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The extraction and purification of a cysteine transfer ribonucleic acid from baker's yeast.

ABSTRACT: 1. A modification of the RPC 1 system of A.D. Kelmers, G.D. Novelli & M.P. Stulberg (1965) (J. Biol. Chem. 240, 3979-3983) is described in which the support medium is a Celite of narrow range particle size treated with dichlorodimethylsilane. 2. By using this system an apparently pure preparation of tRNA Cys was isolated from baker's yeast tRNA. 3. This preparation accepted at least 60% of the theoretical quantity of [3-14C]cysteine in a conventional assay and failed to accept isoleucine, phenylalanine, proline, serine or tyrosine. 4. A theoretical countercurrent-distribution curve calculated by assuming a distribution coefficient K of 2.03 was in excellent agreement with the profiles of E260 and cysteine-acceptor ability after 537 transfers in the 1.85 M-phosphate/formamide/propan-2-ol system of C.M. Connelly & B.P. Doctor (1965) (J. Biol. Chem. 241, 715-719). 5. Chromatography of tRNA Cys on Bio-Gel P100 polyacrylamide beads afforded two components one of which was far less efficient than the other in accepting cysteine. The base compositions of the two were similar.

SUBMITTER: Holness NJ

PROVIDER: S-EPMC1172590 | biostudies-other | 1976 Feb

REPOSITORIES: biostudies-other

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The nucleotide sequence of cysteine transfer ribonucleic acid from baker's yeast. Products of complete digestion with pancreatic ribonuclease and ribonuclease T1.

Project description:1. The nucleotide chain of tRNA Cys from baker's yeast was readily split at the anticolon into two large fragments by brief treatment with ribonuclease T1.2. The whole molecule and the two derived large fragments were completely digested with (a) pancreatic ribonuclease and (b) ribonuclease T1. The fragments present in each of the digests were separated and sequenced by conventional methods. 3. The groups of fragments derived from the two methods of digestion were entirely compatible with each other. 4. The molecule is 75 nucleotides long, but, as isolated, lacks the terminal adenosine and the neighboring cytidylic acid residue. The minor nucleotides 1-methyladenylic acid, 7-methylguanylic acid, 5-methylcytidylic acid and N6 (gamma gamma-dimethylallyl)adenylic acid (isopentenyladenylic acid) were identified.

| S-EPMC1172591 | biostudies-other

The nucleotide sequence of cysteine transfer ribonucleic acid from baker's yeast. Identification of the products from partial degradation of the molecule and derivation of the complete sequence.

Project description:1. A series of large oligonucleotide fragments derived from tRNA Cys, were separated chromatographically and the sequence of each was deduced by examination of the products of digestion with pancreatic and T1 ribonucleases. 2. The location of the specific cleavage points in the nucleotide chain was similar to that produced by brief treatment with pancreatic ribonuclease. 3. The fragments could be arranged into two alternative sequences. The correct sequence was deduced by the sequential removal and identification of the first nine nucleotides from the 3'-end of the terminal half of the molecules.

| S-EPMC1172592 | biostudies-other

Studies on the fractionation of total transfer ribonucleic acid and purification of an alanine transfer ribonucleic acid from chick embryo.

Project description:Chick embryo tRNA, prepared by a simple large-scale method, was fractionated on three different ion-exchange columns. In all cases simple chromatographic patterns for various tRNA species were observed, indicating the presence of only a few major species of tRNA for each amino acid. By repeated chromatography one species of alanine tRNA was purified to approx. 80% purity. T(1) ribonuclease digest of this purified tRNA gave a simple chromatographic pattern. Because of the simplicity of the method of preparation of tRNA from this readily available source and the presence of only a few species of tRNA for each amino acid, chick embryo is suited for the study of tRNA and its various functions in higher systems.

| S-EPMC1168169 | biostudies-other

The infrastructure of valyl-transfer Ribonucleic Acid synthetase from yeast.

Project description:Valyl-tRNA synthetase from yeast is shown to contain a significant proportion of repeated sequence, similar to the enzyme from bacterial sources.

| S-EPMC1165392 | biostudies-other

The extraction and assay of aminoacyl-transfer-ribonucleic acid synthetases of tobacco leaf.

