UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
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Earlier the existence of two conformers of Phe-tRNAPhe of E. coli was demonstrated because one of them yields complexes with 70S-poly(U) of extremely high affinity and the other with at least a 105 lower binding constant. We denote the first conformer as HAC (high affinity conformer) and the second as LAC (low affinity conformer). This high difference in binding constants was used for studying the process of reversible interconversion of conformers of Phe-tRNAPhe. The transition kinetics of LAC to HAC in conditions when the latter is stable (in the presence of magnesium ions) was studied and a high value of activation energy (35 kcal/mole) found. The interconversion is the first order reaction and equilibrium does not depend of overall Phe-tRNA concentration.
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PMID:The interconversion of conformers of phenylalanyl-tRNA with different affinity to 70S ribosomes of Escherichia coli. 35 62

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA results in a binding of 1 mole of the reagent per 1 mole of the enzyme. Exhaustive alkylation of phenylalanyl-tRNA synthetase completely blocks the aminoacylation and partially inhibits the reaction of ATP--[32P]pyrophosphate exchange. Removal of the tRNA moiety of the reagent by hydrolysis of the ester bond N-chlorambucilyl-phenylalanine and terminal adenosine does not result in a restoration of ATP--[32P]pyrophosphate exchange and aminoacylation activity. The latter result may testify a chemical modification of amino acid residues essential for enzymatic activity. Possibility of blocking one of the two tRNA binding sites is discussed.
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PMID:[Modification of one tRNA recognition site of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA]. 36

An unusual class of wheat germ tRNAs has been isolated which completely lacks ribothymidine (rT) and contains an unmodified uridine in its place. We discuss here the isolation, identification and properties of these tRNAs. The rT-lacking tRNAs of wheat germ are essentially limited to the glycine isoacceptors (a minimum of five identifiable species), three threonine and at least, one tyrosine tRNA. All tRNAs were obtained 70-100% pure by chromatographic methods, and were detected by their ability to be methylated by E. coli rT-forming uracil methyltransferase with methyl-labeled S-adenosyl-L-methionine (SAM) as the methyl donor. In vitro methylation of each of the tRNAs resulted in the formation of 1 mole of rT per mole of tRNA. In the one case analyzed in detail (tRNA1Gly), all of the rT was found to be located at the 23rd position from the 3' end of the tRNA molecule. Following complete digestion of four highly purified glycine isoacceptors (tRNAGly1,4,5,6) to nucleosides and subsequent periodate oxidation and 3H potassium borohydride reduction, all were found to contain an unusually high level of 5-methylcytidine (m5C) (3-4 residues per molecule), and all contained no rT. The possible correlation between the presence of m5C and the absence of rT is discussed. All of the chromatographically purified glycine tRNAs function in a wheat germ cell-free protein synthesizing system and polymerize glycine in response to either poly G or poly (G, U).
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PMID:Wheat germ tRNAs containing uridine in place of ribothymidine: a characterization of an unusual class of eukaryotic tRNAs. 65 15

The binding of adenosine diphosphate-ribosylated elongation factor 2 (ADPRib-EF-2) to ribosomes was inhibited both in the presence and absence of GTP in proportion to the amounts of unmodified EF-2 added. Concomitant with this inhibition, an increase in the activity of ribosome-bound EF-2 in polyphenylalanine synthesis was observed. On the other hand, the addition of ADPRib-EF-2 reduced the rate of poly(Phe) synthesis observed in the presence of a saturating amount of EF-2 and increased the amount of EF-2 required for the half-maximal rate of poly(Phe) synthesis. Phe-tRNA, nonenzymatically bound to the ribosome in the presence of poly(U), inhibited the subsequent binding of ADPHRib-EF-2. The same ribosomal population appeared to preferentially bind either aminoacyl-tRNA or ADPRib-EF-2. The Scatchard plot of the binding of ADPRib-EF-2 to the ribosome in the presence of GTP revealed the presence of two ribosomal binding sites (or ribosomal populations) with apparent different affinities for the modified factor (K371 degrees d,1 = 6.6 nM and K37 degrees d,2 = 126 nM). At saturating concentrations of ADPRib-EF-2, a maximum of about 1 molecule of the factor was bound per ribosome. The binding of ADPRib-EF-2 to the ribosome was stimulated by GTP. The binding of radioactive GTP to the ribosome was observed concomitantly with the binding of ADPRib-EF-2. One mole of GTP was bound per mole of ADPRib-EF-2. No significant difference could be found in the binding of GTP to ribosome required in the presence of either EF-2 or ADPRib-EF-2. The binding of ADPRib-EF-2 to the ribosome required the presence of Mg2+ and reached a maximum at 5 mM. The binding was greatest at K+ concentrations below 20 mM. ADPRib-EF-2 was bound primarily to the large ribosomal subunit. A slight, but reproducible binding to the 40 S subunit was also observed. The addition of 40 S to 60 S subunits stimulated the binding of ADPRib-EF-2. GTP displayed a stimulatory effect on the binding only in the presence of recombined subunits. Human ADPRib-EF-2 was bound to rat liver ribosomes as efficiently as to human tonsil ribosomes, while the binding to Escherichia coli ribosomes was insignificant.
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PMID:Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes. 78 67

