Analogues of SB-203207 as inhibitors of tRNA synthetases

SB-203207 and 10 analogues have been prepared, by elaboration of altemicidin, and evaluated as inhibitors of isoleucyl, leucyl and valyl tRNA synthetases (IRS, LRS, and VRS, respectively). Substituting the isoleucine residue of SB-203207 with leucine and valine increased the potency of inhibition of LRS and VRS, respectively. The leucine derivative showed low level antibacterial activity, while several of the compounds inhibited IRS from Staphylococcus aureus WCUH29 more strongly than rat liver IRS.     

Fidelity in the aminoacylation of tRNA(Val) with hydroxy analogues of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli

Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli. Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA. However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed. Thus, valyl-tRNA synthetase from S. cerevisiae shows a high fidelity in forming valyl-tRNA. Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated. This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.     

Ambiguity in a Polypeptide-Synthesizing Extract from Saccharomyces cerevisiae

Environmental factors known to induce ambiguity in bacterial extracts were tested in an in vitro cytoplasmic polypeptide-synthesizing system derived from Saccharomyces cerevisiae. Increasing concentrations of magnesium, spermine, and spermidine resulted in extensive leucine-phenylalanine ambiguity in polyuridylic acid-directed polypeptide synthesis. Kinetic studies showed that spermine-mediated stimulation of leucine incorporation occurred when phenylalanine was being actively incorporated. In addition to leucine, the amino acids isoleucine and serine were incorporated in the presence of added magnesium and spermine. Ambiguity in the presence of a high Mg(2+) concentration was decreased when the pH of the reaction mixture was lowered. Ethanol and neomycin enhanced ambiguity to a small, but significant, extent. Streptomycin and temperature had no effect on ambiguity. Leucine, isoleucine, and serine were not attached to phenylalanine transfer ribonucleic acid (tRNA) when the aminoacylation reaction was performed at increasing Mg(2+) and spermine concentrations. On the other hand, increasing levels of Mg(2+) and spermine stimulated the incorporation of leucine from tRNA into polypeptide during the transfer reaction. The formal similarity between the findings in the yeast and Escherichia coli systems implies the existence of a tRNA-screening site on the yeast ribosome comparable to that suggested for bacteria. A proposal is made as to the manner in which this site may function to produce the ambiguous codon translation observed.     

The plant aminoacyl-tRNA synthetases. Effect of sodium chloride on tRNA aminoacylation and aminoacyl-tRNA decomposition catalysed by aminoacyl-tRNA synthetases from yellow lupin seeds.

The initial velocity and the extent of aminoacylation are affected by sodium chloride in the lupin aminoacylation systems involving serine, isoleucine, lysine, leucine, phenylalanine and valine. Pyrophosphorolysis and enzymatic hydrolysis of [14C]Val-tRNA catalysed by lupin valyl-tRNA synthetase are inhibited by sodium chloride nearly to the same extent. Evidence is presented that when a limiting amount of synthetase is used, the equilibrium of the aminoacylation reaction in the lupin valine system is determined only by the rate of aminoacylation and non-enzymatic deacylation of aminoacyl-tRNA, the former but not the latter reaction being dependent on concentration of the enzyme and monovalent salt.     

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.     

Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.     

Thiaisoleucine and protein synthesis.

Thiaisoleucine is an isoleucine analogue having the gamma-methylene group of the valerianic carbon chain substituted by a sulphur atom. It has been demonstrated that thiaisoleucine is activated and transferred to tRNAIle by rat liver aminoacyl-tRNA synthetase and inhibits isoleucine incorporation into polypeptides in protein synthesizing systems from rat liver or rabbit reticulocytes, whereas it does not affect either leucine incorporation or ribosome run-off or polypeptide chain elongation rate. All tests were performed in comparison with O-methyl-threonine, an isoleucine analogue with the gamma-methylene group substituted by an oxygen atom. In all the reactions studied, both thiaisoleucine and O-methyl-threonine act as competitive inhibitors of isoleucine. With respect to O-methyl-threonine, thiaisoleucine shows higher activity as an isoleucine inhibitor.     

