Insights into the base-pairing preferences of 8-oxoguanosine on the ribosome.

Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoG•C) and near-cognate (8-oxoG•A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed.

New aspects on the kinetics of activation of ribosomal peptidyltransferase-catalyzed peptide bond formation by monovalent ions and spermine.

The effect of NH4+ and K+ ions on the activity of ribosomal peptidyltransferase was investigated in a model system derived from Escherichia coli, in which AcPhe-puromycin is produced by a pseudo-first-order reaction between the preformed AcPhe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin. Detailed kinetic analysis suggests that both NH4+ and K+ ions act as essential activators of peptidyltransferase by filling randomly, but not cooperatively, multiple sites on the ribosome. With respect to the NH4+ effect at 25 degrees C. the values of the molecular interaction coefficient (n), the dissociation constant (KA), and the apparent catalytic rate constant (kmax) of peptidyltransferase at saturating levels of NH4+ and puromycin are 1.99, 268.7 mM and 24.8 min(-1), respectively. The stimulation of peptidyltransferase by K+ ions at 25 degrees C (n = 4.38, KA = 95.5 mM, kmax = 9.6 min[-1]) is not as marked as that caused by NH4+ ions. Furthermore, it is evident that NH4+ at high concentration (200 mM) is effective in filling regulatory sites of complex C, which are responsible for the modulatory effect of spermine. The combination of NH4+ ions (200 mM) with spermine (300 microM) produces an additive increase in peptidyltransferase activity. Taken together, these findings suggest the involvement of two related pathways in the regulation of peptidyltransferase activity, one mediated by specific monovalent cations and the other mediated by spermine.

Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines.

To gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines.

Brugia malayi asparaginyl-transfer RNA synthetase induces chemotaxis of human leukocytes and activates G-protein-coupled receptors CXCR1 and CXCR2.

Background. Lymphatic filariasis is a chronic human parasitic disease in which the parasites repeatedly provoke acute and chronic inflammatory reactions in the host bloodstream and lymphatics. Excretory-secretory products derived from filariae are believed to play an important role in the development of associated immunologic conditions; however, the specific mechanisms involved in these changes are not well understood. Recently, human cytoplasmic aminoacyl-transfer (t) RNA synthetases, which are autoantigens in idiopathic inflammatory myopathies, were shown to activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by recruiting immune cells that could induce innate and adaptive immune responses. Filarial (Brugia malayi) asparaginyl-tRNA synthetase (AsnRS) is known to be an immunodominant antigen that induces strong human immunoglobulin G3 responses.Methods. Recombinant B. malayi AsnRS was used to perform cellular function assays--for example, chemotaxis and kinase activation assays.Results. Unlike human AsnRS, parasite AsnRS is chemotactic for neutrophils and eosinophils. Recombinant B. malayi AsnRS but not recombinant human AsnRS induced chemotaxis of CXCR1 and CXCR2 single-receptor-transfected HEK-293 cell lines, blocked CXCL1-induced calcium flux, and induced mitogen-activated protein kinase.Conclusions. Our findings suggest that a filarial parasite chemoattractant protein may contribute to the development of chronic inflammatory disease and that chemokine receptors may be therapeutic targets to ameliorate parasite-induced pathology.

The initiator tRNA acceptance assay as a short-term test for carcinogens. 5. Results with 42 cytostatic drugs.

The activity of 42 cytostatic drugs used for the treatment of human cancer was tested by the initiator tRNA acceptance assay for carcinogens. Of 17 drugs carcinogenic for rodents, 16 (94.1%) gave a positive response in the assay and six (85.7%) out of seven non-carcinogens showed no activity. The predictive value of the test for cytostatics was 91.7%. Treatment of tRNA with several cytostatics resulted in an inhibition of its acceptance for L-methionine. Cyclophosphamide, dibromdulcitol, 5-deoxy-5-fluorouridine and vincristine also inhibited, in addition to this, the charging of unfractionated tRNA from rat liver with L-alanine, L-lysine, L-phenylalanine and L-valine. Some drugs apparently react with the same target nucleoside which is common for all species of tRNA (probably the terminal adenosine residue that is esterified with amino acids). Such compounds do not yield reliable results in the initiator tRNA acceptance assay since this inhibitory effect interferes with the stimulating effect characteristic for carcinogens. However, results of the present study agree well with those obtained earlier with different classes of compounds (N-nitroso compounds, mycotoxins, etc.) and indicate that this newly developed assay may be a useful alternative also for the testing of carcinogenicity of cytostatic drugs.

Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome.

The 3'-terminal -A-C-C-A sequence of yeast tRNA(Phe) has been modified by replacing either adenosine-73 or adenosine-76 with the photoreactive analogue 8-azidoadenosine (8N3A). The incorporation of 8N3A into tRNA(Phe) was accomplished by ligation of 8-azidoadenosine 3',5'-bisphosphate to the 3' end of tRNA molecules which were shortened by either one or four nucleotides. Replacement of the 3'-terminal A76 with 8N3A completely blocked aminoacylation of the tRNA. In contrast, the replacement of A73 with 8N3A has virtually no effect on the aminoacylation of tRNA(Phe). Neither substitution hindered binding of the modified tRNAs to Escherichia coli ribosomes in the presence of poly(U). Photoreactive tRNA derivatives bound noncovalently to the ribosomal P site were cross-linked to the 50S subunit upon irradiation at 300 nm. Nonaminoacylated tRNA(Phe) containing 8N3A at either position 73 or position 76 cross-linked exclusively to protein L27. When N-acetylphenylalanyl-tRNA(Phe) containing 8N3A at position 73 was bound to the P site and irradiated, 23S rRNA was the main ribosomal component labeled, while smaller amounts of the tRNA were cross-linked to proteins L27 and L2. Differences in the labeling pattern of nonaminoacylated and aminoacylated tRNA(Phe) containing 8N3A in position 73 suggest that the aminoacyl moiety may play an important role in the proper positioning of the 3' end of tRNA in the ribosomal P site. More generally, the results demonstrate the utility of 8N3A-substituted tRNA probes for the specific labeling of ribosomal components at the peptidyltransferase center.

The effectiveness of the bonding of amino acid by tRNA of livers of rats experimentally exposed to cadmium and barium.

The process of amino acid activation takes place in the presence of specific aminoacyl-tRNA synthetases. These enzymes, together with ATP, catalyse the bonding of tRNA and amino acids (5). The effectiveness of binding amino acid by tRNA has a direct influence on the intensity of protein biosynthesis. This effectiveness varies in various metabolic states of the cell (16). Heavy metals influence cellular metabolism. They usually reduce enzyme activity, block chemical reactions, bind with proteins, interact with other elements and even break certain bonds (1, 8, 9, 11, 14). Experiments in the present work were conducted in order to determine changes in the effectiveness of amino acid binding by tRNA of the livers of rats receiving heavy metals such as cadmium and barium in their feed.

5-Fluorouracil induces structural changes in rat liver tRNA.

5-fluorouracil (FUra) has been shown to modulate the aminoacylation function of rat liver tRNA. The present study was aimed at studying the structure-function relationship of FUra-substituted tRNA. Male Wistar rats (2-3 month old) were given a single i.p. injection of FUra at 50, 250, or 500 mg/kg body wt. and FUra-substituted total liver tRNA, i.e. tRNA(FUra50, 250, and 500, respectively, were isolated 3 h later. Normal tRNA (tRNA(N)) was isolated from saline-treated control rats. Thermal denaturation studies showed higher melting temperatures for tRNA(FUra) compared to tRNA(N). Heat denaturation followed by renaturation of total tRNA did not affect the activity of tRNA(N) and tRNA(FUra50), where as tRNA(FUra250 and 500) lost 35% and 72% of activity, respectively, compared to the corresponding group of non-denatured tRNA. Antibodies specific to rat liver tRNA recognized normal and FUra-substituted tRNA in the order of tRNA(N) > tRNA(FUra50) > or = tRNA(FUra250) > tRNA(FUra500) in an avidin-biotin micro-enzyme linked immunosorbant assay. tRNA(N) or tRNA(FUra50) preincubated with tRNA antiserum showed 74% and 59% of aminoacylation activity, respectively, compared to that of corresponding tRNA preincubated with normal rabbit IgG. However, activities of similarly treated tRNA(FUra250 and 500) were not affected. The observations of possible changes in the secondary structure of rat liver tRNA upon incorporation of FUra are discussed.