A 7-methylguanine (m7G) specific tRNA methyltransferase from E. coli MRE 600 was purified about 1000 fold by affinity chromatography on Sepharose bound with normal E. coli tRNA. The purified enzyme catalyzes exclusively the formation of m7G in submethylated bulk tRNA of E. coli K12 met- rel-. The purified enzyme transfers the methyl group from S-adenosyl-methionine to initiator tRNA of B. subtilis and 0.8 moles m7G residues are formed per mole tRNA. It is suggested that the enzyme specifically recognizes the extra arm unpaired guanylate residue.
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PMID:7-Methylguanine specific tRNA-methyltransferase from Escherichia coli. 79 33

The digestion of EF-Tu-GDP (or EF-Tu-GTP) by trypsin [EC 3.4.21.4] under native conditions has been shown to proceed through two different and characteristic stages. 1. In the first phase, the protein is transformed into a fragment (Fragment A) with a molecular weight of 39,000 by exposure to trypsin for a relatively short period of time. Fragment A is unable to catalyze the binding of aminoacyl-tRNA to ribosomes. The ability to promote two partial steps of the binding reaction, i.e., formation of the aminoacyl-tRNA-EF-Tu-GTP ternary complex as well as the methanol-stimulated, ribosome dependent GTPase reaction, was rapidly destroyed. On the other hand, the ability to interact with guanine nucleotides as well as EF-Ts survived well during prolonged digestion. 2. In the second phase of digestion, a nick is introduced in Fragment A to yield two subfragments (Fragments B and C). These two fragments exist as a hybrid molecule which migrates as a single peak on a Sephadex G-75 column, and which dissociates into Fragments B and C only in the presence of 6 M guanidine hydrochloride or 5% sodium dodecyl sulfate. The molecular weights of Fragments B and C, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, were 22,000 and 12,000 respectively. The hybrid molecule still retained one mole of bound guanine nucleotide and was resistant to further tryptic digestion. 3. Three sulfhydryl groups of EF-Tu were found to be present in Fragment B, both by amino acid analysis of the purified fragments and also by electrophoresis of tryptic digests labeled with N-ethyl[14C]maleimide. 4. The tryptic digestion of EF-Tu-GDP (or EF-Tu-GTP) labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) at SH2 (the second SH), caused a 30% decrease in the fluorescence emission during the first rapid phase of digestion. This indicates that destruction of the hydrophobic environment near SH2 of EF-Tu occurred in the early phase of tryptic digestion. 5. The kinetic studies on the reaction of ANM with EF-Tu before and after tryptic digestion indicated that both Fragment A and the hybrid molecule reacted with ANM in the presence of GTP three to four times more rapidly than in the presence of GDP. Thus, it appears that the ability to induce conformational transition near SH2 by a change of nucleotide ligands is still retained in the hybrid molecule consisting of Fragments B and C.
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PMID:Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. 93 63

N6-(delta2-isopentenyl)adenosine was found both as a component of tRNA and as the cytoplasmic mononucleotide in human leukemic lymphoblasts and myeloblasts from peripheral blood and bone marrow samples. This hypermodified nucleotide was also found in the tRNA and as a mononucleotide in human (MRC-5 and KB) and mouse (A9, FLV, LM, and RAG) cell lines. The relative amounts of this hypermodified nucleotide in the tRNA of the cell lines and the human leukemias were similar (the mean value being 0.06 +/- 0.03 mole % of the total tRNA nucleotide content); whereas the amounts occurring as the free cytoplasmic mononucleotide were more varied but still comparable (the mean value being 0.53 +/- .09 mole % of all cytoplasmic nucleotides) for all cells investigated with the notable exception of all normal, diploid cell lines under study (0.04 mole%). A possible relationship of the free cytoplasmic mononucleotide with the nucleotide in the tRNA for control of mammalian cell protein synthesis in vivo was investigated by addition of N6-(delta2-isopentenyl)adenosine to the culture medium. The exogenously added nucleoside caused inhibition of cell growth within 3 h and cell death within 36 h at concentrations as low as 0.4 muM. No comparable effects were seen when adenosine, adenine, or N6-(delta2-isopentenyl)-adenine were added to the cultures. The simultaneous presence of adenosine in cultures containing N6-(delta2-isopentenyl)adenosine did not alter the detrimental effects of the hypermodified nucleoside on cell growth even when the concentration of adenosine was 50-fold that of N6-(delta2-isopentenyl)adenosine. Addition of N6-(delta2-isopentenyl)adenosine to cell cultures caused within the first 6 h a significant reduction in the rates of RNA and protein synthesis; whereas DNA synthesis continued at a rate comparable to control and adenosine-treated cells for 18 h before decreasing.
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PMID:Natural occurrence of an inhibitor of mammalian cell growth in human and mouse cells of normal and tumor origin. 103 27