Project description:1. Aminoacyl-transfer-RNA synthetase activity in extracts prepared from tobacco leaf was increased 3-5-fold when sodium thioglycollate (30mm) and magnesium chloride (16mm) were included in the extraction medium. Omitting sucrose (0.45m) from the extraction medium did not alter the activity. 2. Activity was a linear function of enzyme concentration up to 1 disk (30mg. fresh wt.)/ml. and was not affected by dialysis at any concentration. 3. Activity increased about 13-fold above control values when a mixture of 21 amino acids and amides (1mm) was added to the reaction mixture. 4. Under the conditions used in the standard assay for aminoacyl-transfer-RNA synthetase activity K(m) (ATP) was 0.65mm and K(m) (l-amino acids) was 70mum. 5. Activity above the control value was found with all amino acids and amides tested except alanine, arginine, glutamic acid, glutamine and hydroxyproline. Activity was highest with leucine, isoleucine, valine, cysteine and histidine. Total activity with a mixture of 21 amino acids and amides was 20% lower than the total activity of the enzymes assayed separately.

| S-EPMC1270057 | biostudies-other

The separation of oligonucleotides of baker's-yeast value transfer ribonucleic acid 2b by high-voltage electrophoresis on DEAE-paper and by thin-layer chromatography.

Project description:A modified procedure for the separation of oligoribonucleotides is described that is based on the combination of t.l.c. on cellulose and electrophoresis on DEAE-paper at 4000 V on a cooling plate. The technique is relatively rapid and allows the analysis of larger quantities than is possible by electrophoresis on cellulose acetate.

| S-EPMC1164718 | biostudies-other

Sugar stress during fermentation (baker's yeast)

Project description:Functional genomic analysis using different types of baker's yeast. Keywords: fermentation

2007-02-23 | GSE4295 | GEO

Purification and properties of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli.

Project description:1. Methionyl-t-RNA synthetase (where t-RNA denotes ;soluble' or transfer RNA) has been purified to apparent homogeneity from a ribonuclease I-free strain of Escherichia coli. Polyacrylamide-gel electrophoresis of the final product revealed a single band. The purified enzyme catalyses the exchange of 450mumoles of pyrophosphate into ATP/mg. in 15min. at 37 degrees . 2. Methionyl-t-RNA synthetase is specific for the l-isomer of methionine, but appears to catalyse the methionylation of two distinct species of t-RNA, both of which are specific for methionine, but only one of which may be subsequently formylated. 3. The Michaelis constant for l-methionine is 2x10(-4)m in the ATP-PP(i) exchange assay and 2x10(-5)m for the acylation of t-RNA. 4. Gel filtration of both crude and highly purified preparations of methionyl-t-RNA synthetase on Sephadex G-200 indicates that the active species of enzyme has a molecular weight of about 190000. The amino acid composition of the enzyme is similar to those reported for the isoleucine and tyrosine enzymes from E. coli.

| S-EPMC1198268 | biostudies-other

Mineralization induced by phosphorylated dry baker's yeast.

Project description:We found the mineralization of Cu during long-term Cu2+ adsorption onto dry baker's yeast cells phosphorylated using sodium cyclo-triphosphate. Field emission scanning electron microscopy (FESEM) with energy-dispersive X-ray spectroscopy confirmed that the elemental composition of minerals were copper, phosphorus, and oxygen. Synchrotron-based X-ray absorption fine structure showed that the local structure around Cu atoms deposited on the mineral was almost identical to that of commercial copper (II) phosphate Cu3(PO4)2∙3H2O. However, the crystallinity was low, and the structure was slightly distorted. Time profile analysis using FESEM revealed that copper phosphate mineralization was first apparent on Day 3 of adsorption, whereas mineral formation plateaued at around Day 7. It seems that mineralization occurs by the local saturation of phosphate and Cu2+ on the yeast cells. Mineralization of the rare earth ion Dy3+ was also demonstrated during long-term adsorption. Mineralization on phosphorylated yeast cells appears to follow a common path for various types of metal ions and provides a promising technique for metal recovery via irreversible adsorption.

| S-EPMC7518573 | biostudies-literature

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

Project description:The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

| S-EPMC1183673 | biostudies-other

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