Inhibition of nucleoside Q formation in transfer ribonucleic acid during methionine starvation of relaxed-control Escherichia coli.

The elution profiles of Asp-tRNA from unstarved and starved cultures of a relaxed-control (Rel-) strain of Escherichia coli were compared by reversed-phase chromatography. Methionine starvation results in the appearance of several additional species of Asp-tRNA which are not observed with starvation for leucine or histidine. By the criterion of cyanogen bromide-effected shifts in chromatographic elution position, a large portion of the tRNAAsp synthesized in methionine-starved cells lacks the normal Q nucleoside. By the same criterion, virtually all of the tRNAAsp from unstarved, leucine-starved, and histidine-starved cells contain Q. We conclude that methionine starvation prevents the formation of the norma Q nucleoside in Rel- E. coli.     

Enzymatic synthesis of Q* nucleoside containing mannose in the anticodon of tRNA: isolation of a novel mannosyltransferase from a cell-free extract of rat liver

The Q nucleosides isolated from rabbit liver tRNA are known to have sugars (mannose or galactose) linked to their cyclopentene diol moiety. A Q nucleoside containing mannose (manQ) was synthesized by a cell-free system from rat liver, using purified E. coli tRNAAsp as an acceptor and GDP-mannose as a donor molecule. The novel mannosyltransferase catalyzing this reaction was purified from a particulate-free soluble enzyme fraction and found to be strictly specific for tRNAAsp. These results, together with the anomeric configuration of mannose in Q nucleoside, indicate that no lipid intermediate is involved in the biosynthesis of Q nucleoside.     

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.     

Relation of cell type and cell density in tissue culture to the isoaccepting spectra of the nucleoside Q containing tRNAs: tRNATyr, tRNAHis, tRNAAsn and tRNAAsp.

An examination, using reversed-phase chromatography and cyanogen bromide treatment, of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp from SV40-transformed mouse fibroblasts grown to different cell densities, untransformed cells grown to confluence, and mouse liver indicates that: (1) The tissue cultured mouse fibroblasts examined here are hypomodified with respect to nucleoside Q, while liver tRNA is almost completely modified with respect to Q. (2) Cell density and/or proliferative state do not present as major variables in controlling the expression of Q in the present system. (3) SV40 virus transformation is not a major variable controlling the expression of Q in the present system. The present results support previous use of cyanogen bromide effected shifts in chromatographic elution as an assay for nucleoside Q.     

Specific replacement of Q base in the anticodon of tRNA by guanine catalyzed by a cell-free extract of rabbit reticulocytes.

Guanylation of tRNA by a lysate of rabbit reticulocytes was reported previously by Farkas and Singh. This reaction was investigated further using 18 purified E. coli tRNAs as acceptors.Results showed that only tRNATyr, tRNAHis, tRNAAsn and tRNAAsp which contain the modified nucleoside Q in the anticodon acted as acceptors. Analysis of the nucleotide sequences in the guanylated tRNA showed that guanine specifically replaced Q base in these tRNAs.     

Isolation of mammalian tRNAAsp and tRNATyr by lectin-Sepharose affinity column chromatography.

tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.     

Evidence that the nucleic acid base queuine is incorporated intact into tRNA by animal cells

Queuine (the base of queuosine, Q) catalytically reduced with tritium or deuterium yields a derivative in which the proton at C-8 (purine numbering system) has been exchanged and the cyclopentene ring has been reduced to a cyclopentane ring. Mouse fibroblast tRNA has been labeled by culturing the cells in medium supplemented with [3H]- and [2H]dihydroqueuine. Such tRNA yields, upon hydrolysis, the nucleoside dihydroqueuosine and a saccharide derivative of dihydroqueuosine. Each product has been identified unambiguously by mass spectrometry and chromatography. Both the 3H- and 2H-labeled material coeluted, and no unlabeled Q nucleoside was found. Therefore, dihydroqueuine is incorporated intact into tRNA in mammalian cells. Furthermore, fractionation of the labeled tRNA on concanavalin A-agarose, which specifically binds the mannosyl-Q-containing tRNAAsp, has shown that the dihydroqueuosine-containing tRNAAsp is mannosylated. This is the first direct evidence that queuine is incorporated intact into mammalian tRNA in vivo.     

Structure determination of a nucleoside Q precursor isolated from E. coli tRNA: 7-(aminomethyl)-7-deazaguanosine.

A precursor of modified nucleoside Q isolated from E. coli methyl-deficient tRNA was determined to be 7-(aminomethyl)-7-deazaguanosine. The structure was deduced by means of its chromatographic and electrophoretic mobilities, and UV and mass spectra, in addition to comparison with the synthesized authentic compound. The same molecule is also found in tRNA of an E. coli mutant selected for deficient synthesis of modified nucleosides.     

