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.     

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.     

Isoaccepting species differences between polysome-bound and total cellular tRNA in SVT2 cells.

Reversed-phase chromatographic comparisons of total cellular tRNAs with tRNAs isolated from a polysome enriched cell fraction establish significant enrichments for specific isoaccepting species of tRNAIle, tRNALeu, tRNALys, tRNAMet and possibly tRNAA la. Similar comparisons of tRNAAsn, tRNAAsp, tRNAHis, tRNAPhe, tRNASer and tRNATyr have been performed as well; however, no prominent differences were observed.     

Queuosine modification in tRNA and expression of the nitrate reductase in Escherichia coli.

In eubacteria the modified nucleoside queuosine is present in tRNAAsn, tRNAAsp, tRNAHis and tRNATyr. A precursor of queuine, pre-queuine, is synthesized from GTP, inserted into the first position of the anticodon of the corresponding tRNAs by a specific tRNA-guanine transglycosylase and further modified to queuosine. Isogenic pairs of Escherichia coli, containing or lacking the tRNA-transglycosylase (JE 7335, tgt+ lacZ+ and JE 7337, tgt- lacZ+; JE 7334, tgt+ lacZ- and JE 7336, tgt- lacZ-), have been employed to study the function of queuosine in tRNA. Compared with the tgt+ strain (JE 7335), the tgt- mutant (JE 7337) grown under anaerobic conditions, is defective with respect to the nitrate respiration system, in which electrons are transported from D(-)-lactate via quinone and cytochrome bNO3-(556) to nitrate. Low temperature cytochrome spectra of the anaerobically grown tgt- mutant show a lowered amount of type b cytochromes involving the spectrum of cytochrome bNO3-(556). In the case of the anaerobically grown tgt- mutant three proteins are missing in the protein pattern of cytoplasmic membranes. Their mol. wts. correspond to those of the subunits of the nitrate reductase complex. In contrast to the tgt+ strains (JE 7334, JE 7335) both tgt- mutants (JE 7336, JE 7337) cannot grow on lactate under anaerobic conditions with nitrate offered as electron acceptor and NO3- is not reduced to NO2-. A possible link between Q-modification of tRNAs, the synthesis of proteins of the nitrate reductase complex and the synthesis of menaquinone or ubiquinone is discussed.     

Specific changes in lactate levels, lactate dehydrogenase patterns and cytochrome b559 in Dictyostelium discoideum caused by queuine

Higher eukaryotes contain tRNA transglycosylases that incorporate the guanine derivative queuine from the nutritional environment into specific tRNAs by exchange with guanine at position 34. Alterations in the queuosine content of specific tRNAs are suggested to be involved in regulatory mechanisms of major routes of metabolism during differentiation. Dictyostelium discoideum has been applied as a model to investigate the function of queuine or queuine-containing tRNAs. Axenic strains are supplied with queuine by peptone, but they grow equally well in a defined queuine-free medium. Queuine-lacking amoebae, starved in suspension culture for 24 h, lose their ability to differentiate into stalk cells and spores, whereas amoebae sufficiently supplied with queuine will overcome this metabolic stress and undergo further development when plated on agar. The results presented here show that D(-)-lactate occurs in the slime mould in millimolar amounts and that its level is remarkably decreased in queuine-lacking cells after 24 h of starvation in suspension culture. On isoelectric-focusing polyacrylamide gels, nine different forms of NAD-dependent D(-)-lactate dehydrogenase can be separated from extracts of vegetative cells, and six forms from extracts of the starved cells. Under queuine limitation, one form is missing in the starved cells. Low amounts of L(+)-lactate are usually found in vegetative amoebae but significantly less in queuine-lacking cells. Five forms of NAD-dependent L(+)-lactate dehydrogenase are detectable in extracts from vegetative, queuine-treated cells, and slight alterations occur in queuine-deficient amoebae. In the starved cells only one form of L(+)-lactate dehydrogenase is found, irrespective of the supply of queuine to the cells. A cytochrome of type b with an absorption maximum at 559 nm accumulates during starvation only in queuine-lacking cells; it might be a component of an NAD-independent lactic acid oxidoreductase as is cytochrome b 557 in yeast and be responsible for the reduced level of lactate in cells lacking queuine in tRNA.     

Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence

The eukaryotic tRNA-guanine transglycosylases (queuine insertases) catalyse an exchange of guanine for queuine in position 34, the wobble nucleoside, of tRNAs having a GUN anticodon where N (position 36) stands for A, U, C or G. In tRNAAsp (anticodon QUC) and tRNATyr (anticodon Q psi A) from certain eukaryotic cells, the nucleoside Q-34 is further hypermodified into a glycosylated derivative by tRNA-queuine glycosyltransferase. In order to gain insight into the influence of the nucleosides in position 36, 37 and 38 of an anticodon loop on the potential of a tRNA to become a substrate for the two modifying enzymes, we have constructed several variants of yeast tRNAs in which the normal anticodon has been replaced by one of the synthetic anticodons GUA, GUC, GUG or GUU. In yeast tRNAAsp, the nucleosides 37 (m1G) and 38(C) have also been replaced by an adenosine. These reconstructed chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and tested for their ability to react with the oocyte maturation enzymes. Our results indicate that the nucleosides in positions 36, 37 and 38 influence the efficiencies of conversion of G-34 to Q-34 and of Q-34 to glycosyl Q-34; the importance of their effects are much more pronounced on the glycosylation of Q-34 than on the insertion of queuine. The effect of the nucleoside in position 37 is of particular importance in the case of yeast tRNAAsp: the replacement of the naturally occurring m1G-37 by an unmodified adenosine (as it is in X. laevis tRNAAsp), considerably increases the yield of the glycosylation reaction catalysed by the X. laevis tRNA-queuine glycosyltransferase.     

Presence of queuine in Drosophila melanogaster: correlation of free pool with queuosine content of tRNA and effect of mutations in pteridine metabolism.

Queuine, a modified form of 7-deazaguanine present in certain transfer RNAs, is shown to occur in Drosophila melanogaster adults in a free form and its concentration varies as a function of age, nutrition and genotype. In several, but not all mutant strains, the concentrations of queuine and the Q(+) (queuine-containing) form of tRNATyr are correlated. The bioassay employs L-M cells which respond to the presence of queuine by an increase in their Q(+)tRNAAsp that is accompanied by a decrease in the Q(-)tRNAAsp isoacceptors. The increase in Q(+)tRNATyr in Drosophila that occurs on a yeast diet is accompanied by an increase in queuine. Similarly the increase of Q(+)tRNAs with age also is accompanied by an increase in free queuine. In two mutants, brown and sepia, these correlations were either diminished or failed to occur. Indeed, the extract of both mutants inhibited the response of the L-M cells to authentic queuine. When the pteridines that occur at abnormally high levels in sepia were used at 1 x 10(-6)M, the inhibition of the L-M cell assay occurred in the order biopterin greater than pterin greater than sepiapterin. These pteridines were also inhibitory for the purified guanine:tRNA transglycosylase from rabbit but the relative effectiveness then was pterin greater than biopterin greater than sepiapterin. Pterin was competitive with guanine in the enzyme reaction with Ki = 0.9 x 10(-7)M. Also when an extract of sepia was chromatographed on Sephadex G-50, the pteridine-containing fractions only were inhibitory toward the L-M cell assay or the enzyme assay. These results indicate that free queuine occurs in Drosophila but also that certain pteridines may interfere with the incorporation of queuine into RNA.     

Queuine-containing isoacceptor of tyrosine tRNA in Drosophila melanogaster. Alteration of levels by divalent cations

Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(-), ones of tRNATyr, tRNAHis and tRNAAsp of Drosophila melanogaster. Of the other divalent cations examined, Sr2+, Ni2+, Cu2+, Zn2+ and Hg2+, only Hg2+ failed to cause an increase in Q(+)tRNATyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responded to divalent ions; Hg2+ as well as Cd2+, Sr2+ and Zn2+ caused an increase in Q(+)tRNATyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd2+-containing diet, they formed significantly more Q(+)tRNATyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the 'hard-soft' classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(-) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation.     

Isolation and characterization of a guanine insertion enzyme, a specific tRNA transglycosylase, from Escherichia coli.

A guanine insertion enzyme (tRNA transglycosylase) was purified to a homogeneous state from Escherichia coli B by ammonium sulfate fractionation and DEAE-cellulose, DEAE-Sephadex A-50, phosphocellulose, and Sephadex G-200 column chromatographies. The molecular weight of the enzyme, which appeared to be a single polypeptide, was 4.6 X 10(4) by sodium dodecyl sulfate gel electrophoresis. The enzyme catalyzes exchange of guanine with guanine located in the first position of the anticodon of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp, but unlike the enzymes isolated from rabbit reticulocytes and Ehrlich ascites tumor cells it does not catalyze the exchange of guanine with queuine (7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) present in these tRNAs. The pH optimum of the reaction was 7.0, and the pH1 value was 4.6 to 4.8. The reaction required Mg2+ ion. 7-Methylguanine inhibited guanine insertion, but the other purine analogues tested were not inhibitory and could not replace guanine.20     

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.     

Synthesis of transfer ribonucleic acids with uridine or 2'-O-methylribothymidine at position 54 in developing Dictyostelium discoideum

In amoebae of Dictyostelium discoideum the ribothymidine (rT) content of tRNA is 0.9 mol%, but decreases progressively during development into spores. To elucidate which nucleosides replace rT at position 54 in developmental tRNA we have characterized 'vegetative' and 'developmental' tRNAs from the slime mould. Specific tRNAs were separated by two-dimensional gel electrophoresis. During early developmental stages, all tRNA species that could be separated by this method were newly synthesized. A new tRNA with uridine in place of rT and having an electrophoretic mobility similar to 'vegetative' tRNAAsn was detected during the early preaggregation stage. This 'development' tRNA was also extracted from purified polysomes. When development proceeds from preaggregation to postaggregation, tRNAs accumulate with 2'-O-methylribothymidine in place of rT. We suggest that these developmental tRNAs are important for the synthesis of specific developmental proteins.     

Queuine metabolism and cadmium toxicity in Drosophila melanogaster

Queuine can replace guanine in the anticodon of certain tRNAs and is a hypermodified guanine derivative that can be synthesized by bacteria but not by mice. The study demonstrates that Drosophila can incorporate dietary queuine into tRNA but cannot synthesize it de novo for this purpose. Since an earlier study had shown that dietary CdCl2 caused Drosophila to increase greatly the proportion of queuine-containing tRNA over non-queuine tRNA the ability of dietary queuine to counteract cadmium toxicity was evaluated. When queuine was present in the cadmium-containing medium more pupae matured into adults than when queuine was absent. Other studies had demonstrated that the transglycosylase enzyme, that catalyzes the replacement of guanine in the anticodon of tRNA by queuine, is present in Drosophila larvae but the tRNA is virtually devoid of queuine. This study shows that in the presence of dietary queuine the larval tRNA contains abundant amounts of queuine. Therefore, we postulate a significant role for bacteria in supplying queuine to Drosophila for its incorporation into tRNA and that the control of this process by Drosophila is passive, i.e. is not an essential feature in differentiation.     

The presence of pseudouridine in the anticodon alters the genetic code: a possible mechanism for assignment of the AAA lysine codon as asparagine in echinoderm mitochondria

It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNAAsn with the anticodon GUU. Nucleotide sequencing of starfish mt tRNAAsn revealed that the anticodon is GPsiU, U35 at the anticodon second position being modified to pseudouridine (Psi). In contrast, mt tRNALys, corresponding to another lysine codon, AAG, has the anticodon CUU. mt tRNAs possessing anti-codons closely related to that of tRNAAsn, but responsible for decoding only two codons each-tRNAHis, tRNAAsp and tRNATyr-were found to possess unmodified U35 in all cases, suggesting the importance of Psi35 for decoding the three codons. Therefore, the decoding capabilities of two synthetic Escherichia coli tRNAAla variants with the anticodon GPsiU or GUU were examined using an E.coli in vitro translation system. Both tRNAs could translate not only AAC and AAU with similar efficiency, but also AAA with an efficiency that was approximately 2-fold higher in the case of tRNAAlaGPsiU than tRNAAlaGUU. These findings imply that Psi35 of echinoderm mt tRNAAsn actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria.     

A mammalian tRNAHis-containing antigen is recognized by the polymyositis-specific antibody anti-Jo-1

The mammalian cell antigen reactive with the autoantibody anti-Jo-1 has been shown to contain tRNAHis. The RNA sequence of this human and mouse cell tRNA was determined in a search for unusual features that might be related to antigenicity. The 5' terminal nucleotide is unique among other sequenced tRNAs in that it is a methylated guanine. The presence of the hypermodified base queuine, which occurs in the wobble position of the anticodon of tRNAHis from several species, was not detected in the tRNAHis immunoprecipitated by anti-Jo-1 from either human HeLa or mouse Friend erytholeukemia cell extracts. The binding of protein(s) appears to confer antigenicity on tRNAHis since either proteinase K treatment or phenol extraction resulted in the loss of immunoprecipitability. However, we have not succeeded in identifying an antigenic protein, and we find that the antigenic complex is not resolved from purified tRNAHis by Sephacryl S-200 column chromatography. Immunofluorescence studies indicate that the antigenic form of tRNAHis is located preferentially in the mammalian cell cytoplasm. The results presented here are discussed in light of an earlier report (1) on the nature of the Jo-1 antigen.     

Queuine in plants and plant tRNAs: Differences between embryonic tissue and mature leaves

In eubacterial and eukaryotic tRNAs specific for Asn, Asp, His and Tyr the modified deazaguanosinederivative queuosine occurs in position 34, the first position of the anticodon. Analysis of unfractionated tRNAs from wheat and from tobacco leaves shows that these tRNAs contain high amounts of guanosine (G) in place of queuosine (Q). This was measured by the exchange of G34 for [³H]guanine catalysed by the specific tRNA guanine transglycosylase from E. coli. Upon gel electrophoretic separation of the labeled tRNAs, seven Q-deficient tRNA species including isoacceptors are detectable. Two are identified as cytoplasmic tRNAs^Tyr and tRNA^Asp and two represent chloroplast tRNA^Tyr isoacceptors. In contrast to leaf cytoplasm and chloroplasts, wheat germ has low amounts of tRNAs with G34 in place of Q.A new enzymatic assay is described for quantitation of free queuine in cells and tissues. Analysis of queuine in plant tissues shows that wheat germ contains about 200 ng queuine per g wet weight. In wheat and tobacco leaves queuine is present, if at all, in amounts lower than 10 ng/g wet weight. The absence of Q in tRNAs from plant leaves is therefore caused by a deficiency of queuine. Tobacco cells cultivated in a synthetic medium without added queuine do not contain Q in tRNA, indicating that these rapidly growing cells do not synthesize queuine de novo.     

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.     

Novel salvage of queuine from queuosine and absence of queuine synthesis in Chlorella pyrenoidosa and Chlamydomonas reinhardtii.

Partially purified extracts from Chlorella pyrenoidosa and Chlamydomonas reinhardtii catalyze the cleavage of queuosine (Q), a modified 7-deazaguanine nucleoside found exclusively in the first position of the anticodon of certain tRNAs, to queuine, the base of Q. This is the first report of an enzyme that specifically cleaves a 7-deazapurine riboside. Guanosine is not a substrate for this activity, nor is the epoxide a derivative of Q. We also establish that both algae can incorporate exogenously supplied queuine into their tRNA but lack Q-containing tRNA when cultivated in the absence of queuine, indicating that they are unable to synthesize Q de novo. Although no physiological function for Q has been identified in these algae, Q cleavage to queuine would enable algae to generate queuine from exogenous Q in the wild and also to salvage (and recycle) queuine from intracellular tRNA degraded during the normal turnover process. In mammalian cells, queuine salvage occurs by the specific cleavage of queuine from Q-5'-phosphate. The present data also support the hypothesis that plants, like animals, cannot synthesize Q de novo.     

Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase

We have tested as inhibitors or substrates of tRNA-guanine ribosyltransferase (EC 2.4.2.29) a number of compounds, including derivatives of 7-deazaguanine, pteridines, purines, pyrimidines and antimalarials. Virtually all purines and pteridines that are inhibitors or substrates of the rabbit reticulocyte enzyme have an amino nitrogen at the 2 position. In addition the 9 position and the oxygen at the 6 position may be important for recognition by the enzyme. Saturation of the double bond in the cyclopentenediol moiety of queuine reduces substrate activity and queuine analogs that lack the cyclopentenediol moiety, such as 7-deazaguanine and 7-aminomethyl-7-deazaguanine, are relatively poor substrates for the enzyme. While adenosine is not an inhibitor, neplanocin A (an adenosine analog in which a cyclopentenediol replaces the ribose moiety) is a poor inhibitor. The incorporation of 7-aminomethyl-7-deazaguanine into the tRNA of L-M cells results in a novel chromatographic form of tRNAAsp, indicating that L-M cells cannot modify this Q precursor (in Escherichia coli) to queuosine. The specific incorporation of 7-deazaguanine and 8-azaguanine into tRNA by L-M cells also results in novel chromatographic forms of tRNAAsp. With intact L-M cells, the enzyme-catalyzed insertion into tRNA of queuine, dihydroqueuine, 7-aminomethyl-7-deazaguanine, or 7-deazaguanine is irreversible, while guanine or 8-azaguanine incorporation is reversible; suggesting that it is the substitution of C-7 for N-7 which prevents the reversible incorporation of queuine into tRNA.