Aminoacyl Synthetases Research Articles

Aminoacylation of Escherichia coli cysteine tRNA by selenocysteine

Unfractionated Escherichia coli B tRNAs have been aminoacylated with selenocysteine by using homologous aminoacyl synthetases. Cochromatography of [ 3H]cysteyl-tRNA and [ 75Se]selenocysteyl-tRNA on reverse-phase chromatography-5 columns revealed nearly coincident radioactive elution profiles for the two charged tRNAs. Acylation of a mixture of tRNAs with cysteine protected selenocysteine-acceptor activity from inactivation by periodate oxidation. Likewise, preacylation with selenocysteine protected cysteine acceptor from oxidation. Levels of charging with cysteine are reduced about 50% by the presence of a 40-fold excess of selenocysteine. These results indicate that selenocysteine is bound to cysteine-accepting tRNAs, although it does have considerably lower affinity for the ligase than cysteine. The ester linkage of selenocysteyl-tRNA was shown to be somewhat more stable than that of cysteyl-tRNA under the same conditions. These experiments show that selenocysteine can participate in the early steps leading to peptide-bond formation and provide a possible pathway for selenocysteine incorporation into protein.

The three-dimensional structure of yeast phenylalanine transfer ribonucleic acid and its interaction with aminoacyl synthetases.

The three-dimensional structure of yeast phenylalanine transfer ribonucleic acid and its interaction with aminoacyl synthetases.

Transfer RNA and protein synthesis

The three-dimensional structure of yeast phenylalanine transfert RNA has been determined in orthorhombic crystals. The current status of this work is reviewed together with the relationship of the transfer RNA structure in the crystal to its biologically active form. In addition some speculations are put forward regarding the mode of interaction of tRNA molecules in the ribosome and the manner in which tRNA interacts with aminoacyl synthetase.

Conformation and functioning of tRNAs: cross-linked tRNAs as substrate for tRNA nucleotidyl-transferase and aminoacyl synthetases

Summary The behaviour of mixed E. coli tRNAs «cross-linked by irradiation with near ultraviolet light (310–400 nm) has been compared to that of the intact molecules in two enzymatic processes. No change in the rate and extent of the repair of the pCpCpA 3′ terminus of tRNA by purified E. coli tRNA nucleotidyltransferase can be detected. In contrast, complex data were obtained in the acylation reaction. They can be understood using other tRNA specific modifications as well as our present knowledge of E. coli tRNA sequences and rare base content. Hence: 1) Alteration of the acylation rate for a given amino-acid in general reflects the ability of the corresponding tRNA(s) to be «cross-linked. Yet tRNAs acylated by Lys, Glu and one Gln isoacceptor are specifically inactivated by 310–350 nm light: this effect corresponds to a target distinct fom 4-thiouridine. 2) Introduction of the 8–13 link variably affects the rate of acylation according to the nature of the amino-acids. The results obtained here under uniform conditions parallels those already described with some purified systems. 3) All the tRNAs known to contain 4-thiouridine 8 and cytidine 13 in their sequence (i. e. the tRNA respectively acylated by Trp, Phe, Arg, Val, Asp, Gly, Met, His) can be cross-linked. On the basis of their performance in the acylation test the tRNAs of unknown sequences (or at least the major corresponding isoacceptors) respectively acylated by Pro, Lys, Asn and Thr appear to be cross-linkable. 4) Since all cross-linked tRNAs remain functional (with the possible exceptions of the Phe and Pro tRNA species) and since no evidence for an eventual repair of the 8–13 link was detected, it can be concluded that no large separation of the 8 and 13 residues is required in the normal functioning of the tRNAs in the processes examined here.

