reduced to 50% during amino acid deprivation.

The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600.

1. The technique of DNA-RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25-45 degrees C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate-salts or glucose-salts media the ratio was 0.046+/-0.005 and in enriched broth it was 0.087+/-0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate-salts or glucose-salts media, the messenger RNA fraction constituted 2.2+/-0.3% of the total cellular RNA. In enriched broth 3.6+/-0.3% of the total RNA was messenger.

The Control of Ribonucleic Acid Synthesis in Escherichia coli: V. CHARACTERIZATION OF A NUCLEOTIDE ASSOCIATED WITH THE STRINGENT RESPONSE

Abstract The preceding paper in this series presented evidence implicating an unusual phosphorylated compound (MS I) as a possible inhibitor of RNA accumulation during amino acid starvation of stringent strains of Escherichia coli. Here a method is described for isolating MS I from batch cultures of a stringent valyl transfer RNA synthetase mutant. Chemical analyses indicate that MS I is a tetraphosphate of guanosine. However, in contrast to previously described isomers of guanosine tetraphosphate (ppppG and pppGp), MS I is labile in alkali. Charge considerations, enzyme degradation studies, and labilities in acid and alkali lead to a structural assignment for MS I of guanosine 5'-diphosphate or 3'- or 2'-diphosphate (ppGpp).

Methyl-deficient transfer ribonucleic acid and macromolecular synthesis in methionine-starved Saccharomyces cerevisiae.

Haploid methionine auxotrophs of Saccharomyces cerevisiae continue to multiply for several hours after withdrawal of a required amino acid from the medium. Macro-molecular synthesis continues during this period of residual growth, although the net ribonucleic acid (RNA) and protein content is constant during the later part of this period. In this study, growth after withdrawal of methionine was in some cases accompanied by accumulation of transfer RNA (tRNA), which was shown by methylation in vitro to be deficient in methyl groups. This phenomenon was shown by only four of nine methionine auxotrophs tested, but no evidence could be found that these four strains had "relaxed" control of RNA synthesis. The nine methionine-requiring strains represent mutations in five different positions in the methionine biosynthesis pathway, and only mutants blocked at two of these five positions accumulated methyl-deficient tRNA. This accumulation therefore appears to be correlated with the position of the strain's block in the pathway of methionine biosynthesis.

Coordinate Control of Ribonucleic Acid Synthesis during Uracil Deprivation

Abstract When Escherichia coli 15 TAU is deprived of uracil, the accumulation of RNA abruptly ceases. RNA synthesis, however, continues and is balanced by an equal degradation of RNA. The rate of synthesis of total (messenger and stable) RNA, measured by the incorporation of adenosine, slowly declines during the course of uracil deprivation, attaining a value of 16% of the control after 1 hour. Synthesis of stable RNA, measured as the incorporation of methyl residues into RNA, also slowly declines during uracil starvation and attains a value of 12% after 1 hour. Consequently, the syntheses of message and stable RNA are controlled in an approximately coordinate manner during uracil deprivation.

Control of ribonucleic acid synthesis in Escherichia coli cells with altered transfer ribonucleic acid concentration

1. 1. This investigation tests the hypothesis that amino acid-free transfer RNA mediates the amino acid regulation of RNA synthesis by the inhibition of RNA polymerase (nucleosidetriphosphate: RNA nucleosidyltransferase, EC 2.7.7.6). 2. 2. Isoleucine-requiring Escherichia coli cells were incubated with chloramphenicol for 4 h, and their acceptor RNA level thereby raised 4–5-fold. Meanwhile, the protein content and wet-cell volume were unchanged, the RNA polymerase level fell, and the DNA level rose only 60 %. The cells were then starved for isoleucine and reincubated with various concentrations of chloramphenicol, in order to vary the internal concentration of isoleucine. 3. 3. Under these conditions, the rate of RNA synthesis remained subject to regulation by the amino acid supply. Most of the RNA formed did not turn over during incubation in the presence of 2,4-dinitrophenol. The rate of synthesis of sRNA varied directly with the rate of total RNA synthesis, as the chloramphenicol concentration was changed, both in these and in control cells. 4. 4. At any given rate of RNA synthesis, the concentration of isoleucine-free transfer RNA was five times as high in the chloramphenicol-pretreated samples as in untreated control samples. The proportion of phenylalanine-free to total phenylalanine-specific tRNA was the same in control and chloramphenicol-pretreated samples. 5. 5. In a similar experiment, histidine-requiring cells were incubated 3 h in the presence of chlortetracycline and fluorodeoxyuridine. The protein level increased 10 % and the DNA level 25 %. At any given rate of RNA synthesis, the concentration of histidine-free tRNA was twice as high in the chlortetracycline-pretreated samples as in the control samples. 6. 6. We conclude that the amino acid-free transfer RNA does not mediate the amino acid regulation of RNA synthesis by inhibiting RNA polymerase.

Synthesis of Ribonucleic Acid by Escherichia coli during Inhibition by Azetidine-2-carboxylic Acid

IN bacteria, amino-acids are needed in small amounts for the synthesis of ribonucleic acid (RNA) to occur1 and, if an amino-acid auxotroph is starved of its required amino-acid, both protein and RNA synthesis cease2. However, under certain conditions this ‘stringency’ is not maintained, in that protein synthesis can be sharply retarded while synthesis of RNA is less affected. In Escherichia coli, this differential synthesis of RNA can be brought about by incubation of cells with chloramphenicol3, puromycin4, or chlortetracycline5, and mutants in which the control of RNA synthesis by amino-acids is ‘relaxed’ have also been described6,7. Relaxed mutants which require an amino-acid for growth continue to make RNA when starved of their amino-acid requirement; synthesis of RNA in a relaxed strain during inhibition of protein synthesis by 5-methyl tryptophan has also been, demonstrated8.

Requirement for p-fluorophenylalanine activation in control of ribonucleic acid synthesis

1. 1. Fangman and Neidhardt 1,2 used mutant Escherichia coli strains unable to activate p-fluorophenylalanine (PFPA) to show that amino acid activation is required for ribonucleic acid synthesis. Their findings were confirmed. 2. 2. It was shown directly that the mutant cells do not charge phenylalanine-specific transfer ribonucleic acid with PFPA. 3. 3. A possible criticism of the interpretation is that PFPA might create a shortage of tyrosine and tyrptohan by false feedback inhibition of 3-deoxy-d- arabino-heptulosonic acid 7-phosphate synthetase, and inhibit ribonucleic acid synthesis thereby, rather than by failure of PFPA activation. PFPA inhibition in the mutant was indeed reversed by shikimate, and therefore presumably affects the 3-deoxy- d- arabino-heptulosonic acid 7-phosphate synthetase and decrease the supply of shikimate. However, in the mutant cultures, the limitation of ribonucleic acid synthesis occuring in presence of PFPA was not overcome by supplementation with tyrosine and tryptophan at concentrations adequate for permeation in presence of PFPA. 4. 4. It is concluded that amino acid activation is required for ribonucleic acid synthesis.