Biological activity of tRNA in rat liver during cycloheximide-blocked translation

The aminoacylation of tRNA was investigated with respect to protein synthesis in the rat liver. No correlation was found between the 85-90% inhibition of protein synthesis 2 h after cycloheximide injection and aminoacylation level of some tRNAs both in vivo and in vitro. A decrease in aminoacylation (28%) was established only for lysine. During the recovery phase of protein synthesis 12 and 24 h after cycloheximide treatment the aminoacylation maximal level of mixture with 14C amino acids, 14C leucine, 14C glutamic acid was unchanged.     

The mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena are indistinguishable.

The mitochondrial and cytoplasmic valyl tRNA synthetases from Tetrahymena pyriformis are indistinguishable. These synthetases cannot be differentiated through hydroxylapatite, DEAE-cellulose, or phosphocellulose column chromatography. Both enzymes show the same mean sedimentation coefficient of 5.9 S in sucrose gradient centrifugation analysis; when bound with tRNA, they are relatively stable and sediment at 7.8 S. The temperature optimum for aminoacylation reaction is 27.5 °C, the optimum Mg²⁺ concentration is 4.4 mm, and substrate affinities (K_m values) for valine and ATP in aminoacylation are the same for both enzymes at 1.0 μm and 2.5 mm, respectively. These enzymes show identical specificities for acylation of different tRNA species, i.e., Tetrahymena and rat liver tRNAs can be equally well recognized, but no significant acylation can be observed with Escherichia coli and Saccharomyces tRNAs. These observations suggest the probable molecular identity of mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena.     

The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo. A mechanism for the inhibition of neural protein synthesis by phenylalanine.

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.     

Purification of mammalian histidyl-tRNA synthetase and its interaction with myositis-specific anti-Jo-1 antibodies

Histidyl-tRNA synthetase is purified to near homogeneity from rat liver. The subunit molecular weight of histidyl-tRNA synthetase is 50,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The Stokes radius and the sedimentation coefficient of histidyl-tRNA synthetase are 38 A and 6.0 S, respectively. The native molecular weight of histidyl-tRNA synthetase is calculated to be 96,000 on the basis of its hydrodynamic properties. The purified histidyl-tRNA synthetase reacts with the myositis-specific anti-Jo-1 antibodies. Anti-Jo-1 immunoglobulin G reacts with the native form of histidyl-tRNA synthetase and does not react or only weakly reacts with the denatured form. The anti-Jo-1 antibodies exhibit stronger inhibition toward histidyl-tRNA synthetase that has been preincubated with tRNA than that without preincubation. Anti-Jo-1 antibodies behave as a noncompetitive inhibitor with respect to tRNA in the aminoacylation reaction catalyzed by histidyl-tRNA synthetase. The structural features of the antigen of the anti-Jo-1 antibodies in light of these results are discussed.     

Functionally impaired tRNA from ethionine treated rats as detected in injected Xenopus oocytes.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant.In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.     

Comparison of porcine liver tRNA preparations: Purification of tRNA and its separation from RNA-peptidyl complexes

Six fractions of soluble RNA were obtained from phenol extracts of porcine liver and were tested for their acceptance of 14 amino acids under aminoacylation conditions and for their effects on the aminoacylation of tRNA. Two of the fractions contained appreciable amounts of tRNA, and three of the fractions affected the aminoacylation of tRNA. Based on these observations a revised method of tRNA preparation was developed that includes essentially all the tRNA in one fraction but that excludes the RNA-peptidyl complexes. The revised method is rapid and convenient and provides better quality tRNA than three alternate methods to which it is compared.     