Anticodon-anticodon interactions in solution. Studies of the self-association of yeast or Escherichia coli tRNAAsp and of their interactions with Escherichia coli tRNAVal

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNAAsp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNAAsp (GUC)-Escherichia coli tRNAVal (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNAAsp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant (Kass = 10(4)M-1 at 0 degrees C) is characteristic of a rather weak complex, when compared with that between tRNAAsp and tRNAVal (Kass = 4 X 10(6) M-1 at 0 degrees C), the effect being essentially linked to differences in the rate constant for dissociation. tRNAAsp split in the anticodon by T1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNAGly (GCC), E. coli tRNAVal (GAC) or E. coli tRNAAla (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNAAsp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNAAsp but its complex with E. coli tRNAVal was found to be only 1.5 times less stable than that between yeast tRNAAsp and E. coli tRNAVal. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNAAsp (in the presence of 1.6 M-ammonium sulphate and 3mM-spermine) revealed an important stabilization of the yeast and E. coli tRNAAsp duplexes or of their complexes with E. coli tRNAVal. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.     

Isolation of Q nucleoside precursor present in tRNA of an E. coli mutant and its characterization as 7-(cyano)-7-deazaguanosine.

One of the E. coli mutants selected for deficiency of modified nucleoside Q was found to contain an analogue of Q and normal guanosine in place of Q. The analogue of Q, designated as preQo, was isolated on a large scale from purified tRNATyr containing preQo. The structure of preQo was determined to be 7-(cyano)-7-deazaguanosine by comparison of its ultraviolet absorption spectra, thin-layer chromatographic mobility and mass spectrum with those of synthetic material.     

Comparison of rat liver and Walker 256 carcinosarcoma tRNAs.

The complete nucleotide sequences of both rat liver and Walker 256 mammary carcinosarcoma tRNAAsn reveal that they are identical except for the nucleotide present in the wobble position of the anticodon loop. The rat liver tRNAAsn contains the Q nucleoside, whereas the tumour tRNAAsn contains an unmodified guanosine. The tRNAs from both tissues also show significant quantitative differences in the chromatographic mobilities for isoaccepting species of tRNAAsp, tRNAAsn, tRNAHis and tRNATyr. In addition, chromatographic shifts upon cyanogen bromide treatment and analyses of the alkaline hydrolysates of these tRNAs demonstrate that those of tumour origin contain significantly less Q and Q nucleoside than their normal rat liver counterparts.     

Distribution of the modified nucleoside Q and its derivatives in animal and plant transfer RNA''s.

The modified nucleoside, 7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, designated as Q, and its derivative, Q*, were found in tRNA's from various organisms, including several mammalian tissues, other animals such as starfish, lingula and hagfish, and wheat germ. Q isolated from rat liver tRNA was found to be identical with E. coli Q by mass spectrometry and thin-layer chromatography. Thus the rare modified nucleoside Q originally isolated from E. coli tRNA, is widely distributed in various organisms. Analysis of the mass spectrum of Q* suggested that it has a different side chain from Q.     

Regulation of Ribonucleic Acid Synthesis by Histidine and Methionine During Recovery of Escherichia coli from Magnesium Starvation

During magnesium starvation of Escherichia coli B, most of the ribosomes break down to low-molecular-weight components. When magnesium is restored to the medium, the cells recover. The rate of recovery can be increased greatly by supplementing the growth medium with a mixture of 21 amino acids. This increased rate of recovery is shown to be due to the effect of only two amino acids, histidine and methionine, which initially stimulate accumulation of cellular ribonucleic acid without increasing the rate of protein synthesis. In contrast, histidine and methionine supplementation to logarithmically growing E. coli B is not as effective in stimulating growth as is the complete amino acid mixture. Since cells recovering from magnesium starvation preferentially synthesize ribosomes, it is possible that histidine and methionine play a special role(s) in ribosomal ribonucleic acid synthesis or stability.     

Modified nucleosides and the chromatographic and aminoacylation behavior of tRNA(Ile) from Escherichia coli C6

Transfer RNA from Escherichia coli C6, a Met-, Cys-, relA- mutant, was previously shown to contain an altered tRNA(Ile) which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676-7683). We now report the purification of this altered tRNA(Ile) and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA(Ile) purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA(Ile) (from cysteine-starved cells) was found to have a 5-fold increased Vmax in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl approximately AMP.Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA(Ile) bound more efficiently to its synthetase compared to normal tRNA(Ile). Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA(Ile) contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA(Ile) contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA(Ile) without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA(Ile) has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA(Ile) from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA(Ile) for aminoacylation is discussed.     

