aminoacyl-tRNA Formation Research Articles

EFFECTS OF TEMPERATURE EXTREMES ON PROTEIN SYNTHESIS IN LIVER OF TOADFISH,OPSANUS TAU, IN VIVO

Amino acid incorporation and polypeptide chain elongation rates were determined in toadfish at the upper and lower ends of their range of temperature tolerance. The method was based on pulse injection of radioactive amino acids into the hepatic portal vein and analysis of ribosome-bound and completed chains at various times after injection. Elongation rates at low temperatures were obtained from the rate of completion and release of polypeptide chains pre-labeled at 20°C. Average polypeptide chain assembly time at 4°C was 6 h; that at 37°C was 1 min. Comparison with rates of total protein synthesis obtained in previous studies indicate a coordinated slowing of all protein synthetic reactions at low temperatures. A 10-fold decline in elongation rate from 7°C to 4°C suggests a specific temperature sensitivity in elongation factors or in formation of aminoacyl-tRNA. At high temperatures (32°-40°C) protein synthetic reactions show a loss of coordination, with elongation rate increasing normally (Q10 about 2) ...

Increase of degree of spermidine stimulation of polypeptide synthesis in the presence of phosphate

The addition of phosphate caused an increase in the degree of spermidine stimulation of polypeptide synthesis in an Escherichia coli and a wheat germ cell-free system. Optimal stimulation of polypeptide synthesis was observed at 20 m m phosphate for both systems, but concentrations of phosphate up to 40 m m had no additional effect. The increase of degree of spermidine stimulation in the presence of phosphate in an E. coli cell-free system occurred at the level of aminoacyl-tRNA binding to ribosomes and not at the level of peptide bond formation, translocation, or aminoacyl-tRNA formation. From the results of studies on RNase A sensitivity of ribosomal subunits and the effect of antibiotics known to act on the 30 S ribosomal subunits, it is suggested that the nature of the 30 S ribosomal subunits is changed by phosphate so that the degree of spermidine stimulation of polypeptide synthesis is increased.

Trypanosoma cruzi: Inhibition of protein synthesis by nitrofuran SQ 18,506

SQ 18,506 is a nitrofuran compound related to the trypanocide Lampit. In vitro, radiolabeled leucine, uridine, and thymidine were incorporated into macromolecular protein, RNA, and DNA in order to study growth inhibition of Trypanosoma cruzi. Our findings suggest that the primary effect of the drug is on protein synthesis and not mediated solely by inhibition of RNA synthesis as indicated by prior studies. The drug was also found to reduce markedly the uptake of uridine into the nucleotide precursor pool but to affect only slightly the formation of aminoacyl-tRNA.

The mechanism of action of polychlorosubtilin.

Polychlorosubtilin (PCS) inhibits the growth of Escherichia coli. The antibiotic affected neither respiration nor glycolysis while the synthesis of nucleic acids and proteins were feebly hindered. Formation of aminoacyl-tRNA, peptide bonds and translocation from A to P sites of ribosomes were insignificantly influenced by the drug. The antibiotic exerted its effect(s) on ribosomes by interfering with the 30 S subunits. The 23 S and 30 SP were both sensitive to the drug but the latter was more obviously affected. Changes after developing resistance to the drug by the bacteria were localized in the 30 SP, 23 S and accordingly the 30 S subunits. The principal action of PCS was to cause multisited miscoding upon the incorporation of labeled aminoacyl-tRNA, therefore, malformed protein fractions (abnormal) were synthesized. As a natural consequence such abnormal fractions would not be expected to manifest the vital metabolic activities in the normal way.

Both positional isomers of aminoacyl-tRNA's are bound by elongation factor Tu.

