tRNA Fraction Research Articles

Specific hybridization of tyrosine transfer ribonucleic acids with DNA from a transducing bacteriophage φ80 carrying the amber suppressor gene suIII

Transducing phages of φ80 have been isolated which carry the su III gene. We have used the technique of DNA-RNA hybridization to test the hypothesis that a tRNA Tyr molecule is the direct product of this gene. The following results strongly support this hypothesis: 1. (1) Upon infection of Escherichia coli by φ80 d su III , there is a tenfold increase in the fraction of the tRNA which will hybridize with φ80 d su III DNA. This is to be compared with a similar increase in the fraction of tRNA which will accept tyrosine following infection. 2. (2) Saturation experiments show that there is a single nucleotide sequence (a gene) which will hybridize with tRNA. 3. (3) As tRNA is purified for tyrosine-accepting activity, there is an increase in the fraction of the tRNA which will hybridize with φ80 d su III DNA. This has been shown both in saturation and in competition experiments. Our view of suppression dictates that there be at least two genes specifying tyrosine tRNA's in E. coli. One of the tRNA's, the major species, is not involved in suppression; the other, a minor species, recognizes the normal tyrosine codons in the su − cell, and UAG, a chain-terminating codon in the su + III cell. The present evidence shows that these two tRNA molecules must be very similar, as they both hybridize efficiently with φ80 d su III DNA.

Electrophoretic separation of complexes of aminoacyl-tRNA synthetase and transfer RNA

Stable complexes of tRNA and aminoacyl-tRNA synthetase were demonstrated and isolated from the bulk of unbound protein by sucrose density-gradient electrophoresis, using a crude enzyme preparation from thermophilic bacteria. Glycyl-tRNA synthetase formed a stable complex with tRNA from both the homologous and a heterologous (yeast) species. Valyl- and arginyl-tRNA synthetases also complexed with homologous tRNA, but not, or only weakly, with yeast tRNA, in spite of the cross reactivity of the valine enzyme with the latter. Periodate-oxidized homologous tRNA formed complexes with glycyl- and valyl-tRNA synthetases, but not with arginyl-tRNA synthetase. That the binding between the enzyme and tRNA is specific was shown by the use of two tRNA fractions, one enriched and the other depleted with respect to glycine-accepting activity.

Ribonucleic acid synthesizing activity of spinach chloroplasts and nuclei

Purified chloroplast and nuclei preparations from spinach leaves possess RNA polymerase activity, as measured by the incorporation of 3H-ATP into an acid-insoluble product. In both types of organelle the polymerase reaction is dependent on the presence of all four nucleoside triphosphates, Mg ++, and an ATP-generating system. The reaction is inhibited by RNase, DNase, and actinomycin D. No stimulation was found with spermidine, with ammonium sulfate, or with the plant growth regulators kinetin, indoleacetic acid, and gibberellic acid. RNA polymerase activity was higher in leaf extracts from young plants. The product of the in vitro RNA polymerase reaction was isolated by phenol extraction and sucrose density gradient centrifugation. The chloroplast product had a major peak at about 11S, and was somewhat polydisperse in the range 11–23S. The product of the in vitro nuclear reaction had a maximum at about 8S and was less polydisperse than the chloroplast product. Short-term incorporation of 3H-uridine by intact spinach plants revealed the presence of a rapidly labelled RNA fraction in the chloroplasts with sedimentation properties similar to the product of the in vitro chloroplast polymerase reaction. The corresponding rapidly labelled RNA fraction in the nuclei resembled the in vitro nuclear product but was more polydisperse in the high molecular weight regions of the gradient. In long-term, in vivo incorporation experiments the labelled RNA pattern of both chloroplasts and nuclei corresponded closely to the ribosomal and transfer RNA fractions.

Isolation of “Renaturable” Transfer Ribonucleic Acids

Abstract A new type of fractionating step involving gel filtration is introduced for the purification of certain transfer ribonucleic acids (tRNAs). It is based on the increase that occurs in the molecular dimensions of these tRNAs when they are trapped, reversibly, in a denatured state under conditions where the other tRNA components of the mixture are in the native conformation. This step, coupled to countercurrent distribution, is used to obtain in a near homogeneous state a leucine-specific tRNA that cannot be so purified by countercurrent distribution alone, as several other tRNA species have very similar partition coefficients in the two-phase solvent system employed. The applicability of the same techniques for the partial purification of a methionine-, an arginine-, and a glutamine-specific tRNA from yeast is also demonstrated. The potential of this combination of methods for large scale fractionation of tRNA is discussed.

