Methyl-Deficient Transfer Ribonucleic Acid in Saccharomyces cerevisiae

Methyl-deficient transfer ribonucleic acid (tRNA) is found in certain methionine auxotrophs of Saccharomyces cerevisiae during logarithmic growth (at one generation time before the late growth phase) and during residual growth in the absence of exogenous methionine. The former effect seems to be accounted for by the general increase in RNA synthesis that occurs at the time; there is no specific synthesis of tRNA in the absence of ribosomal RNA synthesis, nor is the methyl group deficiency limited to a single tRNA species. During methionine starvation, all species of tRNA are methyl-deficient, but this occurs only in strains with certain blocks in the methionine pathway. The kinetics of disappearance of the methyl group donor, S-adenosylmethionine, during starvation of D73 (which accumulates methyl-deficient tRNA), do not differ from other strains, but D73 loses the methylase inhibitor, S-adenosylhomocysteine, much more slowly.     

Influence of Methionine Pool Composition on the Formation of Methyl-Deficient Transfer Ribonucleic Acid in Saccharomyces cerevisiae

Methionine auxotrophs of Saccharomyces cerevisiae continue to synthesize ribonucleic acid (RNA) after methionine withdrawal. The newly synthesized transfer RNA (tRNA) is methyl-deficient in some strains, but not in all. Whether such tRNA will accumulate depends on the position of the block in the methionine pathway that is carried by the mutant strain. Free methionine rapidly decreases in the intracellular pool of all strains after its removal from the medium. Certain metabolites derived from methionine are removed from the pool relatively slowly after methionine withdrawal. Notable among these is S-adenosylhomocysteine, which is depleted less rapidly from those strains that accumulate methyl-deficient tRNA than from others. S-adenosylhomocysteine is a potent inhibitor of tRNA-methylating enzymes in vitro.     

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.     

Transfer Ribonucleic Acid Nucleotidyltransferase and Transfer Ribonucleic Acid in Sendai Virions

Sendai virions contain both transfer ribonucleic acid (tRNA) nucleotidyltransferase and its substrate, tRNA missing its CCA-OH end.     

Selective methylation of newly synthesized transfer RNA from an rel mutant of Escherichia coli during methionine starvation

The observation that the change in concentration of several methylated nucelosides from normal to methyl-deficient tRNA populations is not the same has been explained on the basis of continued methylation of newly synthesized tRNA by several but not all tRNA methyltransferases.     

Methyl-deficient mammalian transfer RNA: II. Homologous methylation in vitro of liver tRNA from normal and ethionine-fed rats: ethionine effect on 5-methyl-cytidine synthesis in vivo

Following hydroxyapatite chromatography, rat liver tRNA methylase activity was assayed with liver tRNA from normal rats and with methyl-deficient liver tRNA from ethionine-fed rats. The difference in homologous methylation between normal and methyl-deficient tRNA was maximal in certain fractions in presence of cadaverine, and much less in presence of Mg⁺⁺ or Mg⁺⁺ plus cadaverine. These methylase fractions, which contained endogenous tRNA, were used for preparative homologous methylation of added normal and methyl-deficient tRNA in presence of 30 mM cadaverine. The ¹⁴C-methylated tRNA was digested with RNase T₂ and the resulting methylated mononucleotides were characterized and quantitated after twodimensional thinlayer chromatography and autoradiography. The major products of homologous tRNA methylation were m⁵C and m¹A. However, the methylase fraction used here did not catalyze the formation of m⁶₂A with m⁶₂A-deficient tRNA as substrate.- In addition to the previously described, analytically detectable m⁶₂A-deficiency, a partial m⁵C-deficiency was demonstrated in liver tRNA from ethionine-fed rats by measuring the methylacceptance in vitro. In presence of cadaverine, with the methylase fraction used here, methyl-deficient tRNA from ethionine-fed rats was a twofold more efficient methyl-acceptor in vitro than normal liver tRNA, while endogenous tRNA isolated from the methylase fraction was a threefold more efficient methyl-acceptor than normal liver tRNA. Homologous methylation of normal tRNA, as observed here, has not been described before.     