Methionineless Death in Escherichia coli

Methionine auxotrophs of strains derived from Escherichia coli 15 lose their colony-forming ability when deprived of this amino acid. Late addition of methionine to liquid cultures did not restore plating efficiency but permitted growth of surviving cells. This phenomenon, termed methionineless death (mld), was not observed with methionine auxotrophs of E. coli strains B, W, or K(12), nor was a similar amino acidless death observed with corresponding auxotrophs of E. coli 15 for arginine, tryptophan, proline, isoleucine, and leucine. Mld was not dependent upon the genetic site determining methionine auxotrophy, nor did it affect the decarboxylation of methionine or the stability of methionyl-transfer ribonucleic acid synthetase activity of starved cells. Death was not altered by the presence of spermine or spermidine but was abolished by the methionine analogue, alpha-methylmethionine. Simultaneous starvation of another amino acid in a multiple auxotroph also significantly reduced mld, suggesting a possible role of protein synthesis. The onset of mld is correlated with a lower net increase of deoxyribonucleic acid.     

Isolation of Escherichia coli precursor tRNAs containing modified nucleoside Q.

Affinity chromatography based on the complex formation of the modified nucleoside Q with boronic acid has been applied to the isolation of specific tRNA precursors containing this modified nucleoside. When [32P]RNA isolated from an Escherichia coli strain containing a thermolabile ribonuclease P was chromatographed on dihydroxyboryl-substituted cellulose, the precursors for asparagine, aspartate, histidine, and tyrosine tRNA were specifically retained. All precursors were monomeric. The nucleotide sequences of four asparagine tRNA precursors were determined.     

The twenty aminoacyl-tRNA synthetases from Escherichia coli. General separation procedure, and comparison of the influence of pH and divalent cations on their catalytic activities.

A general separation procedure of the twenty E. coli aminoacyl-tRNA synthetases including either a 105 000 g centrifugation or a poly-ethyleneglycol-dextran two-phases partition fractionation, and chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite is described. The specific activities of the synthetases have been determined after each chromatographic step and compared to their respective activities in the 105 000 g supernatant. Some aminoacyl-tRNA synthetases were obtained at 80 per cent purity. The presence of phenylmethylsulfonyl fluoride does not significantly modify either the elution patterns of the synthetases during the various chromatographic steps or their specific activities. Thus, contrarily to enzymes from various eukaryotic organisms no significant inactivation of the E. coli aminoacyl-tRNA synthetases occurs via proteolytic processes during the purification procedure. The effects of various factors: pH, magnesium, and other bivalent cations including spermidine, were tested on the aminoacylation and the [32P] PPi-ATP isotope-exchange reactions, and the optimal aminoacylation and isotope-exchange conditions determined for 18 of the 20 E. coli aminoacyl-tRNA synthetases.     

Biosynthesis of thiazole from thiamine in Escherichia coli]

The incorporation into the thiazole moiety of thiamine of several labeled compounds has been studied on short time incubations of washed-cells suspensions. No incorporation of radioactivity from [G-14C] methionine was found in a mutant auxotrophic for methionine. No radioactivity was incorporated from [U-14C] aspartate or from [U-14C] serine. The incorporation of 35S from sulphate was lowered by cysteine or glutathione but was unaffected by methionine or homocystine. Although the synthesis of thiazole is dependent on methionine, neither the sulphur atom nor the carbon chain of thiazole originate from methonine in E. coli. No carbon originates from cysteine which is the likely direct donor of sulphur.     

Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli.

N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli. Using E. coli WP2 (trp), we measured the incorporation of [5-3H]N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction. First, we observed that N4-aminocytidine uptake by the E. coli cells was as efficient as cytidine uptake. High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained [3H]N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucleoside; the content was dependent on the dose of N4-aminocytidine. There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed. These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E. coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine. We also found that the major portion of radioactivity in DNA of cells that had been treated with [5-3H]N4-aminocytidine was in the deoxycytidine fraction. We propose a metabolic pathway for N4-aminocytidine in cells of E. coli. This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine. In support of this proposed scheme, a cytidine deaminase preparation obtained from E. coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.     

Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus.

Nucleoside triphosphate phosphohydrolase [EC 3.6.1.15] activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification. This enzyme releases orthophosphate from the gamma-position in a nucleoside triphosphate, leaving nucleoside diphosphate. The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate. Deoxy ATP is hydrolyzed rather faster than ATP. However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E. coli RNA polymerase [EC 2.7.7.6] using calf thymus DNA as a template, and the phage Q beta RNA (30S), are not effective substrates for this enzyme. Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity. The phosphohydrolase must therefore be associated firmly with the virion. This enzyme does not require the presence of nucleic acid for its function. Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus.