anticodon and queuine

Queuine, 7-(( (4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]-methyl)-7-deazagu ani ne is synthesized de novo only in eubacteria and is preseent in place of guanine 34 in specific tRNAs containing anticodones GUN where N is one of the four canonical nucleotides. The biosynthetic pathway starting with GTP shares common steps with that of pteridines and riboflavin, and involves iron ions and a 'vitamin B12' coenzyme. Lower and higher eukaryotes are supplied with queuine by nutrition or the intestinal flora. The modification of tRNA with queuine is tissue specific and depends on the metabolic state of cells and tissues. Starvation for queuine and/or Q-deficiency in tRNA causes a few specific changes in the pattern of protein synthesis involving lactate dehydrogenases and cytochromes.

Topics: Animals; Anticodon; Base Sequence; Guanine; RNA, Transfer

In eukaryotes, the wobble position of tRNA with a GUN anticodon is modified to the 7-deaza-guanosine derivative queuosine (Q34), but the original source of Q is bacterial, since Q is synthesized by eubacteria and salvaged by eukaryotes for incorporation into tRNA. Q34 modification stimulates Dnmt2/Pmt1-dependent C38 methylation (m5C38) in the tRNAAsp anticodon loop in Schizosaccharomyces pombe. Here, we show by ribosome profiling in S. pombe that Q modification enhances the translational speed of the C-ending codons for aspartate (GAC) and histidine (CAC) and reduces that of U-ending codons for asparagine (AAU) and tyrosine (UAU), thus equilibrating the genome-wide translation of synonymous Q codons. Furthermore, Q prevents translation errors by suppressing second-position misreading of the glycine codon GGC, but not of wobble misreading. The absence of Q causes reduced translation of mRNAs involved in mitochondrial functions, and accordingly, lack of Q modification causes a mitochondrial defect in S. pombe. We also show that Q-dependent stimulation of Dnmt2 is conserved in mice. Our findings reveal a direct mechanism for the regulation of translational speed and fidelity in eukaryotes by a nutrient originating from bacteria.

Topics: Animals; Anticodon; Asparagine; DNA (Cytosine-5-)-Methyltransferases; DNA, Mitochondrial; Eukaryota; Guanine; Methylation; Mice; Micronutrients; Protein Biosynthesis; Ribosomes; RNA, Transfer; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Tyrosine

Constant generation of Reactive oxygen species (ROS) during normal cellular metabolism of an organism is generally balanced by similar rate of consumption by antioxidants. Imbalance between ROS production and antioxidant defense results in increased level of ROS causing oxidative stress which leads to promotion of malignancy. Queuine is a hyper modified base analogue of guanine, found at first anti-codon position of Q- family of tRNAs. These tRNAs are completely modified with respect to queuosine in terminally differentiated somatic cells, however hypomodification of Q-tRNAs is close association with cell proliferation. Q-tRNA modification is essential for normal development, differentiation and cellular functions. Queuine is a nutrient factor to eukaryotes. It is found to promote cellular antioxidant defense system and inhibit tumorigenesis. The activities of antioxidant enzymes like catalase, SOD, glutathione peroxidase and glutathione reductase are found to be low in Dalton's lymphoma ascites transplanted (DLAT) mouse liver compared to normal. However, exogenous administration of queuine to DLAT mouse improves the activities of antioxidant enzymes. The results suggest that queuine promotes antioxidant defense system by increasing antioxidant enzyme activities and in turn inhibits oxidative stress and tumorigenesis.

Topics: Animals; Anticodon; Antioxidants; Gene Expression Regulation; Guanine; Liver; Lymphocytes; Male; Mice; Neoplasm Transplantation; Neoplasms; Oxidative Stress; Reactive Oxygen Species; Time Factors

The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5.

Topics: Anticodon; Bacterial Proteins; Base Sequence; Cloning, Molecular; Colicins; Escherichia coli; Escherichia coli Proteins; Guanine; Molecular Sequence Data; Ribonucleases; Ribosomes; RNA, Bacterial; RNA, Ribosomal, 16S; RNA, Transfer, Amino Acid-Specific; RNA, Transfer, Asn; RNA, Transfer, Asp; RNA, Transfer, His; RNA, Transfer, Tyr

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.

Topics: Amino Acid Sequence; Anticodon; Catalysis; Crystallography, X-Ray; Guanine; Metalloproteins; Models, Molecular; Molecular Sequence Data; Nucleic Acid Precursors; Pentosyltransferases; Pyrimidinones; Pyrroles; Recombinant Proteins; RNA, Transfer; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Zinc; Zymomonas

The eukaryotic tRNA:guanine transglycosylase (TGT) catalyses the base-for-base exchange of guanine for queuine (the q-base)--a nutrition factor for eukaryotes--at position 34 of the anticodon of tRNAsGUN (where 'N' represents one of the four canonical tRNA nucleosides), yielding the modified tRNA nucleoside queuosine (Q). This unique tRNA modification process was investigated in HeLa cells grown under either aerobic (21% O2) or hypoxic conditions (7% O2) after addition of chemically synthesized q-base to q-deficient cells. While the q-base was always inserted into tRNA under aerobic conditions, HeLa cells lost this ability under hypoxic conditions, however, only when serum factors became depleted from the culture medium. The inability to insert q into tRNA did not result from a lack of substrate, because the q-base accumulated within these cells against the concentration gradient, suggesting the presence of an active transport system for this base in HeLa cells. The activity of the TGT enzyme was restored after treatment of the cells with the protein kinase C activator, TPA, even in the presence of mRNA or protein synthesis inhibitors. The results indicate that the eukaryotic tRNA modifying enzyme, TGT, is a downstream target of activated protein kinase C.

Topics: Anticodon; Blood; Culture Media; Enzyme Reactivators; Epidermal Growth Factor; Guanine; HeLa Cells; Humans; Kinetics; Oxygen; Pentosyltransferases; Platelet-Derived Growth Factor; Protein Kinase C; RNA, Transfer; Tetradecanoylphorbol Acetate

Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.

Topics: Anticodon; Aspartate-tRNA Ligase; Base Composition; Base Sequence; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Guanine; Molecular Sequence Data; Nucleic Acid Conformation; RNA, Transfer, Asp; Saccharomyces cerevisiae; Structure-Activity Relationship; Transfer RNA Aminoacylation

6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells.

Topics: Anticodon; Blotting, Northern; Cell Differentiation; Cell Division; Cell Line; Gene Expression; Genes, myc; Guanine; Humans; Hypoxanthine Phosphoribosyltransferase; Kinetics; Leukemia, Promyelocytic, Acute; RNA, Messenger; RNA, Neoplasm; Thioguanine