Six purified Escherichia coli and yeast tRNA's were converted to positionally defined tRNA's terminating in 2'- and 3'-deoxyadenosine; the modified (amino-acyl) tRNA's were compared for their abilities to bind to elongation factor Tu (EF-Tu) in the presence both of GTP and guanylylimidodiphosphate (GMP-P(NH)P). Formation of aminoacyl-tRNA . EF-Tu . guanine nucleotide ternary complexes was monitored by gel filtration on Sephadex G-100 and Ultrogel ACA 44 columns and also by measurement of the ability of the factor to diminish the rate of chemical hydrolysis of the aminoacyl-tRNA's. The apparent positional specificity of the factor was found to be affected substantially both by the choice of guanine nucleotide and gel filtration resin utilized, but not in any systematic fashion. Likewise, assay of ternary complex formation by diminution of the rate of chemical deacylation failed to reveal any consistent positional preference from one isoacceptor to another. It is worthy of note that each modified aminoacyl-tRNA tested did form a ternary complex with EF-Tu under each of the experimental conditions used for assay, but that in each case the difference in affinity of the factor for isomeric aminoacyl-tRNA's was less than that between either of the modified aminoacyl-tRNA's and the corresponding unmodified species. On the basis of the experiments performed, we conclude that (i) EF-Tu has remarkable conformation flexibility, possibly reflecting its physiological role in recognizing 20 tRNA isoacceptors and (ii) the factor has no obvious preference for a single positional isomer of aminoacyl-tRNA and it is not clear that any preference that might exist could be established convincingly using tRNA's terminating in 2'- and 3'-deoxyadenosine.

The determination of binding parameters from protection experiments: A quantitative assay for ternary complex formation of elongation factor Tu, GTP, and aminoacyl-tRNA

A procedure is described by which binding parameters for the interaction between proteins and their substrates can be derived from protection experiments. The only prerequisite for its use is that the interaction of protein and ligand leads to a measurable change in the rate of that reaction that leads to the removal of one of the reactants. The applicability of this procedure is demonstrated for the binding of aminoacyl-tRNAs to the elongation factor Tu. It involves the determination of the rates of hydrolysis for free and complex-bound aminoacyl-tRNA, as well as the evaluation of initial rates in a series of protection experiments with varying concentration of protein and nucleic acid. The experimental data are used for a Scatchard plot.

Effect of phenylpyruvate and homogentisate on the formation of aromatic aminoacyl-tRNAs

Effect of phenylpyruvate and homogentisate on the formation of aromatic aminoacyl-tRNAs

Role of divalent cations and polyamines in aminoacyl-tRNA formation

Role of divalent cations and polyamines in aminoacyl-tRNA formation

Aminoacy1 Transfer RNA Formation. VI. Binding of Cations to Transfer RNA and Its Role in Aminoacy1 Transfer RNA Formation1

The role of cations (polyamines and Mg2+) in isoleucyl-tRNA formation catalyzed by purified isolecuyl-tRNA synthetase [EC 6.1.1.5] from Escherichia coli was studied. It was found that spermine, spermidine, and Mg2+ bind to tRNA and that when bound to these cations, tRNA acts as substrate of aminoacylation without requiring further cations. These findings suggest that the primary function of cations in aminoacyl-tRNA formation is to bind to tRNA to stabilize its structure, not to bind to the enzyme to activate it.

Interaction of aminoacyl-tRNA synthetases and tRNA: Positive and negative cooperativity of their active centres

The influence of tRNA on the kinetics of PP-ATP exchange and aminoacyl-tRNA formation catalysed by leucyl-, phenylalanyl-, and tryptophanyl-tRNA synthetases has been investigated. These enzymes were chosen because they belong to three main classes of quaternary structure alpha1, alpha2beta2 and alpha2, respectively. The present paper shows that the investigated synthetases manifest kinetic cooperativity of the active centres which is negative in the case of AAA formation and positive in the case of leucyl- and tryptophanyl-tRNA synthesis. The obtained data were interpreted with the aid of the trigger model of the enzyme.

A low-resolution model of crystalline methionyl-transfer RNA synthetase from Escherichia coli

When submitted to a controlled proteolysis by trypsin, native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) yields a well-defined fragment of molecular weight 64,000 composed of one single polypeptide chain. This fragment retains full specificity towards methionine and tRNA met, and has unimpaired activity in both the activation reaction and aminoacyl-tRNA formation. Crystals of this active fragment have been studied by X-ray crystallography and, using two isomorphous heavy-atom derivatives, a 4 Å electron density map has been calculated. The molecule appears as an elongated ellipsoid of overall dimensions 90 Å × 43 Å × 43 Å. It is clearly built of two parts separated by a large cleft. The volume of one of these “domains” is approximately twice that of the other; these results are consistent with our present knowledge of the chemistry of the protein.

