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.     

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.     

Studies on Microbial Ribonucleic Acid VI. Appearance of Methyl-deficient Transfer Ribonucleic Acid During Logarithmic Growth of Saccharomyces cerevisiae

Transfer ribonucleic acid (tRNA) that is deficient in methyl groups may be detected in logarithmically growing Saccharomyces cerevisiae. The amount of methyl-deficient tRNA is not constant throughout the logarithmic phase, but is maximal about one generation before the onset of the late growth phase. During this latter phase, the tRNA is fully methylated. The methyl-deficient tRNA is present during a period of high metabolic activity of the cell, characterized by increased RNA and protein content.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.     

Isolation and Partial Characterization of Escherichia coli Mutants with Low Levels of Transfer Ribonucleic Acid Nucleotidyltransferase

To determine the function of the enzyme transfer ribonucleic acid (tRNA) nucleotidyltransferase in vivo, five mutants of Escherichia coli containing low levels of this enzyme were isolated. Since no selection procedure for such mutants existed, these strains were isolated by assay of large numbers of colonies from a heavily mutagenized stock. A procedure employing cells made permeable to tRNA and ATP was used to screen the large number of colonies required for the isolation. All the mutants contained less than 20% of the normal level of the AMP-incorporating activity of tRNA nucleotidyltransferase in extracts prepared by several methods, and the best mutant contained only about 2% of this activity. Three of the mutants also had equally low levels of the cytidine 5'-monophosphate-incorporating activity of the enzyme. Despite these low activities, the mutant strains displayed relatively normal growth characteristics at all temperatures examined. The enzyme in the mutant strains was not temperature sensitive, nor were any other abnormal biochemical properties detected. tRNA isolated from the mutant strains was missing significant amounts of its 3' terminal adenosine 5'-monophosphate residue, amounting to 10 to 15% in the best mutant. However, only small amounts of the terminal cytidine 5'-monophosphate residue were missing. The results indicate that tRNA nucleotidyltransferase is involved in some aspect of synthesis or repair of the 3' terminus of tRNA, and that the enzyme is present in large excess over its requirements for this function.     

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.     

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.     

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Changes in Chromatography of Transfer Ribonucleic Acids Associated with Stage of Development

Changes in chromatographic profiles of tyrosyl-, leucyl-, tryptophanyl-, and lysyl-transfer ribonucleic acids (tRNAs) are presented as a function of the growth stage in Bacillus subtilis. All of the tRNA groups investigated expressed different temporal patterns of change in isoaccepting species. Tyrosyl-tRNAs were the earliest to change and were followed by changes in leucyl- and then tryptophanyl-tRNAs. Lysyl-tRNAs were unique in having two times of change: one early and one very late. As an aid in understanding the temporal aspect of tRNA alterations during sporulation, the chromatographic profiles of aminoacyl tRNAs from an early blocked asporogenous mutant were studied. The asporogenous mutant used was blocked at the axial filament stage, stage 0 of sporulation. Nevertheless, those tRNAs which showed differences between the spore and cells in exponential growth exhibited similar changes in the asporogenous mutant after 24 h of growth. The data suggest that several tRNA changes occur during development in B. subtilis but that the events leading to these changes are either independent of, or occur before, stage 0 of sporulation, except in the case of lysyl-tRNA.     

Specificity of Interaction Between the First Enzyme for Histidine Biosynthesis and Aminoacylated Histidine Transfer Ribonucleic Acid

The specificity of the interaction between phosphoribosyltransferase and partially purified preparations of various species of transfer ribonucleic acid (tRNA) was investigated with the use of a filter binding assay. The enzyme showed a higher affinity for histidyl-tRNA than for arginyl- or glutamyl-tRNA. Competition experiments revealed that the enzyme does not distinguish between the aminoacylated and deacylated forms of arginine tRNA or glutamic acid tRNA, since all the binding of the aminoacylated tRNA could be inhibited by deacylated tRNA. The enzyme does, however, distinguish between the aminoacylated and deacylated forms of histidine tRNA. Approximately 70% of the binding of aminoacylated histidine tRNA is specific, since only 30% of the binding could be inhibited by deacylated tRNA. The possibility that the regulatory role of phosphoribosyltransferase is carried out as a complex with histidyl-tRNA is consistent with these data.     

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Chromatographic Differences Between Transfer Ribonucleic Acids from Spores and Cells in Exponential Growth

Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs.     

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.     

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 in KB Cells Infected with Adenovirus Type 2

Populations of transfer ribonucleic acid (tRNA) extracted from control and type 2 adenovirus (Ad2)-infected KB cells were compared. No consistent differences in acceptor activity for 11 amino acids were observed. Comparison of methylated albumin-kieselguhr (MAK) elution profiles of arginyl-tRNA from control and infected cells revealed a minor modification in that the proportion of arginyl-tRNA eluting at high salt concentration was somewhat greater in infected cells. No similar differences were observed in MAK elution profiles of aspartyl-, isoleucyl-, leucyl-, phenylalanyl-, seryl-, tyrosyl-, and valyl-tRNA. Hybridization of 4S RNA from infected cells labeled by incorporation of ³H-uridine with Ad2 deoxyribonucleic acid revealed the presence of a complementary species of RNA in this preparation. Hybridization of ³H-arginyl-tRNA and of ³H-aminoacyl-tRNA labeled by charging with ³H-arginine or a ³H-mixture of amino acids, respectively, failed to detect the presence of virus-specific tRNA in Ad2-infected cells.