Yeast Alanine Transfer RNA Research Articles

Chemical modification of yeast alanine transfer ribonucleic acid with a radioactive carbodiimide.

Chemical modification of yeast alanine transfer ribonucleic acid with a radioactive carbodiimide.

Electron microscopic study of base sequence in nucleic acids VI. Guanine sites in yeast alanine transfer RNA

Yeast alanine t-RNA was reacted with 2-diazo-p-benzene-disulphonic acid, which is known to attach with high specificity to the guanosine monophosphate nucleotides. The guanine marked RNA molecules were extended on thin carbon film, stained with uranyl acetate, and examined in the electron microscope. In high resolution micrographs most of the markers made visible by the uranyl acetate staining, could be distinguished from the background noise on the basis of contrast and size. They were found in linear arrays with a length not exceeding that of an extended t-RNA molecule and their number not exceeding that expected from chemical reactivity data as well as from the known number of guanosine residues in ala t-RNA molecule.

Reinvestigation of the primary structure of yeast alanine tRNA

AbstractThe primary structure of alanine transfer RNA reported by Holley and his colleagues contained a number of inconsistencies between the data and the proposed sequence. In order to resolve these inconsistencies, alanine tRNA was isolated from yeast by using countercurrent techniques similar to those employed by Holley. The alanine accepting RNA obtained by this procedure was found by further purification employing reverse phase partition chromatography, to contain two different species of alanine tRNA. One of these, tRNAala I, could be shown to be similar to Holley's proposed structure with the addition of a guanine between positions 47 and 48 and only 2 dihydrouridines instead of the 2.5 reported by Holley.

The modification of nucleosides and nucleotides: X. Evidence for the important role of inosine residue in codon recognition of yeast alanine tRNA

Fractionated alanine tRNA of baker's yeast was treated with acrylonitrile in the presence of 0.5 M NaCl or 2 mM MgCl 2. It was found that inosine residue was the most reactive with the reagent, while one of the two pseudouridine (Ψ) residues, which was contained in CpMeIpΨpGp sequence was cyanoethylated slowly. The other Ψ residue in TpΨpCpGp sequence was entirely resistant to the modification in both the conditions. The alanine tRNA thus modified in the presence of 2 mM MgCl 2 for 10 h, in which 1 mole of inosine and 0.4 mole of Ψ residues contained in UpIpGpCpMeIpΨpGp sequence were cyanoethylated, was fully active in the alanine acceptor activity, but almost inactive in the binding ability to ribosomes in the presence of GpCpU, GpCpC and GpCpA. The tRNA, cyanoethylated in the presence of 0.5 M NaCl, revealed the similar properties as those found for the tRNA modified in 2 mM MgCl 2. Thus, the experimental evidence is provided for the hypothesis that inosinecontaining sequence, IpGpCp, is anticodon of yeast alanine tRNA.

Transfer RNA, I. Isolation and characterization of a new yeast alanine transfer RNA.

Transfer RNA, I. Isolation and characterization of a new yeast alanine transfer RNA.

Studies on polynucleotides, 88. Enzymatic joining of chemically synthesized segments corresponding to the gene for alanine-tRNA.