We report a method for predicting the most stable secondary structure of RNA from its primary sequence of nucleotides. The technique consists of a series of three computer programs interfaced to take the nucleotide sequence of any RNA and (a) list all possible helical regions, using modified Watson-Crick base-pairing rules; (b) create all possible secondary structures by forming permutations of compatible helical regions; and (c)evaluate each structure for total free energy of formation from a completely extended chain. A free energy distribution and the base-by-base bonding interactions of each possible structure are catalogued by the system and are readily available for examination. The method has been applied to 62 tRNA sequences. The total free-energy of the predicted most stable structures ranged from -19 to -41 kcal/mole (-22 to -49 kJ/mole). The number of structures created was also highly sequence-dependent and ranged from 200 to 13,000. In nearly all cases the cloverleaf is predicted to be the structure with the lowest free energy of formation.
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PMID:Method for predicting RNA secondary structure. 105 9

The binding of spermidine and ethidium bromide to mixed tRNA and phenylalanine tRNA has been studied under equilibrium conditions. The numbers and classes of binding sites obtained have been compared to those found in complexes isolated by gel filtration a low ionic strength. The latter complexes contain 10-11 moles of either spermidine or ethidium per mole of tRNA; either cation is completely displaceable by the other. In ethidium complexes, the first 2-3 moles are bound in fluorescent binding sites; the remaining 7-8 molecules bind in non-fluorescent form. At least one of the binding sites for spermidine appears similar to a binding site for fluorescent ethidium. Similar results are found with E. coli formylmethionine tRNA. Spermine, in excess of 18-20 moles per mole tRNA, causes precipitation of the complex. Putrescine does not form isolable complexes with yeast tRNA and displaces ethidium less readily from preformed ethidium-tRNA complexes. Under equilibrium conditions, in the absence of Mg++, there are 16-17 moles of spermidine bound per mole of tRNA as determined by equilibrium dialysis. Of these, 2-3 bind with a Ksence of 9 mM Mg++, the total number of binding sites is decreased slightly and there appears to be only one class of sites with a Ka = 600 M(-1). Quantitatively similar results are obtained for the binding of spermidine to yeast phenylalanine tRNA. When the interaction between ethidium bromide and mixed tRNA is studied by equilibrium dialysis or spectrophotometric titration, two classes of binding sites are obtained: 2-3 molecules bind with an average Ka = 6.6 x 10(5) M(-1) and 14-15 molecules bind with an average Ka = 4.1 x 10(4) M(-1). Spermidine, spermine, and Mg++ compete effectively for both classes of ethidium sites and have the effect of reducing the apparent binding constants for ethidium. When the binding of ethidium is studied by fluorometry, there are 3-4 highly fluorescent sites per tRNA. These sites are also affected by spermidine, spermine and Mg++. Putrescine has little effect on any of the classes of binding sites. These data are consistent with those found under non-equilibrium conditions. They suggest that polyamines bind to fairly specific regions of tRNA and may be involved in the maintenance of certain structural features of tRNA.
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PMID:The binding of polyamines and of ethidium bromide to tRNA. 109 21

Complexes between tRNAPhe (yeast), tRNASer (yeast) and tRNATyr (Escherichia coli) and their cognate aminoacyl-tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast-moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results. 1. Two moles of tRNAPhe can be bound to one mole of phenylalanyl-tRNA synthetase with a binding constant greater than 10(6) M-1. The binding constants for both tRNAs are very similar; the binding sites are independent of each other. Omission of Mg2+ does not prevent binding. 2. Two moles of tRNASer can be bound to one mole of Seryl-tRNA synthetase; the binding of the first and second tRNA is non-equivalent, K1 greater than 10(6) M-1, K2 is determined to be 1.3 X 10(5) M-1 at pH 7.2. Omission of Mg2+ prevents complex formation. 3. Tyrosyl-tRNA synthetase behaves very similarly to seryl-tRNA synthetase. The binding constant for the weakly bound tRNA is 2.3 X 10(5) M-1 at pH 7.2, and 2.5 X 10(6) M-1 at pH 6.0. No complexes are observed in the absence of Mg2+. 4. Unspecific binding was only obtained with phenylalanyl-tRNA synthetase. It binds tRNASer (yeast), tRNAAla (yeast) and tRNATyr (E. coli) with a binding constant about 100 times lower compared to its cognate tRNA. The binding data are discussed with respect to the tertiary structure of the tRNAs, the subunit structure of the synthetases and the possible physical basis for the non-equivalence of binding sites.
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PMID:Equivalent and non-equivalent binding sites for tRNA on aminoacyl-tRNA synthetases. 110 Mar 84

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