Studies on Ribonucleic Acid Synthesis in Tadpole Liver during Metamorphosis Induced by Thyroxine: II. ANALYSIS OF RIBONUCLEIC ACID FRACTIONS

Abstract A method of preparing ribonucleic acid from tadpole liver has been described. It was found that tadpole liver RNA is unstable during the isolation procedure. Various factors, including temperature, the concentration of Mg++ in the homogenizing medium, and the presence of ribonuclease inhibitors, were found to be of importance in protecting RNA during its preparation. Three peaks with sedimentation coefficients of 29 S, 18 S, and 6 S were obtained by zonal sucrose density gradient centrifugation. The 6 S RNA was found in the purified ribosomes from mitochondrial and postmitochondrial fractions. The base ratios of 6 S RNA were different from those of the 18 S and 29 S fractions and the bulk RNA, showing in particular a low concentration of UMP. Some properties of RNase from tadpole liver have been described and discussed in relation to the base composition of 6 S RNA. The most rapidly labeled RNA appeared in the transfer RNA fraction when orotic-acid-14C was administered to untreated tadpoles. The RNA heavier than transfer RNA has the highest specific radioactivity 2 days after thyroxine treatment. The base composition of this RNA was analyzed with the use of 32Pi and found to be high in uridine 5'-phosphate (25 to 35%) in spite of the low value of the UMP in bulk RNA in this area. Rapidly labeled RNA which appears in the 6 to 10 S area has the properties of messenger RNA needed for induction of the enzymes in liver associated with metamorphosis.

Specificity in the Structure of Transfer RNA

This chapter discusses the recent work on the specificity embodied in the structure of transfer RNA (tRNA). The chapter also discusses the base composition of tRNA. The numerous analyses of the base composition of bulk tRNA preparations from different sources have been reviewed in this chapter. The various arrangements of nucleotides in tRNA are also discussed in this chapter. Counter-current distribution, column chromatography, and other separation techniques have been used in the fractionation of tRNA's. Some of them are highly specific for one particular amino acid. The base composition of these fractionated tRNA's is characteristic. The arrangement of nucleotides in tRNA, such as (a) nucleotide distribution, (b) anticodon sequence, and (c) terminal nucleotide sequences, are discussed in this chapter. Although a definite tertiary structure of tRNA has not yet been defined, each specific tRNA, which has a specific base composition, may have a specific tertiary configuration, because the melting profiles of a few specific tRNA's differ from each other. The chapter also describes various three-dimensional structure of tRNA. The chapter concludes with the modification of nucleic acid bases, without scission of the polynucleotide chain, and the resulting change in the biological activities of nucleic acids from viruses or bacteria. In order to elucidate the functional sites of tRNA, it seems logical to examine the change in the activity of such modified tRNA.

An analysis of arginine codon multiplicity in rabbit hemoglobin

Two arginyl tRNA's from yeast (Arg tRNA I and Arg tRNA II) are separable by chromatography. Arg tRNA I which binds to the Escherichia coli ribosomes in response to the triplets CpGpU, CpGpC and CpGpA, transfers arginine only into the C-terminal position of the α -chain of rabbit hemoglobin (tryptic peptide α T17, position 141). Arg tRNA II, which binds to E. coli ribosomes in response to the triplets ApGpA and ApGpG, transfers arginine into position 31 of the α -chain (corresponding to peptide α T4). Codon assignments to positions 31 and 141 of the α -chain, based on the utilization of arginine from either of the two tRNA fractions, are consistent with known amino acid replacements at these positions, and could have been produced by a change of a single base. No labeled arginine was transferred into position 92 (corresponding to peptide α T11) nor into the two β -chain arginine positions. The lack of labeling of position 92 may be due to its specification by the triplet CpGpG, since neither of the tRNA fractions was bound to ribosomes in the presence of CpGpG. This codon assignment is consistent with the observed replacement of arginine at position 92 by leucine and glutamine in mutant human hemoglobins. The lower level of β -chains synthesis precluded a determination of the transfer specificity of β -chain arginine residues. Nevertheless, only the triplet ApGpPu at positions 31 and 41 of the β -chain can be interrelated with observed amino acid replacements, by a change of a single base.