A principal aim of current work in this laboratory is the development of methods for the synthesis of long-chain bihelical DNA's with specific nucleotide sequences. An immediate objective is the total synthesis of the gene for yeast alanine transfer RNA (tRNA).1 2 In approaching this problem, we wanted to exploit the ability of polynucleotide chains to form ordered bihelical complexes by virtue of base-pairing. Thus, it was hoped to join end-to-end relatively short chemically synthesized deoxyribopolynucleotides while these were held together in properly aligned bihelical complexes. In chemical work, syntheses of two icosanucleotides and several shorter deoxypolynucleotide chains corresponding to segments of the ala-tRNA gene (Fig. 1) have recently been completed.3 For the joining reactions, the recently discovered DNA-joining enzymes4 appeared to be highly promising, and a recent study5 on them using deoxyribopolynucleotides with repeating nucleotide sequences showed that very short oligonucleotide chains were adequate for the joining reaction to occur. In extending this work, we have now demonstrated the enzymatic joining of the chemically synthesized deoxyribopolynucleotides with specific sequences (Fig. 1) when these are provided in appropriate combinations. The present paper documents these findings. Materials and Methods.-Deoxyribopolynucleotides: All of the deoxyribo-, oligo-, and polynucleotides shown in Figure 1 are chemically synthesized products. Except for the tetranucleotide, d-T-C-T-C,6 syintheses of these polynucleotides remain to be published.3 In the incubation mixtures for the DNA-joining enzymes, only the short oligonucleotidic components, e.g., d-T-C-T-C, d-T-C-T-C-C, contained 5'-phosphate end groups and these were labeled with P32 as shown in Figure 1. The 5'-P32-labeled products were prepared by phosphorylation of the 5'-OH groups using y-P32-labeled adenosine 5'-triphosphate (ATP) and polynucleotide kinase as described previously.5 We are grateful to Dr. S. Chang for preparation of the y-labeled ATP and of the polynucleotide kinase. Assay of the joining reactions and isolation of the products: The standard incubation mixture (0.015-0.06 ml) contained per ml: 8 mM tris(hydroxymethyl)aminomethane (Tris)-Cl buffer, pH 7.6, 5 mM MgCl2, 64 AiM ATP, 8 mM dithiothreitol, 0.4 OD2E each of Icosa-I and Icosa-1I, and equivalent amounts of 5'-P32-labeled short oligonucleotides. In the experiments with T4-joining enzyme, the concentration of the enzyme was 1100 units/ml and in some experiments subsequent additions of comparable level were made. The E. coli joining enzyme was used at 200 units/ml. (Both enzymes were tested for specific activity side by side by usinlg the assay described previously.5) Inicubations were at 5-20g. Aliquots (I -5 Al) rernioved at differernt intervals were t,akent to 0.08 ml with (.1 M Tris-Cl buffer, pH 8.0, aad after the solutions had beent boiled at 100' for 2 inlIl tlhev were inieubate(d with 25 Ag of bacteriat alkalinie phosphlatase at 70' for 30 min. The mixtures were then applied to O-(diethylaminoethyl)cellulose paper strips for chromatography and the strips were counted for radioactivity as described before.5 The joined product which remained at the origin was eluted with 1 M triethylammonium bicarbo

Studies on the secondary structure of yeast alanine tRNA: Reaction with N-bromosuccinimide and with nitrous acid

Studies on the secondary structure of yeast alanine tRNA: Reaction with N-bromosuccinimide and with nitrous acid

Analyses of Large Oligonucleotide Fragments Obtained from a Yeast Alanine Transfer Ribonucleic Acid by Partial Digestion with Ribonuclease T1

Abstract Procedures are described for the isolation of large oligonucleotide fragments after partial digestion of a yeast alanine transfer ribonucleic acid with Taka-Diastase ribonuclease T1. Quantitative analyses of the large fragments are presented. This paper completes the description of experimental methods used in the determination of the nucleotide sequence of this alanine transfer ribonucleic acid.

Nucleotide Sequences in the Yeast Alanine Transfer Ribonucleic Acid

Nucleotide Sequences in the Yeast Alanine Transfer Ribonucleic Acid

The effect of ribonucleic acid and its hydrolytic products and of desoxyribonucleic acid on succinic dehydrogenase and cytochrome oxidase : Charles Zittle ( Journal of Biological Chemistry, 162: 287, 1946)

In the reaction of yeast alanine transfer RNA with nitrous acid, interior nucleotides deaminate relatively slowly with the order of reactivity CMP > AMP ≅ GMP instead of GMP > AMP > CMP, the order found for mononucleotides. Approximately 20% of the cytidylic acid residues in yeast tRNAAla are deaminated before there is detectable reaction of guanylic acid or interior adenylic acid residues. The 3′-terminal adenylic acid residue is reactive, and this reaction leads to the loss of alanine acceptor activity. If the terminal adenylic residue is missing from the RNA, deamination of the 3′-terminal cytidylic acid leads to loss of acceptor activity for adenylic acid, as well as alanine. Interior deamination at certain positions leads to loss of alanine acceptor activity, but yeast tRNAAla deaminated in the anticodon or in the “dihydrouracil loop” is still active. Deamination in the anticodon, which changes the IGC sequence to IGU, alters the specificity of the transfer reaction of tRNAAla to that expected for tRNAThr.