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.     

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.     

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.     

Methyldeficient mammalian 4S RNA: evidence for L-ethionine-induced inhibition of N6-dimethyladenosine synthesis in rat liver tRNA

The nucleotide composition of 4s RNA from livers of rats fed with a diet containing 0.3% D-ethionine was found to be identical with that from untreated animals. In contrast, one single modified nucleotide was absent in 4s RNA from livers of rats fed with a 0.3% L-ethionine diet. The minor nucleo=tide was also absent in liver 4s RNA from rats fed with a 0.3% L-ethionine diet followed by ten days of normal food. It was identified after dephosphorylation by ultraviolet absorption spectra, cochromatography with authentic material and mass spectra as N(6)-dimethyladenosine. It is concluded that S-adenosylethionine, the primary product of L-ethionine in the liver, causes strong and selective inhibition of the specific RNA-methylase responsible for adenosine to N(6)-dimethyl=adenosine methylation in rat liver 4s RNA. Compared to the strong inhibition of N(6)-dimethyladenosine formation described here, L-ethionine-dependent ethylation of liver 4s RNA is far less efficient. The quantitation of l-methyladenosine, ribothymidine and 3'-terminal adenosine in this 4s RNA as well as its aminoacid acceptor activity is typical for tRNA; hence it may be concluded that N(6)-dimethyladenosine is a component of rat liver tRNA. This may demonstrate the first evidence for the existence of specifically methyl-deficient mammalian tRNA. A possible correlation between the activity of L-ethionine as a liver carcinogen and its ability to induce the formation of methyl-deficient tRNA by selectively inhibiting the synthesis of N(6)-dimethyladenosine on the tRNA level in the same organ is discussed.     

Partial derepression of the isoleucine-valine enzymes during methionine starvation is Salmonella typhimurium.

Methionine starvation of methionine auxotrophs in the presence of excess branched-chain amino acids results in a partial derepression of the isoleucine and valine enzymes. Reversed-phase chromatography indicated that isoleucine, valine and leucine tRNA were altered during methionine starvation. In addition, the total tRNA isolated from cells under these conditions were undermethylated. The observed derepression may be caused by the inability of methyl-deficient tRNA's to participate adequately in normal regulatory functions.     

Transfer ribonucleic acid methylase deficiency found in UGA supressor strains.

Extracts of recessive UGA suppressor strains, designated supK, are deficient in transfer ribonucleic acid (tRNA)-methylating activity when compared to wild-type extracts. Moreover, the tRNA from suppressor strains is methyl deficient when compared to wild-type tRNA. This deficiency is due to the lack of a single tRNA methylase activity in suppressor strains. UGA suppressor activity may be caused by the miscoding of one or more methyl-deficient tRNA's in supK strains.     

A rapid method for determining tRNA charging levels in vivo: analysis of yeast mutants defective in the general control of amino acid biosynthesis.

We describe a simple method to quantitate the intracellular levels of charged tRNA species representing all 20 amino acids. Small RNA species are isolated from yeast cells under conditions where amino acids remain bound to their cognate tRNAs. After chromatographic removal of free amino acids, the tRNAs are discharged, and the amounts of the released amino acids are then quantitated. This method was applied to yeast cells from a wild type strain and from three mutant strains that are defective both in the general control of amino acid biosynthesis and in protein synthesis. Two of these mutant strains, previously shown to be defective in the methionine or isoleucine tRNA synthetases, respectively contain undetectable amounts of charged methionine or isoleucine although their levels of the remaining 19 amino acids are similar to a wild type strain. In contrast, a gcd1 mutant strain has normal levels of all 20 amino-acyl tRNA species. Thus, gcd1 strains are defective in general control of amino acid biosynthesis for reasons other than artifactual starvation of an amino acid due to a failure in tRNA changing.     

Selective inhibition of uracil tRNA methylases of E. coli by ethionine.

L-ethionine has been found to inhibit uracil tRNA methylating enzymes in vitro under conditions where methylation of other tRNA bases is unaffected. No selective inhibitor for uracil tRNA methylases has been identified previously. 15 mM L-ethionine or 30 mM D,L-ethionine caused about 40% inhibition of tRNA methylation catalyzed by enzyme extracts from E. coli B or E. coli M3S (mixtures of methylases for uracil, guanine, cytosine, and adenine) but did not inhibit the activity of preparations from an E. coli mutant that lacks uracil tRNA methylase. Analysis of the 14CH3 bases in methyl-deficient E. coli tRNA after its in vitro methylation with E. coli B3 enzymes in the presence or absence of ethionine showed that ethionine inhibited 14CH3 transfer to uracil in tRNA, but did not diminish significantly the 14CH3 transfer to other tRNA bases. Under similar conditions 0.6 mM S-adenosylethionine and 0.2 mM ethylthioadenosine inhibited the overall tRNA base methylating activity of E. coli B preparations about 50% but neither of these ethionine metabolites preferentially inhibited uracil methylation. Ethionine was not competitive with S-adenosyl methionine. Uracil methylation was not inhibited by alanine, valine, or ethionine sulfoxide. It is suggested that the thymine deficiency that we found earlier in tRNA from ethionine-treated E. coli B cells, resulted from base specific inhibition by the amino acid, ethionine, of uracil tRNA methylation in vivo.     

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.     

Detection of nucleoside Q precursor in methyl-deficient E.coli tRNA.