Presence and Function of Sulfur-containing Transfer Ribonucleic Acid of Bacillus subtilis

Bacillus subtilis transfer ribonucleic acid (tRNA) was analyzed for the occurrence of thionucleotides by in vivo labeling with (35)S and fractionation by methylated albumin kieselguhr column chromatography. Alkaline hydrolysates of tRNA were also examined by column chromatography and paper electrophoresis, and the amino acid-accepting ability of thionucleotide-containing tRNA was tested after iodine oxidation. The results showed that B. subtilis tRNA contains 4-thiouridylate, a second nucleotide with properties similar to 2-thiopyrimidine, and a third unidentified thionucleotide. The amino acid-accepting ability for serine, tyrosine, lysine, and glutamic acid was markedly inhibited after oxidation of the tRNA with iodine, suggesting the presence of thionucleotides in these tRNA species. This inhibition could be reversed by thiosulfate reduction. The iodine treatment totally inactivated all lysine tRNA species, partially inactivated the serine tRNA species, and did not affect the accepting ability for valine. A comparison of tRNA from cells in the log and stationary phases and from spores revealed similar iodine inactivation patterns in all cases. The thionucleotide content in B. subtilis tRNA differed from that in Escherichia coli, both in extent and in distribution. A possible function of the thionucleotides in tRNA is discussed.     

Role of Histidine Transfer Ribonucleic Acid in Regulation of Synthesis of Histidyl-Transfer Ribonucleic Acid Synthetase of Salmonella typhimurium

The role of histidine transfer ribonucleic acid (tRNA) in repression of synthesis of histidyl-tRNA synthetase was examined in two strains of Salmonella typhimurium, one of which was a histidine tRNA (hisR) mutant possessing 52% of the wild-type (hisR(+)) histidine tRNA and a derepressed level of the histidine biosynthetic enzymes during histidine-unrestricted growth. Histidine-restricted growth caused a derepression of the rate of formation of histidyl-tRNA synthetase in both strains. In the case of the wild-type strain, addition of histidine to the derepressed culture caused a repression of synthesis of histidyl-tRNA synthetase for at least one generation of growth. In contrast, when histidine was restored to the derepressed hisR mutant culture, synthesis of histidyl-tRNA synthetase was continued at the initial derepressed rate. These results suggest that histidine must be attached to histidine tRNA for repression of synthesis of histidyl-tRNA synthetase.     

In Vivo Aminoacylation of Transfer Ribonucleic Acid in Bacillus subtilis and Evidence for Differential Utilization of Lysine-Isoaccepting Transfer Ribonucleic Acid Species

The presence or absence of certain amino acids has different effects on the ability of Bacillus subtilis to sporulate, and the intracellular pool size of amino acids has been reported to vary during sporulation. The idea that these variations might exert a regulatory effect through aminoacylation of transfer ribonucleic acid (tRNA) was investigated by studying the levels of aminoacylation in vivo in the logarithmic or stationary phase of growth. Both the periodate oxidation method and the amino acid analyzer were used to evaluate in vivo aminoacylation. The results indicated that in general the level of aminoacylation of tRNA's remained constant through stage III of sporulation, although there were detectable variations for specific amino acid groups. Our studies also showed that periodate oxidation damaged certain tRNA's; therefore, the results obtained by such a method should be interpreted with caution. Because the damage can affect certain isoaccepting species specifically, the periodate oxidation method cannot be used to establish which isoaccepting species are acylated in vivo. We also investigated the possibility of preferential use of particular tRNA species by polyribosomes. These results demonstrated a preferential use of lysyl-tRNA's at different growth stages. Control mechanisms operating during the early stages of sporulation, therefore, do not affect the overall level of aminoacylation. However, there is an effect on the levels of aminoacylation of specific amino acids and on which isoaccepting species are utilized by the polyribosome system.     

Preparation of Cells Permeable to Macromolecules by Treatment with Toluene: Studies of Transfer Ribonucleic Acid Nucleotidyltransferase

Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene. The conditions of toluene treatment necessary for obtaining maximal activity were defined. Toluene treatment was most efficient when carried out for 5 min at 37 C at pH 9.0 on log-phase cells. No activity could be detected if cells were treated at 0 C, or in the presence of MgCl₂, or if the cells were in the stationary phase of growth. However, inclusion of lysozyme and ethylenediaminetetraacetic acid during the toluene treatment did render stationary phase cells permeable. The properties of tRNA nucleotidyltransferase from toluene-treated cells were essentially identical to those of purified enzyme with regard to pH optimum, specificity for nucleoside triphosphates and tRNA, and apparent K_m values for substrates. In addition to tRNA nucleotidyltransferase, a variety of other enzymes which incorporate adenosine 5′-triphosphate into acid-precipitable material could also be detected in toluene-treated cells. Centrifugation of cells treated with toluene revealed that tRNA nucleotidyltransferase leaked out of cells, whereas other activities remained associated with the cell pellets. Chromatography of the material extracted from toluene-treated cells on Sephadex G-100 indicated that toluene treatment selectively extracts lower molecular weight proteins. The usefulness of such a procedure as an initial step in purification of such enzymes, and its application to tRNA nucleotidyltransferase, is discussed.     

Relationships Among Deoxyribonucleic Acid, Ribonucleic Acid, and Specific Transfer Ribonucleic Acids in Escherichia coli 15T− at Various Growth Rates

The levels of macromolecules in Escherichia coli 15T(-) growing in broth, glucose, succinate, and acetate media were determined to compare relationships among deoxyribonucleic acid (DNA), ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA), and protein in cells at different growth rates. DNA and protein increased in relative amounts with decreasing growth rate; relative amounts of rRNA and tRNA decreased, tRNA making up a slightly larger proportion of RNA. For several amino acid-specific tRNAs studied, acceptor capacities per unit of DNA increased with increasing growth rate. The syntheses of tRNA and rRNA are regulated by similar, yet different, mechanisms. Chromatographic examination on columns of benzoylated diethylaminoethyl-cellulose of isoaccepting tRNAs for arginine, leucine, lysine, methionine, phenylalanine, serine, and valine did not reveal differences in the isoaccepting profiles for rapidly (broth culture) and slowly growing (acetate culture) cells. Therefore, isoacceptors for individual amino acids appear to be regulated as a group. Lower efficiencies of ribosomal function in protein synthesis can be explained, in part, by a low ratio of tRNA to the number of ribosomes available and by a decreasing concentration of tRNA with decreasing growth rate. Data on the tRNAs specific for seven amino acids indicate that the decreasing concentration of tRNA is a general event rather than a severe limitation of any one tRNA or isoaccepting tRNA.     

Suppression of Glutamic Acid Codons by Mutant Glycine Transfer Ribonucleic Acid

In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.     

Control of Deoxyribonucleic Acid and Ribonucleic Acid Synthesis in Pyrimidine-Limited Escherichia coli

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.     

Role of Isoleucyl-Transfer Ribonucleic Acid Synthetase in Ribonucleic Acid Synthesis and Enzyme Repression in Yeast

Temperature-sensitive mutations in the isoleucyl-transfer ribonucleic acid (tRNA) synthetase of yeast, ilS(-)1-1 and ilS(-)1-2, were used to examine the role of aminoacyl-tRNA synthetase enzymes in the regulation of ribonucleic acid (RNA) synthesis and enzyme synthesis in a eucaryotic organism. At the permissive temperature, 70 to 100% of the intracellular isoleucyl-tRNA was charged in mutants carrying these mutations; at growth-limiting temperatures, less than 10% was charged with isoleucine. Other aminoacyl-tRNA molecules remained essentially fully charged under both conditions. Net protein and RNA syntheses were rapidly inhibited when the mutant was shifted from the permissive to the restrictive temperature. Most of the ribosomes remained in polyribosome structures at the restrictive temperature even though protein synthesis was strongly inhibited. Two of the enzymes of isoleucine biosynthesis, threonine deaminase and acetohydroxyacid synthetase, were derepressed about twofold during slow growth of the mutants at a growth-limiting temperature. This is about the same degree of derepression that is achieved by growth of an auxotroph on limiting isoleucine. We conclude that charged aminoacyl-tRNA is essential for RNA synthesis and for the multivalent repression of the isoleucine biosynthetic enzymes. Aminoacyl tRNA synthetase enzymes appear to play important regulatory roles in the cell physiology of eucaryotic organisms.     

Transfer Ribonucleic Acid Methylases During the Germination of Neurospora crassa

Transfer ribonucleic acid (tRNA) methylases were studied during the germination of spores in Neurospora crassa. The total methylase capacity and base specific tRNA methylase activities were determined in extracts from cells harvested at various stages of germination. Germinated conidia have a 65% higher methylase capacity than ungerminated conidia. Three predominant methylase activities were found in the extracts, and the relative amount of each activity was different at the various stages. Enzymes from vegetative cells catalyzed significant hypermethylation of tRNA from conidia, whereas conidial enzymes were much less active on tRNA from vegetative cells. The results indicate differences in the tRNA methylase content and tRNA species of conidia and vegetative cells.     