Qualitative and quantitative variation of tRNA in certain invertebrates

Total tRNA was isolated, purified and quantitated from earthworm, cockroach, fresh water mussel and rat liver. The total tRNA content of invertebrates was found to be much lower than that of rat liver. When checked for aminoacylation capacity with homologous and heterologous enzymes and algal protein hydrolysate, the tRNA preparation from rat liver and fresh water mussel, a mollusc, were found to be active. On the other hand, the tRNAs from earthworm, an annelid, and cockroach, an arthropod, were completely inactive with the homologous enzymes but showed partial activity with heterologous enzymes. Similar results were obtained with individual amino acids also. The low activity or inactivity of earthworm and cockroach tRNAs appears to be due to certain endogenous aminoacylation inhibitors. This work was carried out at the School of Life Sciences, University of Hyderabad, Hyderabad 500 134, India.     

Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses.

During the isolation of the activator protein for glucosylceramide beta-glucosidase, we found that certain column fractions contained an inhibitor of the enzyme. After separation from the activator protein by a DEAE-Sephacel column, the inhibitor was purified further with a Spehadex G-75 column. The u.v. absorption spectrum of the purified material was similar to that of nucleic acids and the protein content of the purified material was negligible. Furthermore the purified inhibitor reacted with orcinol but not with diphenylamine, and its inhibitory activity was completely destroyed by treatment with RNAases. It seems likely that the purified inhibitor was tRNA. Authentic RNA, tRNA and DNA had similar inhibitory effects on beta-glucosidase (Ki 17 micrograms/ml for tRNA, noncompetitive toward the substrate). The inhibitory effect of nucleic acids was not fully overcome by an excess amount of the activator protein, but phosphatidylserine could restore the activity to normal. Tests with several other hydrolases revealed that the inhibitory effect of nucleic acids was fairly general.     

On dealing with anomalies in the transfer RNA aminoacylation reaction in partially purified systems

Apparent differences in tRNA and aminoacyl-tRNA synthetase complements in tissues undergoing differentiation have frequently been used to support theories of translational control. The validity of at least some of these studies must now be questioned because of anomalies in the tRNA aminoacylation reaction which can lead to incomplete aminoacylation of tRNA. Incomplete acylation of a tRNA mixture could result in different relative amounts of acylated isoaccepting species if acylation rates were not identical for all species. Using common methods of analysis, this situation could lead to misestimation of relative levels of isoacceptors or an inability to detect the presence of minor species. Bonnet and Ebel [Bonnet, J., and Ebel, J. (1972) Eur. J. Biochem.31, 335] used a highly purified valyl-tRNA and valyl-tRNA synthetase from yeast to demonstrate the presence of four reactions that occur simultaneously in that system. Herein, I apply the findings of Bonnet and Ebel to a mammalian system in a manner which is representative of attempts to study relative tissue proportions of tRNA isoacceptors. Total complements of tRNAs and the aminoacyl-tRNA synthetases have been partially purified from rabbit liver according to the methods of Yang and Novelli [Yang, W. K., and Novelli, G. D. (1971) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, p. 44, Academic Press, New York], probably the most commonly used procedures in the literature. Reaction conditions for tRNA acylation are shown to be modifiable so as to influence the extent of tRNA acylation. Procedures for optimizing the extent of tRNA acylation in such systems are demonstrated, and the unfavorable influence of Tris buffer, a factor not discussed by Bonnet and Ebel, is shown. Finally, examples of altered ratios of isoaccepting species in samples incompletely acylated due to suboptimal reaction conditions are provided.     

Cooperativity in low-affinity Mg2+ binding to tRNA

The Pb2+-catalyzed cleavage of tRNAPhe has been used to probe the effect of Na+ and Mg2+ binding to tRNA. Na+ is a noncompetitive inhibitor of the Pb2+-catalyzed cleavage. Millimolar Mg2+ is also a noncompetitive inhibitor. Analysis of the Mg2+ data show that at least two sites are involved in binding and that there is an interaction between the sites (cooperativity). Low-affinity Mg2+ binding is thus different from "weak" and "strong" Mg2+ binding to tRNA characterized previously. We postulate that the alterations induced by low-affinity Mg2+ binding in tRNA mimic to some extent those brought about in RNA by the interaction with a protein factor and that at appropriate [Mg2+] the whole structure of tRNA is able to respond in a concerted way to a signal from the environment such as aminoacylation or codon binding.     

Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.