Effect of polyamines on isoleucyl-tRNA formation by rat-liver isoleucyl-tRNA synthetase.

The effect of polyamines on rat-liver isoelucyl-tRNA formation was studied using isoleucyl-tRNA synthetase purified by column chromatography successively on Sephadex G-200, DEAE-Sephadex A-25, and tRNA-Sepharose 4B. In the presence of 50 mMK+, isoleucyl-tRNA formation was inhibited markedly by 1.5 mM or higher concentrations of Mg2+. However, the addition of spermine to the reaction mixture prevented the inhibitory effect of Mg2+. In the presence of 200 mMK+, the addition of spermine to the reaction mixture stimulated isoleucyl-tRNA formation in the presence of Mg2+ concentrations from 0 to 5 mM. Although the effective concentration was different, spermidine exhibited a similar stimulative effect. The effective concentration of spermine required for stimulation was higher when larger amounts of tRNA were used. The stimulatory effect of isoleucyl-tRNA formation by polyamines was shown to reflect on polypeptide synthesis. When formaldehyde-treated poly(A,U) was used as messenger RNA, polypeptide synthesis from amino acids was stimulated by polyamines, but that from aminoacyl-tRNAs was not stimulated by polyamines.     

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg²⁺, polyamines significantly stimulate the reaction. Of the cations tested, only Mn²⁺, Co²⁺ and Ca²⁺ can partially substitute for Mg²⁺ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg²⁺ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg²⁺ is also prevntedvby spemine.     

Proofreading in trans by an aminoacyl-tRNA synthetase: a model for single site editing by isoleucyl-tRNA synthetase.

Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code. Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. Here, it is shown that isoleucyl-tRNA synthetase (IleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine). That these enzymatic reactions occur between Ile-tRNAIle or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme. Although IleRS.Val-AMP undergoes thiolysis as efficiently as do IleRS.Ile-AMP and IleRS.Ile-tRNAIle, IleRS.Val-tRNAIle does not react with thiols. These and other data suggest that the mischarged valine residue in IleRS.Val-tRNAIle is, most likely, positioned off the enzyme.     

On the role of isoleucyl-tRNA synthetase in multivalent repression

An isoleucine auxotroph of Salmonella typhimurium was derived from a merodiploid strain (containing the F-14 episome from Escherichia coli) that contained two copies of the structural genes concerned with isoleucine and valine biosynthesis. A haploid derivative, strain TU6001, having the same growth properties as the original merodiploid mutant was found to have normal biosynthetic enzymes and an altered isoleucyl-tRNA synthetase. The K _m for isoleucine was increased by about 200-fold over that for the wild-type enzyme. All five enzymes in the isoleucine and valine biosynthetic pathway were derepressed relative to wild-type enzyme levels. A partial revertant of strain TU6001 was isolated which had properties that were intermediate between those of the mutant and the wild type (i.e., intermediate growth dependence on exogenous isoleucine, intermediate activity of isoleucyl-tRNA synthetase, and intermediate derepression of biosynthetic enzymes). The properties of strain TU6001 were demonstrated to be simultaneously transferable by transduction (using P_LT22 H₄ bacteriophage) of a single genetic locus, linked to pyr A, which has been designated ilv S. It is concluded that some function of the isoleucyl-tRNA synthetase is important in repression of the isoleucine and valine biosynthetic enzymes.Supported by grant GM 12522 from the National Institute of General Medical Sciences, U.S. Public Health Service. J. M. B. received a U.S. Public Health Service Postdoctoral Fellowship 1-F02-GM-30, 650-02.     

Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.     

Interacting binding sites of isoleucyl-tRNA synthetase from Escherichia coli studied by equilibrium partition.

The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.     

Analysis of the isoleucyl-tRNA synthetase reaction by total rate equations. Magnesium and spermidine in the tRNA kinetics.

Derivation of a steady-state rate equation for the aminoacyl-tRNA synthetases is described, and its suitability for the analysis of various details of the reaction is tested. The equation is applied to the magnesium and spermidine dependences of the isoleucyl-tRNA synthetase reaction. Earlier work [Airas, R.K. (1990) Eur. J. Biochem. 192, 401-409] is expanded by experiments and calculations of the tRNA kinetics. The analysis suggests the following new details in addition to the earlier results: (a) The binding of tRNA to the enzyme (and not only the rate of the aminoacylation reaction) is affected by the presence of the Mg2+ and spermidine in the tRNA molecule. At least two bound Mg2+ or spermidines are required. (b) tRNA and PPi partly inhibit the binding of each other to the enzyme. (c) The transfer reaction is rather slow, and, at least under some conditions, it participates in rate limitation. (d) A Mg(2+)-induced reduction in the aminoacylation rate seems to be directed to the dissociation of the aminoacyl-tRNA from the enzyme. This dissociation rate is enhanced if a Mg2+ is first dissociated from the enzyme or tRNA. An increase in the Mg2+ concentration shifts the rate limitation from the transfer reaction towards dissociation of the product.     

ATP-induced activation of the aminoacylation of tRNA by the isoleucyl-tRNA synthetase from Escherichia coli

The rate of aminoacylation of tRNA catalyzed by the isoleucyl-tRNA synthetase form Escherichia coli has been measured. A steady-state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile-tRNA from the enzyme.     