32P-Labeled tRNAAsn was isolated from methyl-deficient E. coli tRNA. Nucleotide sequence analysis showed that tRNAAsn contains three derivatives of the Q nucleoside, possibly Q precursors, in addition to guanosine in the first position of the anticodon. One of the Q precursors was isolated on a large scale. Its UV spectra were identical with those of normal Q, indicating that 7-deazaguanosine structure having a side chain at position C-7 is complete in the Q precursor. No radioactivity was incorporated into Q or Q precursors from either [methyl-14C]methionine, [1-14C]methionine or [U-14C]methionine, showing that methionine was not directly involved in the formation of Q.     

Maturation of a hypermodified nucleoside in transfer RNA.

E. coli C6 rel- met- cys- was cultured in a fully supplemented medium and in media lacking cysteine or methionine. tRNA isolated from the three cultures containted, respectively, a normal complement of modified nucleosides; a deficiency in thiolated nucleosides and a deficiency in methylated nucleosides. Both sulfur-deficient tRNA and methyl-deficient tRNA contained large amounts of N-6- (delta-2-isopentenyl) adenosine and small amounts of the 2-methylthio derivative. Methyl-deficient tRNA contained, in addition a large amount of a cytokinin active, differently modified nucleoside that is believed to be a sulfur derivative of N6-(delta-2-isopentenyl) adenosine. The structure of this compound is unknown. When methly-deficient tRNA and the precusor the tRNA-Tyr su3-+ A25 were enzymatically methylated in vitro, methyl groups were incorporated into derivatives of isopentenyladenosine. These results indicate that the biosynthesis of the 2-methylthio derivative of isopentenyladenosine may occur in a sequential manner, i.e., thiolation of isopentenyladenosine followed by methylation.     

RNA quality control: degradation of defective transfer RNA

The distinction between stable (tRNA and rRNA) and unstable (mRNA) RNA has been considered an important feature of bacterial RNA metabolism. One factor thought to contribute to the difference between these RNA populations is polyadenylation, which promotes degradation of unstable RNA. However, the recent discovery that polyadenylation also occurs on stable RNA led us to examine whether poly(A) might serve as a signal for eliminating defective stable RNAs, and thus play a role in RNA quality control. Here we show that a readily denaturable, mutant tRNA(Trp) does not accumulate to normal levels in Escherichia coli because its precursor is rapidly degraded. Degradation is largely dependent on polyadenylation of the precursor by poly(A) polymerase and on its removal by polynucleotide phosphorylase. Thus, in the absence of these two enzymes large amounts of tRNA(Trp) precursor accumulate. We propose that defective stable RNA precursors that are poorly converted to their mature forms may be polyadenylated and subsequently degraded. These data indicate that quality control of stable RNA metabolism in many ways resembles normal turnover of unstable RNA.     

Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver.

A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250,000 x g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP-32PPi exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.     

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 Regulatory Mutations upon Methionine Biosynthesis in Saccharomyces cerevisiae: Loci eth2-eth3-eth10

The effects of mutations occurring at three independent loci, eth2, eth3, and eth10, were studied on the basis of several criteria: level of resistance towards two methionine analogues (ethionine and selenomethionine), pool sizes of free methionine and S-adenosyl methionine (SAM) under different growth conditions, and susceptibility towards methionine-mediated repression and SAM-mediated repression of some enzymes involved in methionine biosynthesis (met group I enzymes). It was shown that: (i) the level of resistance towards both methionine analogues roughly correlates with the amount of methionine accumulated in the pool; (ii) the repressibility of met group I enzymes by exogenous methionine is either abolished or greatly lowered, depending upon the mutation studied; (iii) the repressibility of the same enzymes by exogenous SAM remains, in at least three mutants studied, close to that observed in a wild-type strain; (iv) the accumulation of SAM does not occur in the most extreme mutants either from endogenously overproduced or from exogenously supplied methionine: (v) the two methionine-activating enzymes, methionyl-transfer ribonucleic acid (tRNA) synthetase and methionine adenosyl transferase, do not seem modified in any of the mutants presented here; and (vi) the amount of tRNA^met and its level of charging are alike in all strains. Thus, the three recessive mutations presented here affect methionine-mediated repression, both at the level of overall methionine biosynthesis which results in its accumulation in the pool, and at the level of the synthesis of met group I enzymes. The implications of these findings are discussed.     

Transfer RNAs as genotypic fingerprints of eubacteria

A new method was developed for rapid genotypic identification and classification of bacteria. The method is based on high resolution gel electrophoresis of the stable, low molecular weight (LMW) RNA fraction of single bacterial strains. This fraction comprises the total transfer RNA pool and the 5S ribosomal RNA. On a one-dimensional gel, every eubacterial strain exhibited a distinct LMW RNA profile, a set of bands belonging to three different size classes: 5S rRNAs (110–131 nt), class 2 tRNAs (82–96 nt) and class 1 tRNAs (72–79 nt). LMW RNA profiles of members of five of the ten major eubacterial groups, previously defined by 16S rRNA sequence analysis, were highly diverse. For some major groups, like flavobacteria and planctomyces, the distinctive sizes of their 5S rRNAs allowed the assignment of strains to these groups. More specific taxonomic information was gained from analysis of the tRNA part of the profile. Strains could be grouped as species and genera due to species- and genus-specific tRNA bands. From an evolutionary point of view, this order found in the total tRNA pool of eubacteria could indicate that cytoplasmic tRNA evolution reflects ribosomal RNA evolution. Given the universality of tRNAs, it is to be expected that their electrophoretic mobility profiles may serve as a convenient RNA fingerprint for defining bacterial species operationally and for identifying new genotypes by differing patterns.     

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.     

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.