In vitro protein synthesis at elevated temperature by an extract of an extreme thermophile : Effects of polyamines on the polyuridylic acid-directed reaction

1. 1. It was found that spermine stimulated phenylalanine incorporation by a cell-free extract of Thermus thermophilus at the high, physiological temperature of this extreme thermophile. Without spermine the reaction stopped at an early stage, resulting in poor total incorporation. Addition of spermine increased the rate and the saturating level of phenylalanine incorporation at 50–80 °C. 2. 2. Spermine and a native polyamine extracted from the thermophile were the most effective of the amines tested so far. Spermidine was less effective and other polyamines including putrescine had no effect on the incorporation. The optimum Mg 2+ concentration was lowered to 6–10 m m by addition of 3 m m spermine. 3. 3. Spermine was required in the formation of a ternary complex of 70S ribosomes, polyuridylic acid and phenylalanyl-tRNA and also for chain elongation in polyphenylalanine synthesis at high temperature. Putrescine inhibited the stimulating effect of spermine for the former step. Amino acyl-tRNA formation was not affected by the addition of polyamine. The action of the native polyamine extracted from the thermophile resembled that of spermine. 4. 4. It was also found that preincubation in the cold enhanced polyuridylic acid-directed incorporation of phenylalanine by the thermophile extract without spermine at high temperature. The mechanism of enhancement by preincubation in the cold was compared with that by addition of spermine.

Effect of Alcohols on Polypeptide Chain Elongation and Aminoacyl-tRNA Formation

The effect of alcohols (methanol, ethanol, and propanol) on polypeptide chain elongation was studied. In the E. coli and rat liver cell-free systems, the optimal concentration of Mg2+ decreased with increase of ethanol concentration, although the maximum polyphenylalanine synthesis decreased. Methanol had almost the same effect as ethanol. Propanol decreased the optimal magnesium concentration, but polyphenylalanine synthetic activity was markedly decreased. The shift of optimal Mg2+ concentration by ethanol was also observed in polylysine and polysome-dependent polypeptide syntheses. Even in the presence of spermidine, ethanol caused the shift of optimal Mg2+ concentration. Ribosome-bound Mg2+ was decreased by the addition of ethanol. A study of the effect of alcohols on aminoacyl-tRNA formation with ten amino acids in the absence of added Mg2+ showed that the formation of arginyl-, leucyl-, and valyl-tRNA was stimulated by the alcohols. Valyl-tRNA formation in the presence of alcohols was completely inhibited by EDTA, while that in the presence of Mg2+ was inhibited slightly by EDTA. No PP1-ATP exchange was observed when alcohol was used as the only stimulant of valyl-tRNA formation.

Binding of transfer rna to polyamines in preference to Mg 2+

Binding of spermine and spermidine to tRNA are preferential to that of Mg2+. Polyamines displace Mg2+ bound to tRNA while Mg2+ does not displace previously bound polyamines. These results suggest that polyamine-tRNA complexes can exist in vivo and can act as substrates in aminoacyl-tRNA formation.

Role of N-acetylamino acids in cerebral protein synthesis. II. Incorporation of radioactivity from labelled acetyl and aminoacyl moieties into RNA

Radioactivity from both [ 3H]acetate and [ 14C]aspartate was incorporated into immature rat brain RNA in vivo indicating active de novo RNA synthesis. The incorporation into RNA was 25–30-fold lower than incorporation into protein. In contrast to acetate and aspartate, the acetyl and aspartyl moieties of N-acetylaspartate did not contribute their 3H- and 14C-labels to RNA to a significant extent. This was in conformity with earlier observations based on protein synthesis studies that the metabolic pools receiving acetyl and aspartyl groups from these 2 sources had different dynamic characteristics. Formation of aminoacyl-tRNAs was measured using tRNA, pH 5 enzymes and purified aminoacyl-tRNA synthetases from rat and calf brains in homologous as well as heterologous combinations. Aspartate, methionine, leucine and phenylalanine were incorporated into tRNA at a high level, but no incorporation of N-acetylaspartate was observed under any of the experimental conditions employed. Further, aspartyl- and methionyl-tRNAs were not acetylated by acetate or acetyl-CoA. These results suggest that N-acetylaspartate, which occurs in a high concentration in nervous tissue, is not the initiating amino acid for the synthesis of at least a large majority of brain proteins.

Necessity of polyamines for maximum isoleucyl-tRNA formation in a rat liver cell-free system

From a study of the effect of polyamines on aminoacyl-tRNA formation of nine amino acids in a rat liver cell-free system, it is shown that isoleucyl-tRNA formation in the presence of polyamines is much greater than that in presence of Mg 2+. The data suggest that polyamines may play an important role in protein synthesis by regulating aminoacyl-tRNA formation.

