Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.1.1.20 (phenylalanyl-tRNA synthetase)
358 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction between tRNAPhe (yeast), from which the Y-base has been removed by acid treatment, and phenylalanyl-tRNA synthetase (yeast) has been investigated by fluorescence competition titrations and sedimentation velocity runs. The binding parameters are given under various ionic conditions. The tRNAPhe-Y still can occupy the specific binding sites on the enzyme. Compared to unmodified tRNAPhe, the binding constant is lowered by more than one order of magnitude. It can be concluded that the Y-base is not necessary for specific recognition of tRNAPhe by the cognate synthetase, it rather may represent a point of attachment for the synthetase.
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PMID:Effect of excision of the Y-base on the interaction of tRNAPhe (yeast) with phenylalanyl-tRNA synthetase (yeast). 0 7

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.
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PMID:Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast. 0 88

The preparation of four fluorescent derivatives of tRNAPhe (yeast) and their characterization by chemical, spectroscopic, and biochemical methods is described. The derivatives are prepared by replacing wybutine (position 37 in the anticodon loop) or NaBH4-reduced dihydrouracil (positions 16/17 in the hU loop) with ethidium or proflavine; they are isolated by reversed-phase chromatography (RPC-5). All tRNAPhe-dye derivatives are aminoacylated by yeast phenylalanyl-tRNA synthetase to at least 80% of the charging capacity of the unmodified tRNAPhe with an unchanged Km (0.2 mucroM) and a V lowered by 30--50%. They exhibit good to excellent activity in the aminoacylation assay from synthetase from Escherichia coli. It is concluded that the insertion of the dyes does not seriously disturb essential elements of the native tRNAPhe structure. The dyes are bound via N-ribosylic linkages. The appearance of isomeric tRNAPhe-ethidium derivatives is attributed to the involvement of the different amino groups of ethidium in the condensation. In addition, there are indications for the existence of alpha and beta anomers of the tRNA-dye compounds. The dyes are rigidly fixed to their position in the tRNA molecule by stacking interactions with the neighboring bases. The ethidium probes show Mg2+-induced changes of the tRNA conformation which are paralleled by changes of the rate of aminoacylation. On the basis of this observation it is hypothesized that conformational flexibility of the tRNA molecule is a functionally important feature of the tRNA structure.
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PMID:Fluorescent derivatives of yeast tRNAPhe. 11 93

The influence of modifications of the 3'-terminal adenosine of tRNAPhe (yeast) on the complex formation between this tRNA and phenylalanyl-tRNA synthetase (yeast) has been investigated by using fluorescence titrations and fast kinetic techniques. Subtle changes in the 3' terminus are reflected by distinct alterations in the two-step recognition process which had been demonstrated earlier for the native substrate tRNAPheCCA [Krauss, G., Riesner, D., & Maass, G. (1977) Nucleic Acids Res. 4, 2253--2262]. Binding experiments with tRNAPheCC, tRNAPheCCA-ox-red, tRNAPheCC2'dA, tRNAPheCC3'dA, tRNAPheCC-formycin, and tRNAPheCC-formycin-ox-red confirm that the 3'-terminal adenosine participates in a conformational change of the tRNA--synthetase complex. This is valid in both the absence and presence of phenylalaninyl-5'-AMP, the alkyl analogue of the aminoacyladenylate. As compared to tRNAPheCCA, a slower conformational change is observed with the competitive inhibitor tRNAPheCC-formycin-ox-red. The reaction enthalpy and/or the quench of the Y-base fluorescence that accompany the conformational change are altered upon binding of tRNAPheC2'dA, tRNAPheCC3'dA, and tRNAPheCC-formycin. It is evident that the final adaptation between tRNA and its synthetase in the complex is determined by the chemical nature of the 3'-terminal nucleotide. This is of vital importance for the specificity of the aminoacylation process.
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PMID:Conformation transitions of a tRNA--aminoacyl-tRNA synthetase complex induced by tRNAs bearing different modifications in the 3' terminus. 38 79