Specific Degradation of a Plant Leucyl Transfer Ribonucleic Acid by a Factor in the Homologous Synthetase Preparation

Partially purified aminoacyl synthetase preparations from pea roots (Pisum sativum L. var. Alaska) contain a heat-labile factor which can degrade leucyl-tRNA(6) (leu) to a new species. The singular electrophoretic and chromatographic mobilities, the isoprenoid nucleoside content, and the charging characteristics of the new species (designated leucyl-tRNA(L) (leu)), suggest that it is a fragment of tRNA(6) (leu) containing at least that portion of the original molecule extending from the 3' terminus to the anticodon. Conversion appears to be highly specific since neither bulk tRNA, the other leucine tRNA subspecies, nor tyrosine, phenylalanine, or tryptophan tRNAs are susceptible to degradation during incubation with the synthetase preparation.

Activity of sRNA amino acyl synthetases in ontogenic development of rabbit brain.

The purpose of the study was the estimation of the changes in the activity of 20 individual sRNA amino acyl synthetases in the newborn, 12th, 30th, 48th and 180th days old rabbit brain. The investigations showed that the highest enzyme activity was found in the pH 5 fraction obtained from the CNS of newborn rabbits. In the further period of brain’s development there was a decline in the activity of amino acyl sRNA synthetases beginning from the 12th day of extrauterine life and continuing until the animals were 48 days old, when the activity approximated that measured in the adult rabbit brain. The activity of these enzymes in the developing brain was higher in the cerebral cortex than in the white matter.

TRNA and tRNA aminoacyl synthetases of chloroplasts, mitochondria and cytoplasm from Euglena gracilis

Purified chloroplasts from Euglena gracilis contain specific tRNAs and aminoacyl synthetases different from those found in the mitochondrial and cytoplasmic fractions. Examination of isoleucyl-tRNA by benzoylated DEAE-cellulose (BD-cellulose) chromatography reveals five isoleucyl-tRNA species, one associated with the chloroplast, one with the mitochondria and three with the cytoplasm. Chloroplast isoleucyl-tRNA can be aminoacylated only by chloroplast synthetase. Isoleucyl-tRNAs from mitochondria or from the cytoplasmic fraction can be aminoacylated by either mitochondrial or cytoplasmic aminoacyl synthetases.

A rapid technique for the analytical and preparative isolation of transfer RNA from reaction mixtures

The isolation of enzymically modified tRNA's from enzyme reaction mixtures is usually achieved by one of two general methods. Amino-acyl synthetase reactions are easily assayed by filter paper techniques (1) in which the tRNA is precipitated on filters of materials such as cellulose ester or glass fiber. After drying, the filters can be counted by several techniques. The limitations of this technique include unknown counting efficiency and loss of the sample. In cases in which recovery of the tRNA is desired the usual procedure involves phenol extraction of the tRNA followed by selective precipitation with salts and/or organic solvents. We wish to report a technique for the rapid isolation of tRNA from reaction mixtures which leads to the quantitative recovery of tRNA essentially free of protein. The assay depends on the selective adsorption to and elution from microgranular DEAE in the presence of low and high salt, respectively.

Capacité de charge et formation du complexe ternaire dans un système acellulaire de foie de rats thyréoprives

The charging activity and the formation of on mRNA-ribosome-tRNA complex in an acellular system from livers of thyroidectomized rats 1. 1. The charging activity of the “pH 5 enzyme” from livers of thyroidectomized rats is lower than that of the “pH 5 enzyme” from livers of normal rats. This decrease in activity is constant during the time course of incubation (5 to 60 min). It does not seem to depend on an isotopic dilution of the labeled amino acids. It is not due to a modification of either the tRNA or the aminoacyl synthetase activities. 2. 2. The formation of the ternary complex, mRNA-ribosome-charged tRNA, is altered by thyroidectomy. The binding of [ 3H]poly(U) to 80-S ribosomes from hypothyroid rats is lower. The attachment of Phe-tRNA on these ribosomes is also inhibited. The enzymatic factors required for the attachment of the charged tRNA on the ribosomes do not seem to be responsible for the alteration of the ternary complex. 3. 3. Preincubation of polyribosomes from control animals in the presence of liver “pH 5 enzyme” from thyroidectomized rats lowers their activity. 4. 4. After treatment of both types of ribosomes by trypsin, the activity of ribosomes from livers of normal rats decreases. The inhibition reaches 50 %. On the contrary ribosomes from livers of thyroidectomized rats are not modified by tryptic treatment. 5. 5. The “pH 5 enzyme” from livers of operated animals does not contain higher proteolytic or ribonucleasic activity. 6. 6. In conclusion in the absence of thyroid hormones liver “pH 5 enzyme” seems to contain one (or more) inhibitory factor(s) which acts on the charging activity of the “pH 5 enzyme” and the ribosome activity.