Oligo- und Polynucleotidtrennungen: IV. Chromatographie von löslicher Ribonucleinsäure und deren Spaltprodukten an Sephadex

Chromatography of soluble ribonucleic acid and its cleavage products on Sephadex Chromatographic studies on Sephadex columns with a number of s-RNA preparations, [ 14C]seryl s-RNA, a serine t-RNA fraction, pancreatic and T 1-ribonuclease hydrolysates of RNA, and s-RNA's which were partially degraded by heating, are reported (Figs. 1–7). It was shown that: 1. 1 s-RNA preparations from brewer's yeast, baker's yeast, and Escherichia coli contain varying and sometimes considerable amounts of biologically inactive poly- and oligonucleotides. 2. 2. Seryl and serine t-RNA's are eluted well before many of the other t-RNA's and therefore may differ from them in size and/or secondary structure. 3. 3. The largest oligonucleotides of enzymatic RNA hydrolysates, i.e. octa- to decanucleotides are eluted from columns with Sephadex or polyacrylamide gel immediately after the s-RNA. One of the reasons for this may be aggregation of the oligonucleotides, as was indicated by heating experiments, chromatography in the presence of urea, etc. The isolation of large RNA fragments by standard Sephadex chromatography therefore seems to be difficult.

Enzymatische Spaltungen von Serin-t-RNA-Fraktionen

Enzymatic splitting of serine-specific transfer-RNA Serine-specific t-RNA I and II fractions, which had been largely separated from each other and from other t-RNA's by countercurrent distribution, were investigated. The splitting products obtained with pancreatic and T 1-ribonucleases (EC 2.7.7.16 and EC 3.1.4.8 respectively) were separated by column chromatography (Figs. 1 and 2) and further purified. Some oligonucleotides were further degraded enzymically and the products separated by column chromatography (Tables I–III). One each of N 2-dimethyl-Gp, 5-methyl-Cp, rTp, Ip, 2′- O-methyl-Gp, 2′- O-methyl-Up; three ψ-Up's and two nucleotides with the properties of 4,5-dihydro-Up were found in defined sequences of the serine t-RNA's. Degradation of a serine t-RNA fraction with snake venom phosphodiesterase (EC 3.1.4.1) proceeded quickly at first and then slowed down (Figs. 3 and 4). The 5′-mononucleotides split off in the fast initial phase were analyzed.

Nucleotidsequenzen in serinspezifischen Transfer-Ribonucleinsäuren

Nucleotide sequences in serine-specific transfer ribonucleic acids The results of the preceding papers on the isolation of several serine t-RNA fractions and their splitting with pancreatic and T 1-ribonucleases (EC 2.7.7.16 and EC 3.1.4.8 respectively) are discussed. By countercurrent distribution two serine t-RNA's (I and II) were largely separated from each other; they also differed in behaviour during methylated albumin-kieselgur column chromatography. The oligonucleotide patterns of serine t-RNA's I and II, on the other hand, were very similar. The nucleotide sequences found in the serine t-RNA's are summarized in Table I. Several overlapping sequences were found, with the odd nucleotides and the (Ap) nG-sequences as links. Some of the 11 odd nucleotides occur clustered in the RNA chains. Various possible “anticodons” appear in the RNA. There is very little similarity between the sequence model of serine t-RNA, proposed by Cantoni et al., and the nucleotide sequences of the serine t-RNA's determined here. Possible base pairing between the nucleotide sequences of the serine t-RNA's (Table I) are discussed.

Hemin synthesis in the developing frog embryo and its stimulation by frog liver RNA

Hemin was synthesized from δ-aminolevulinic acid-4-C 14 by intact and minced frog embryos; the isotopic activity of the hemin samples is a measure of the activity of the enzymes of this pathway of porphyrin biosynthesis. Administration of the isotopic hemin precursor to intact embryos revealed low levels of synthesis until stage 25, but this is attributed to the poor penetration of δ-aminolevulinic acid into the embryos due to their relatively impermeable surface coat. Cutting each embryo into 4 or 5 pieces (mincing) allowed penetration of the hemin precursor into the embryo. It appears there is a synthesis of the enzymes of this porphyrin biosynthetic pathway in the minced embryos, since puromycin inhibited the incorporation of labeled amino acids into protein, and also the incorporation of δ-aminolevulinic acid-4-C 14 into hemin of minced stage 20 embryos. The minced embryos showed a slight increase in hemin synthesis from gastrulation to neurulation and a larger increase to the tailbud stage. The level of hemin formation in minced stage 25 larvae was lower than that of intact stage 25 larvae; at the feeding larval stage the labeled compound can enter the organism and is converted to hemin more efficiently than by minced stage 25 larvae. The real course of hemin synthesis in developing frog embryos, as revealed from the results obtained with both minced and intact embryos, is that of a gradual increase from gastrulation to neurulation, followed by an increased rate of hemin synthesis at tailbud and larval stages. The addition of frog liver RNA (1 mg/ml) to minced stage 20 embryos consistently stimulated the level of hemin synthesis above that of untreated minced embryos. Frog liver DNA and RNA of frog spleens, muscle, and red blood cells, as well as mouse ascites tumor cell RNA (1 mg/ml), did not stimulate hemin synthesis. Stimulation is also evoked by a mixture of the four ribonucleosides. Puromycin inhibits both protein and hemin synthesis of minced embryos while liver RNA stimulates the incorporation of labeled amino acids into the protein fraction. It appears that hemin synthesis is linked to the synthesis of the enzymes of porphyrin biosynthesis in these experiments. Phenol extraction of RNA from centrifugal fractions of liver homogenates revealed that there is active RNA in the ultracentrifugal supernatant fraction and that a transfer RNA fraction isolated from the ultracentrifugal supernatant of frog liver retains the ability to stimulate hemin synthesis of minced stage 20 embryos. Hot phenol extracts of the interphase of cold phenol extracts of frog liver also stimulated hemin synthesis of minced gastrulae cultured for 24 hours, but the stimulation by the four ribonucleosides and the RNA of the ultracentrifugal supernatant argue against an effect of messenger-RNA molecules. It is likely that supplying precursors of RNA synthesis, or transfer RNA, to cut frog embryos can stimulate the synthesis of enzymes synthesizing hemin, as well as the synthesis of other proteins.

Spaltung von löslicher ribonucleinsäure und serin-spezifischen transfer-ribonucleinsäure-fraktionen mit pankreas-ribonuclease

Several RNA samples were split with pancreatic RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16). The fragments were separated by chromatography on DEAE-cellulose columns and in some cases by subsequent paper electrophoresis. The amounts of mono- and dinucleotides from various unfractionated and partially fractionated soluble RNA's were compared (Table I). In hydrolysates of highly purified serine transfer RNA fractions 7 di-, 7 tri-, 7 tetra-, 2 penta- and 2 hexanucleotides - coming from 2 serine transfer-RNAs - were identified and quantitatively determined (Tables II and III), as well as some oligonucleotides, which occur in smaller amounts and probably come from contaminations of the serine transfer-RNA fractions. 3 oligonucleotides could not be identified, and one only partially. Several oligonucleotides contain odd nucleotides.

Spaltung einer serinspezifischen transfer-ribonucleinsäure-fraktion mit T 1-ribonuclease

Soluble RNA and a serine specific transfer RNA fraction were digested by highly purified T 1-RNAase (ribonucleate 3′-guanylohydrolase, EC 3.1.4.8) and phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). Conditions for the separation of the oligonucleotides on DEAE-cellulose columns were worked out with digests of soluble RNA (Fig. 1–3). The splitting products of the serine-transfer-RNA fraction were separated (Fig. 4) and further purified by paper electrophoresis. Their nucleotide composition — including the odd nucleotides — was determined by conventional methods and the sequences partially elucidated. 5 Di-, 6 tri-, 7 tetra-, 2 penta- and 1 hexanucleotide of the two serine-transfer-RNA chains were identified (Table I) and quantitatively determined (Table II). One longer oligonucleotide was partially identified. Some oligonucleotides, which are present in small amounts, were due to impurities in the serine-transfer-RNA sample.

Isolierung von serinspezifischen transfer-ribonucleinsäure-fraktionen

Soluble RNA was isolated from brewer's yeast; on the average 30–35 g were obtained from 50 kg. 350 mg of a serine transfer-RNA fraction were isolated from 32 g of soluble RNA by several subsequent countercurrent distributions in the tri- n-butylamin system (Fig. 1, Table I). The serine transfer-RNA fraction (31 mμmoles serine/mg RNA; less than 1 % of acceptor activity for other amino acids was detected) contained two serine transfer-RNA chains. The nucleotide composition of the serine transfer-RNA fraction was determined (Fig. 3, Table II). In a second series of countercurrent distributions the two serine transfer-RNA's were separated (Fig. 2) from each other and partially purified.