Kinetics of inhibition of peptide bond formation on bacterial ribosomes.

A cell-free system derived from Escherichia coli has been used in order to study the kinetics of inhibition of peptide bond formation with the aid of the puromycin reaction in solution. A similar study has been carried out earlier on a solid support matrix with the same inhibitors. We find that the overall pattern of the kinetics of inhibition is the same in the two systems. At low concentrations of inhibitor there is a competitive phase of inhibition, whereas at higher concentrations of inhibitor the type of inhibition becomes mixed noncompetitive. The values of Ki of the competitive phase in the system in solution are: 5.8 microM (amicetin), 0.2 microM (blasticidin S), 0.5 microM (chloramphenicol), and 0.5 microM (tevenel). The inhibitors amicetin, blasticidin S, and tevenel interact with the ribosome in a reaction which is slower than that of the substrate puromycin, showing clear-cut characteristics of slow-onset inhibition in both systems. Chloramphenicol, on the other hand does not easily show such a delay in solution. It interacts with the ribosome relatively faster than the other three antibiotics. Despite this, chloramphenicol too shows characteristics of time-dependent inhibition.

Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA.

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.

Viomycin does not stimulate the dissociation of peptidyl-tRNA.

Peptidyl-transfer RNA normally dissociates at a low rate from the ribosomes of Escherichia coli during protein synthesis but accumulates under nonpermissive conditions in cells with a temperature-sensitive allele (pthts) of the gene encoding peptidyl-transfer RNA hydrolase. The antibiotic-hypersensitive strain E. coli DB-11 with the pthts mutation was exposed to viomycin, then placed at nonpermissive temperatures. Under these conditions in the absence of drugs, peptidyl-tRNA accumulates, protein synthesis is inhibited and pthts cells die. When viomycin was present at sufficient concentration to arrest protein synthesis, cell death was not accelerated, error-inducing effects of streptomycin were not counteracted and, at high doses, cytoplasmic accumulation of peptidyl-transfer RNA was slowed down. Blocking the translocation of peptidyl-transfer RNA with viomycin did not stimulate its dissociation from ribosomes. Erythromycin-enhanced cell death was not affected by viomycin at doses sufficient to block amino acid incorporation, suggesting that short peptidyl-transfer RNAs could still be synthesized and dissociated from ribosomes.

Effects of the active aldehyde group generated by RNA N-glycosidase in the sarcin/ricin domain of rat 28S ribosomal RNA on peptide elongation.

Effects of the active aldehyde group of ribose C1' at position 4324 of rat 28S rRNA, in the inactivated ribosome generated by RNA N-glycosidases (trichosanthin, A-chain of cinnamomin and ricin), on peptide elongation have been studied. The aldehyde group inhibits the activities of eEF1A-dependent aminoacyl-tRNA binding to the inactivated ribosome and eEF1A-dependent GTPase, but increases eEF2-dependent activity. At a high concentration of RNA N-glycosidase, the generated aldehyde group also inhibits aminoacyl-tRNA binding to the inactivated ribosome in the absence of elongation factor and translocation activity. When the aldehyde group is reduced into a hydroxyl group by sodium borohydride or blocked with an amino acid through nucleophilic addition, the activities of eEF1A-dependent aminoacyl-tRNA binding and eEF1A-dependent GTPase of the inactivated ribosome are partially restored, but the altered activities of eEF2-dependent GTPase, translocation and aminoacyl-tRNA binding in the absence of elongation factor are not normalized. Thus, reduction or blockage of the aldehyde group with sodium borohydride or amino acids might change the conformation of the S/R domain in rat 28S ribosomal RNA to meet the requirement for eEF1A-dependent reactions, but not eEF2-involved reactions.