Isoaccepting transfer rna species in differing morphogenetic states of Rhodopseudomonas spheroides

1. Benzoylated DEAE-cellulose chromatography was employed to compare isoaccepting tRNA species from aerobic and anaerobic photosynthetic cultures of Rhodopseudomonas spheroides. 2. Variation in isoaccepting species were observed with phenylalanine and tryptophan tRNAs which were independent of the source of aminoacyl synthetase. 3. Tryptophanyl-tRNA formed in vivo was chromatographed and found to show similar variations to those observed with in vitro charged tRNA.

Phosphorolysis of tRNA. Multiple conformational states of the tRNA in solution.

Polynucleotide phosphorylase was used as a probe to study the conformation of tRNA in solution. This enzyme degrades polyribonucleotide chains in the presence of orthophosphate into nucleoside diphosphate (phosphorolysis reaction) in a sequential and non-dissociating fashion (“processive” mechanism). All natural and biosynthetic polyribonucleotides, except tRNA, are completely degraded by the enzyme. Only a fraction of tRNA molecules can be phosphorolyzed at low temperatures: thus, at a given temperature some tRNA molecules are completely degraded from the 3′OH end, while the remaining molecules are entirely resistant, and their 3′OH terminal adenosine 5′-phosphate stays intact. This phenomenon has been extensively investigated and the results presented in this article lead to the conclusion that the reaction stops before complete phosphorolysis, not because the enzyme has become inactive, but because the limiting factor resides in the substrates themselves, i.e. tRNA. This property of tRNA is not a result of the formation of aggregates resistant to the enzyme; neither is it due to preferential phosphorolysis of certain species of tRNA, since the same phenomenon is observed with several purified specific tRNAs, as well as with bulk tRNA isolated from Escherichia coli, yeast, or rat liver. The results suggest the existence of two classes of tRNA conformations defined by their ability (S class) or inability (R class) to be phosphorolyzed. These conformations are not identical to the native and denatured forms, as defined by the ability or inability of tRNA to be charged with amino acid by aminoacyl synthetases. The effects of temperature, of mono-or divalent ions, and of denaturing agents were studied. Purine has been found to markedly enhance the percentage of phosphorolysis without any concommittant effect on the enzyme. Pre-heating of tRNA at a temperature higher than 90°C, in the presence of divalent ion can modify the percentage distribution of the two types of molecules. The conformational change of each molecule is temperature-dependent. The apparent non-interconvertibility of the two classes of conformations at low temperature is an unusual dynamic behavior which cannot be explained by a “frozen” equilibrium. It can, however, be accounted for in a multiple equilibrium model with a slow rate of interconversion involving large entropic changes.

Protein synthesis at high temperatures: Aminoacylation of tRNA

This paper presents data on the structure and function of tRNA and aminoacyl synthetases at high temperatures. The aminoacylating system from Thermus aquaticus was found to be more thermostable and biologically active at much higher temperatures than the aminoacylating sytem from Escherichia coli. The optimum temperature for the formation of phenylalanyl- and isoleucyl-tRNA from T. aquaticus was 70°, whereas the optimum temperature in E. coli was 45°. Both the tRNA and the aminoacylating enzymes of T. aquaticus were more thermostable than those of E. coli. The temperature optima in heterologous systems reflected the heat sensitivity of the E. coli component. Both reversible and irreversible effects of heat on the E. coli tRNA could be detected. These data provide further evidence that thermophily is achieved by virtue of inherently thermal stable constituents which have high temperature optima for biological function.