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

Purified yeast phenylalanyl-tRNA synthetase can aminoacylate (yeast) tRNA(Phe), (wheat) tRNA(Phe), and (Escherichia coli) tRNA(1) (Val) (1, 2). We now report that this synthetase can also aminoacylate (E. coli) tRNA(Phe) and (E. coli) tRNA(1) (Ala). Highly purified (E. coli) tRNA(Phe) is heterologously aminoacylated to approximately 90% of the extent achieved with the homologous enzyme (crude E. coli phenylalanyl-tRNA synthetase). Pure (E. coli) tRNA(1) (Ala) (the major species) is heterologously aminoacylated to 70% of the extent achieved with the homologous synthetase (crude E. coli alanyl-tRNA synthetase).(E. coli) tRNA(Phe) is the fourth purified transfer RNA of known sequence to be shown to be an acceptable substrate for purified yeast phenylalanyl-tRNA synthetase. A comparison of these sequences shows that only one region is extremely similar in all four tRNAs. This region is located adjacent to the dihydrouridine loop, and consists of the nucleotides [Formula: see text] We conclude that this is the synthetase recognition site for yeast phenylalanyl-tRNA synthetase. This conclusion is further supported by partial fragment analysis of (E. coli) tRNA(1) (Ala).
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PMID:The yeast phenylalanyl-transfer RNA synthetase recognition site: the region adjacent to the dihydrouridine loop. 527 81

Unlike phenylalanyl-tRNA synthetase from lower eukaryotes, the corresponding enzyme from higher eukaryotes displays a pronounced tendency to associate with ribosomes in vitro. To attempt to uncover the structural features responsible for this difference in behavior, a comparative study of the enzymes purified to homogeneity from sheep liver and yeast was undertaken. The two alpha 2 beta 2-type enzymes displayed remarkably similar subunit molecular masses (71 and 63 kDa for sheep, 74 and 63 kDa for yeast), yet differed markedly in their isoelectric points (8.0 and 5.6 pH units, respectively). Mild tryptic digestion of the enzyme from sheep led to preferential degradation of the 63-kDa beta subunit into two major fragments of 35 and 25 kDa, respectively, with concomitant loss of activity. The isoelectric points of the denatured fragments were found to be distinctly lower than that of the denatured beta subunit, implying that the residues responsible for the basic net charge of the original beta subunit are mainly clustered in a small portion of the polypeptide chain which was excised during proteolysis. Despite their different isoelectric points, the enzymes from yeast and sheep displayed identical requirements for aminoacylation of tRNA at optimal rates. Moreover, the incidence of variations in pH and ionic strength on the kinetic parameters of the two enzymes was indistinguishable. Interpreted in terms of the polyelectrolyte theory, these results support the view that the residues responsible for the basic net charge of the mammalian enzyme are located in a region distal from the active site. It is suggested that the cationic charge of the enzyme allows anchorage to a cellular component carrying negative charges, possibly the ribosome.
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PMID:Phenylalanyl-tRNA synthetases from sheep liver and yeast. Correlation between net charge and binding to ribosomes. 639 97

The diffusion constant of phenylalanyl-tRNA synthetase has been measured by laser light scattering under conditions of complex formation with Mg2+, L-phenylalanine, MgATP, tRNAPhe, modified tRNAPhe, tRNAPhe (yeast), and noncognate tRNA. The diffusion constant (pH 7.5, 20 degrees C) of the free enzyme is (2.85 +/- 0.005) x 10(-7) cm2 s-1, of the enzyme . Mg2+ complex (2.40 +/- 0.05) x 10(-7) cm2 s-1 and of the enzyme . Mg2+ . tRNAPhe complex (2.95 +/- 0.06) x 10(-7) cm2 s-1. The effect of tRNAPhe is only seen when the enzyme is saturated with Mg2+. The smaller substrates exhibit no effect besides a small increase of the value of the diffusion constant under conditions where the enzyme-phenylalanyladenylate is synthesized. Of the noncognate tRNATyr and tRNAIle, the latter is able to associate with the enzyme, causing the value of the diffusion constant to increase. tRNAPhe (yeast) and tRNAhvPhe (photo-cross-linked tRNAPhe) exhibit similar effects. The observed variation of the diffusion constant is attributed to conformational changes of the enzyme. The opposite effects of Mg2+ and tRNAPhe are interpreted as an expansion and recontraction, respectively, of the enzyme molecule. In several cases, the effects were used to follow a titration of the enzyme with a ligand. Dissociation constants were calculated from the resulting titration curves, yielding values which are in agreement with those obtained by other techniques. It is established by comparison that of the two possible binding sites for each Mg2+ and tRNAPhe the diffusion constant reflects occupation of only a single class of sites.
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PMID:Detection of ligand-induced conformational changes in phenylalanyl-tRNA synthetase of Escherichia coli K10 by laser light scattering. 701 76

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