Complexes of Aminoacyl‐tRNA Synthetases with tRNAs as Studied by Partial Nuclease Digestion

Hörz, Wolfram; Zachau, Hans G.

doi:10.1111/j.1432-1033.1973.tb02571.x

Cited by 51 publications

(28 citation statements)

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“…Blanquet In equilibrium dialysis experiment the affinity for phenylalanine was somewhat higher ; as before, one binding site per synthetase molecule was found by dialysis in the presence of 0, 0.5, and 1.0 mole Phe-01-PA per mole of synthetase. In nuclease protection studies (W. Horz, personal communication) the 1 : 1 stoichiometry of the tRNA-synthetase complexes remained as previously reported [8]. The two group of experiments indicate that the previously used phenyhlanyl-tRNA synthetase preparations contained inactive but ligand binding enzyme molecules.…”

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confidence: 62%

“…Competition with respect to ATP was not investigated. I n studies of phenylalanyl-tRNA synthetase protection of tRNAPhe against nuclease digestion, additional tRNAPhe protection was found when Phe-ol-pA was present [8]. This additional protection disappeared when the tRNA was aminoacylated, while in the absence of Phe-ol-pA no difference in protection was detected between charged and uncharged tRNA.…”

Section: Experiments With Arninmll$ Adenyhtatesmentioning

confidence: 91%

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Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

Hertz¹,

Zachau²

1973

European Journal of Biochemistry

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The kinetics of phenylalanyl-tRNA and seryl-tRNA formation were investigated with tRNAs and aminoacyl-tRNA synthetases from yeast.Phenylalanyl-tRNA synthetase yielded linear Lineweaver-Burk plots with tRNAPhe, phenylalanine, and 1 ,Ne-ethenoadenosine triphosphate ( EATP) as variable substrates. According to equilibrium dialysis in the absence or presence of phenylalaninyl adenosine 5'-phosphate, phenylalanyl-tRNA synthetase possesses one binding site for phenylalanine. For ATP as variable substrate, the deviation from linearity in the Lineweaver-Burk plot, observed by other investigators, was confirmed. The slope of the curve indicates the presence of more than two ATP binding sites.Seryl-tBNA spthetase yielded a linear Lineweaver-Burk plot only with EATP as variable substrate. The Lineweaver-Burk plots for serine and tRNAS-9' were non-linear ; the interpretation we favor involves positive cooperativity between amino acid binding sites and between tRNA binding sites. Hill plots of the kinetic data showed that the enzyme possesses a t least two binding sites for each of these substrates. The kinetic data for ATP could be interpreted as showing more than two binding sites with negative and positive cooperativity in binding of successive ATP molecules.The aminoalkyl adenylates, phenylalaninyl adenosine 6'-phosphate and serinyl adenosine 5'-phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid.EATP functions in place of ATP in phenylalanyl-tRNA and seryl-tRNA formation although with rather Werent kinetic properties. Modified tRNAPhe and tRNASer, in which the 3'-terminal adenosine was replaced by ethenoadenosine, were prepared by a C-C-A transferase-catalyzed reaction of EATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodsed tRNAs, as fluorescent probes in synthetase-tRNA interaction studies.The kinetic behavior of phenylalanyl-tRNA synthetase appears to be much simpler than that of seryl-tRNA synthetaae, despite the fact that the former enzyme is twice as big and contains twice as many subunits as the latter one. The comparative simplicity of the one enzyme relative to the other correlates with previous results on interactions with substrates, which were obtained by fluorescence measurements and nuclease protection studies.

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confidence: 62%

Section: Experiments With Arninmll$ Adenyhtatesmentioning

confidence: 91%

Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

Hertz¹,

Zachau²

1973

European Journal of Biochemistry

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“…methioninyladenylate complex. In the case of phenylalanyl-tRNA synthetase [8,12,13], seryltRNA synthetase [I41 and tyrosyl-tRNA synthetase [ 151 controversial results have been reported as well.…”

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confidence: 99%

“…Widely different techniques have been applied to determine the number of binding sites for tRNA on the synthetase : gel chromatography [16], membrane filtration [I 71, fluorescence spectroscopy [18], sucrose gradient centrifugation [ 191 and partial nuclease digestion [8].…”