Biosynthesis of polypeptide antibiotics.

Abstract

Preparation of aminoacyl synthetases from higher plants

Preparation of aminoacyl synthetases from higher plants

Pyrophosphate as a selective inhibitor of macromolecule synthesis in normal and leukemic leukocytes

Pyrophosphate (PPi) was shown to inhibit RNA, DNA and protein synthesis of intact human leukocytes and HeLa cells and to inhibit such enzymes as DNA polymerase and amino acyl synthetase. The results with intact leukocytes and the demonstration in inhibition of bacterial growth suggest that PPi is readily transported into the cell and is not efficiently degraded by pyrophosphatase. A possible regulatory role for PPi is suggested.

Die Temperaturabhängigkeit der Aminoacylierungsgeschwindigkeit von Phenylalanin- und Serin-spezifischer Transfer-Ribonucleinsäure aus Brauerhefe

It is known that several physical properties in tRNA * are changed between 35°C and 45°C. These changes have been attributed to unfolding of tertiary structure. In this paper the correlation between these changes and the rate of aminoacylation is studied. With purified tRNASer yeast and tRNAPhe yeast and purified aminoacyl synthetases, aminoacylation reaches a plateau within 20 min. (Fig. 2). The rate of charging was determined in the linear region (5 min.) at varying temperatures. With tRNASer yeast there is a sharp drop in the rate of charging at 39° (Fig. 3 a) corresponding to the first melting of Ser yeast (Fig. 6 a). By changing the Mg2⊕ concentration, rate of aminoacylation and first melting are shifted in the same direction. With tRNAPhe yeast no stepwise decrease of aminoacylation with temperature is observed (Fig. 3 b). At the same time this molecule does not exhibit a two step melting profile (Fig. 6 b). The effects observed cannot be due to a change or irreversible inactivation of the enzyme since the thermal inactivation of the enzyme follows a normal pattern above 43°C and 38.5°C respectively (Figs. 4 a and 4 b). Likewise the amino acid activation reaction is not responsible for these effects. This reaction has been measured by the pyrophosphate formation to have a normal temperature profile up to 50°C. Only at this temperature the enzyme begins to become denatured (Fig. 5). The results indicate that the observed changes in aminoacylation are caused by a change in physical properties of the tRNA. This change yields a molecule having a conformation that can no longer be aminoacylated. Since at the higher temperature still the same plateau is reached, however, at a slower rate, an equilibrium between the two forms must exist. This equilibrium could be of the following manner:

Aminoacyl synthetase and transfer ribonucleic acid patterns in normal and neoplastic tissues

Aminoacyl synthetase and transfer RNA fractions were obtained from normal rat liver and from Novikoff ascites tumor cells. The tRNA's were precharged with H 3 or C 14-labeled amino acids and the labeled aminoacyl tRNA's were resolved by reversed phase column chromatography. Elution profiles of the aminoacyl tRNA's of liver and tumor were similar for arginine, the most extensively studied amino acid. Comparisons of the aminoacyl tRNA patterns revealed that the tumor and liver were almost identical with respect to each of the following amino acids: threonine, histidine, methionine, glutamic acid, and tyrosine. Minor deviations in the aminoacyl tRNA patterns of the two cell types were seen in the valine series and with serine. When liver phenylalanyl tRNA was chromatographed on the reversed phase columns, three peaks were observed. In contrast, the tumor phenylalanyl tRNA was lacking in the first peak present in the liver pattern and the second phenylalanyl tRNA peak of the tumor was displaced. The significance of these findings is considered in terms of the role of the aminoacyl synthetases and tRNA's in cancer induction and in the function of malignant tumor cells.