Phenylalanyl-tRNA synthetase from yeast. Steady-state kinetic investigation of the reaction mechanism.

It has been shown that the initial rates, v, of the formation of aminoacyl‐tRNA, catalyzed by phenylalanine‐tRNA synthetase, do not depend on the concentration of the substrates ATP and phenylalanine in a simple way. In contrast a normal Michaelis‐Menten behaviour has been observed for the substrate tRNA. The reciprocal plots of v versus v/[S] for ATP and phenylalanine are curved and only approach linearity at low and high concentrations of the varied substrate. From these limiting slopes two Km and two V values corresponding to low and high concentrations of ATP and phenylalanine have been determined. Since the reciprocal plots for tRNA are linear, only one Km and V value has been obtained for this substrate. In order to show that this kinetic behaviour is an intrinsic property of the enzyme and not an artifact, a new procedure for the purification of the enzyme has been devised and the purity and stability of the preparation carefully checked. In addition the influence of ammonium ions and of spermine on the initial rates has been measured to verify that the non‐linearity of the reciprocal plots is not the result of the substrate acting as a base. Inhibition experiments with tRNAox (a modified tRNA obtained from native tRNA by periodate oxidation of the 2′:3′‐diol in the 3′‐terminal ribose to the corresponding dialdehyde) at low and high phenylalanine concentrations have shown that it is also not a consequence of two fundamentally different reaction mechanisms being followed at the two concentration ranges. On the basis of this and other evidence it is proposed that two mutually interacting sites are engaged, of which one or both are operative, depending on the concentration of the substrates. Studies of pyrophosphate inhibition at low tRNA concentration have provided evidence that the aminoacylation of tRNA proceeds via two catalytic steps in which aminoacyl‐AMP occurs as an intermediate. At high tRNA concentrations it has been found that this substrate is adding prior to the dissociation of pyrophosphate.

Anti-anabolic effects of adenosine 3':5'-cyclic monophosphate. Inhibition of protein synthesis.

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.

Untersuchungen über das Verhalten eines während der Keimung von Agrostemma -Samen gebildeten Inhibitors der Aminoacyl-tRNS-Synthetasen

Summary An inhibitor of aminoacyl-tRNA synthetase activity is described which is produced during germination of Agrostemma seeds. This substance inhibits the ATP-PP exchange reaction as well as the aminoacyl-tRNA formation. All amino acid-specific synthetases (from animals as well as from bacteria) are inhibited, whereas other enzymes are not influenced. By increasing the ATP-concentration the action of the inhibitor can be eliminated. A competitive inhibition mechanism could be excluded. The molecular weight of the inhibitor amounts to 2000 to 4000 daltons. The inhibitor contains sugars with reducing groups, but seems not to contain nucleotides or peptides. While the activity of the synthetases already existing in the dry embryo is only slightly inhibited, there is a strong increase of inhibition of the synthetase activity measured after germination.

Anomalies in the tRNA-aminoacylation reaction which could lead to misinterpretation of evidence for tRNA changes during development and aging

Anomalies in the tRNA-aminoacylation reaction are described and discussed. These include enzyme-dependent tRNA-acylation plateau values at low enzyme to tRNA ratios in the presence of Tris buffer, and the attainment of tRNA-acylation maxima at high enzyme to tRNA ratios. The enzyme-dependent plateaus are believed to represent the steady state resultant of simultaneously occurring enzymatic and non-enzymatic deacylation processes with the acylation reaction. The failure to maintain a level plateau at high enzyme to tRNA ratios is ascribed to another competing reaction — the hydrolysis of ATP to AMP and pyrophosphate (PPi) which is not dependent upon aminoacyl-tRNA formation. Tris buffer appears to accelerate non-enzymatic deacylation of tRNA thus leading to plateau values representing (at low enzyme to tRNA ratios) incomplete charging of tRNA. The relative balance of these competing reactions may be favorably manipulated by avoiding Tris-type buffers and by altering the enzyme to tRNA ratio to obtain an optimal acylation plateau. If increasing the enzyme concentration (keeping the tRNA concentration constant) leads to increasing tRNA acylation plateaus, this is evidence that one is operating below the optimal ratio; if increasing enzyme concentration leads to decreasing plateaus or to charging maxima (no plateau) it is evidence that one is operating above the optimal ratio. Since these precautions have not ordinarily been considered, some reports of changes in tRNA and/or aminoacyl-tRNA synthetase complements during development and aging may be inaccurate.