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confidence: 99%

Equivalent and Non‐equivalent Binding Sites for tRNA on Aminoacyl‐tRNA Synthetases

Krauss

Pingoud

Boehme

et al. 1975

European Journal of Biochemistry

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Complexes between tRNAPhe (yeast), tRNAser (yeast) and tRNATy' (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 % lo6 M-I. 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. K , % lo6 M-' , K2 is determined to be 1.3 x lo5 M-' 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~ lo5 M-' at pH 7.2, and 2 . 5~ lo6 M-' 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 tRNASFr (yeast), tRNAA'" (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.Aminoacyl-tRNA synthetases functionally represent a unique class of enzymes. They differ, however, substantially in their physical properties and the mechanistic details of the reaction they catalyze. It has been of particular interest to correlate the number of binding sites for the various substrates as well as the number of catalytic sites to the size of the synthetases and their subunit structures. So far, molecular weights ranging from 50000 to 250000 have been determined. Subunit structures of the sll, cx2, aB and a2P2 type have been found (review: [l]). Up till now no generalization could be made as to a correspondence of size, number of identical subunits, and number of binding or catalytic sites.In the present paper we will concern ourselves merely with the binding of tRNA to the aminoacyltRNA synthetases. In most cases studied so far only one binding site was found per synthetase. This includes large enzymes like the phenylalanyl-tRNA synthetase from Escherichia coli [2,3], which has an sl,~,-structure, as well as small synthetases like the glutaminyl-tRNA synthetase from E. coli [4], which has an a,-structure. (1975)

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“…Under conditions of 50% inhibition, [E -tRNAPhe-C-C-N] is 50% of the total amount of enzyme, Etotai, added to the assay, since the enzyme has only a single binding site for tRNAPhe (15). The enzyme not complexed with tRNAPhe-C-C-N interacts with tRNAPhe-C-C-A according to…”

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confidence: 99%

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

Haar¹,

Gaertner²

1975

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACTtRNAPhe species from baker's yeast modified at the 3'-terminus in many cases are phenylalanylatable substrates. Out of several tRNAPhe species possessing a modified 3'-end that cannot be phenylalanylated, only two, tRNAPheC-C-2'dA and tRNAPheC-C-formycinoxi-reds are strong competitive inhibitors for tRNAPheC-C-A during phenylalanylation. In the ATP/PPi exchange, both these inhibitors reduce Vman, to about 25%o; but whereas tRNAPheCC_2'dA has no influence on Km ATP and Km Phe during ATP/PPj exchange, tRNAPheCC-formycinox i-red reduces Km AT! from 1430 IAM, found in the absence of tRNAPhe, to 230 jiM, and Km Phe, from 38 to 14 uM. The values found in the presence of tRNAPheC-C-CformycinOxi-re during ATP/PPi exchange, are identical with those determined in the phenylalanylation of tRNAPheC_C-A.All other tRNAPhe species carrying a modified 3'-end that cannot be phenylalanylated exhibit a mixed competitivenoncompetitive inhibition in the phenylalanylation reaction. In the ATP/PPi exchange, they do not influence Km ATP and K,, Phe and only weakly, if at all, Vmax.The results show that the 3'-adenosine of tRNAPhe cannot solely be a passive acceptor for phenylalanine, but must in addition play an active role during enzyme-substrate interaction. The data can be consistently explained by the hypothesis that the 3'-adenosine of tRNAPhe triggers a conformational change of the enzyme.In the living cell part of the genetic information stored in the DNA is translated into proteins. This is achieved by first transcribing the information into mRNAs and then translating the latter into peptide sequences on the ribosome. Prior to translation on the ribosome, the amino acids are activated by attachment to the 3'-terminal ribose of their particular tRNAs.The overall aminoacylation reaction is achieved according to tRNAaa + ATP + aa + E 2 aa-tRNAaa + AMNP + PPj + E, where E is the enzyme and aa is the amino acid. This reaction can be followed experimentally by the incorporation of radioactively labeled amino acid into tRNA.

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Complexes of Aminoacyl‐tRNA Synthetases with tRNAs as Studied by Partial Nuclease Digestion

Cited by 51 publications

References 28 publications

Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

Equivalent and Non‐equivalent Binding Sites for tRNA on Aminoacyl‐tRNA Synthetases

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

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