Purification of isoleucyl-tRNA synthetase from Methanobacterium thermoautotrophicum by pseudomonic acid affinity chromatography

The isoleucyl-tRNA synthetase of the archaebacterium Methanobacterium thermoautotrophicum was purified 1500-fold to electrophoretic homogeneity by a procedure based on affinity chromatography on Sepharose-bound pseudomonic acid, a strong competitive inhibitor of this enzyme. The purified enzyme is a monomer with a molecular mass of 120 kDa. In this respect and in its Km values for the PPi-ATP exchange, and aminoacylation reactions, it resembles the isoleucyl-tRNA synthetases from eubacterial and eukaryotic sources. Its aminoacylation activity is optimal at pH 8.0 and at 55 degrees C. Pseudomonic acid is a strong competitive inhibitor of the aminoacylation reaction with respect to both L-isoleucine (KiIle 10 nM) and ATP (KiATP 20 nM).     

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.     

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl-tRNA synthetase from Escherichia coli.

A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E.aa-AMP possible. If the E.Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E.Ile-AMP with E.Ile and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E.Ile-AMP and the corrected (*E)tRNA(Ile-AMP). If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be beneficial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.     

Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro.

Using a protein synthesizing in vitro system programmed with MS 2-RNA, the relative efficiency (in the presence of each other) of valine tRNAs with the anticodons U*AC (U* represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC to read the valine codons was investigated. An anticodon which can read all three positions of the codon according to the rules of Watson-Crick base-pairing and the wobble hypothesis is an order of magnitude more efficient than an anticodon which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon. There are two seeming exceptions to this behavior: the anticodon U*AC reads the codon GUU quite efficiently and IAC is as effective as U*AC in reading the codon GUG. The significance of these exceptions is evaluated with respect to the organization and evolution of the genetic code.     

Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase

We describe the heterologous expression of a recombinant Saccharomyces cerevisiae isoleucyl-tRNA synthetase (IRS) gene in Escherichia coli, as well as the purification and characterization of the recombinant gene product. High level expression of the yeast isoleucyl-tRNA synthetase gene was facilitated by site-specific mutagenesis. The putative ribosome-binding site of the yeast IRS gene was made to be the consensus of many highly expressed genes of E. coli. Mutagenesis simultaneously created a unique BclI restriction site such that the gene coding region could be conveniently subcloned as a "cassette." The variant gene was cloned into the expression vector pKK223-3 (Brosius, J., and Holy, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6929-6933) thereby creating the plasmid pKR4 in which yeast IRS expression is under the control of the isopropyl-thio-beta-galactopyranoside (IPTG)-inducible tac promoter. Recombinant yeast IRS, on the order of 10 mg/liter of cell culture, was purified from pKR4-infected and IPTG-induced E. coli strain TG2. Yeast IRS was purified to homogeneity by a combination of anion-exchange and hydroxyapatite gel chromatography. Inhibition of yeast IRS activity by the antibiotic pseudomonic acid A was tested. The yeast IRS enzyme was found to be 10(4) times less sensitive to inhibition by pseudomonic acid A (Ki = 1.5 x 10(-5) M) than the E. coli enzyme. E. coli strain TG2 infected with pKR4, and induced with IPTG, had a plating efficiency of 100% at inhibitor concentrations in excess of 25 micrograms/ml. At the same concentration of pseudomonic acid A, E. coli strain TG2 infected with pKK223-3 had a plating efficiency less than 1%. The ability of yeast IRS to rescue E. coli from pseudomonic acid A suggests that the eukaryotic synthetase has full activity in its prokaryotic host and has specificity for E. coli tRNA(ile).     

Integrated function of a kinetic proofreading mechanism: double-stage proofreading by isoleucyl-tRNA synthetase

Experimental measurements for isoleucyl-tRNA synthetase proofreading valyl-tRNAIle in Escherichia coli previously have been incorporated into the conventional Michaelis-Menten model for this system. This model was augmented to include two stages of proofreading--the aminoacyl adenylate and aminoacyl-tRNA stages--and used to predict the values of four additional rate constants that have been determined experimentally. The results suggest that two stages of conventional kinetic proofreading with binding sites designed for isoleucine (the "correct" substrate) are inconsistent with the experimental data, that a double-stage mechanism in which one stage (the "double-sieve") involves a binding site designed for valine (the "incorrect" substrate) and the other involves a binding site designed for isoleucine is consistent with all the experimental data, and that the experimental data are not sufficiently accurate to distinguish the stage at which the double-sieve mechanism operates in vivo. Furthermore, analysis of the model suggests that four parameters have the most questionable values and that experimental refinement of their estimates will be needed to determine which of the two stages involves the double-sieve mechanism.     

On the binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

The binding of nine aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 was measured and compared with the binding of the cognate amino acids. It was found that they bind rather tightly to the enzyme, the Kd's ranging from 3.1.10(-4) M with glycinol-AMP ester to 3.7.10(-9) M with L-isoleucinol-AMP ester. The binding is not affected by magnesium. It is shown that the free energies of binding of the esters can be calculated adding a constant contribution of the AMP-moiety of about - 4.1 (- 17) kcal/mole (kJ/mole) to the free energies of binding of the cognate amino acids, which we have reported earlier (19, 25, 26).