Chloramphenicol and the Stimulation of Ribonucleic Acid Synthesis in Escherichia coli

Data have been obtained which imply that chloramphenicol stimulation of ribonucleic acid (RNA) synthesis is a result of the accumulation of aminoacyl transfer RNA (tRNA) molecules. The data also support the hypothesis that chloramphenicol exerts an additional effect upon the stimulation of RNA synthesis. This effect may be at the level of the ribosome or the aminoacyl tRNA, or of both. It is this effect combined with the presence of aminoacyl tRNA that results in stimulation by chloramphenicol of RNA synthesis.     

Correlation Between the Rate of Ribonucleic Acid Synthesis and the Level of Valyl Transfer Ribonucleic Acid in Mutants of Escherichia coli

By use of a mutant of Escherichia coli with a partially thermolabile transfer ribonucleic acid (tRNA) synthase, it was possible to regulate the rate of RNA synthesis over a 10-fold range. The addition of chloramphenicol to cultures kept at the nonpermissive temperature stimulated RNA synthesis. The longer the culture was kept at the nonpermissive temperature prior to addition of chloramphenicol, the lower was the resulting rate of RNA synthesis. The decrease in the rate of incorporation of labeled uracil into RNA was correlated with the decrease in the level of valyl tRNA. Additional experiments provided evidence which may be interpreted as indicating that valyl tRNA does not, by itself, react with the RNA-forming system.     

Characterization of Altered Forms of Glycyl Transfer Ribonucleic Acid Synthetase and the Effects of Such Alterations on Aminoacyl Transfer Ribonucleic Acid Synthesis In Vivo

The glycyl transfer ribonucleic acid (tRNA) synthetase (GRS) activities of several Escherichia coli glyS mutants have been partially characterized; the K(m) for glycine and the apparent V(max) of several of the altered GRS differ significantly from the parental GRS. Paradoxically, some of the altered forms exhibit more activity in vitro than the GRS from a prototrophic strain (GRS(L)); several parameters of these activities have been studied in an attempt to resolve this problem. The amount of acylated tRNA(Gly) in vivo was examined to assess the GRS activities inside the cells. During exponential growth in media containing glycine, moderate amounts of acylated tRNA(Gly) occur in the glyS mutants; glycine deprivation leads to a dramatic drop in the amount of acylated tRNA(Gly). An alternative measure of the in vivo activities of the altered enzymes is the efficiency of suppression of the trpA36 locus by su(36) (+); glyS mutants grown with added glycine exhibit one-third to one-fourth the suppression efficiency of the prototrophic glyS(H) parent, presumably because they are less efficient, even in the presence of high levels of glycine, in charging the tRNA(Gly) species which functions as the translational suppressor.     

Stimulation of Ribonucleic Acid Synthesis by Chloramphenicol in a rel+ Aminoacyl-Transfer Ribonucleic Acid Synthetase Mutant of Escherichia coli

Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel(+) strain, it cannot carry out net RNA synthesis at high temperature. A 100-mug amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 mug of CAP or 100 mug of CAP plus 50 mug of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNA(val) at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases.     

Different Arginine Transfer Ribonucleic Acid Species Prevalent in Shaken and Unshaken Cultures of Neurospora

When the arginyl-transfer ribonucleic acid (tRNA) species isolated from unshaken and from shaken cultures of Neurospora were compared by co-chromatography, a marked change in the relative abundance of the two main tRNA(arg) species was found. The two arginine tRNA species had different codon responses in ribosome binding assays. The tRNA(arg) eluting first (prevalent in shaken cultures) bound strongly to polyadenylic-guanylic acid [poly(A,G)] and to a lesser extent to polycytidylic-guanylic-adenylic acid [poly(C,G,A)]. The second tRNA(arg) species (prevalent in unshaken cultures) bound to poly(C,G,A) but not to poly(A, G). The possible significance of these observations is briefly discussed. Several modifications that improve the yield of tRNA from Neurospora were introduced in a standard isolation procedure.