On the Specificity of Interactions between Transfer Ribonucleic Acids and Aminoacyl‐tRNA Synthetases

Pachmann, Ulrich; Cronvall, E.; Rigler, Rudolf; Hirsch, Rolf D.; Wintermeyer, Wolfgang; Zachau, Hans G.

doi:10.1111/j.1432-1033.1973.tb03123.x

Cited by 37 publications

(39 citation statements)

References 22 publications

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“…In fluorescence studies [5], cooperativity has been previously indicated for the binding of tRNAser to seryl-tRNA synthetase. I n studies of protection against nuclease digestion [S] and in fluorescence measurements [5,7,39] two sites for tRNAser were obtained. Unfortunately attempts to determine serine binding sites by equilibrium dialysis were unsuccessful, due to the low binding constant (which correlates with the relatively high "Rm").…”

Section: Discussionmentioning

confidence: 99%

“…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. In initial fluorescence polarization studies no change in binding of modified tRNAs to the cognate synthetase was observed, when up to I mM Phe-ol-pA or Ser-ol-pA was added [7], although the degree of polarization was influenced by ATP.…”

Section: Experiments With Arninmll$ Adenyhtatesmentioning

confidence: 92%

“…It seemed particularly interesting to use tRNA,, in fluorescence studies with aminoacyl-tRNA synthetase since the fluorescent label must be a t the catalytic site of the enzyme during the aminoacylation process. Initial experiments along these lines show an increase in fluorescence intensity when seryl-tRNA synthetase is added to a solution of tRNAE;' [7].…”

Section: Anda-containing Trivas and Fatpmentioning

confidence: 99%

“…Experiments showed that the C-C-A transferase was not limiting in the incubation mixture; no attemph were made to obtain a tRNAfy preparation with full amino acid acceptor activity, The fluorescence emission spectra of tRNAfF and tRN&te are the same as that of EATP [11,25]. A corrected emission spectrum of tRNAt2 is presented elsewhere [7]. Results of kinetic experiments with the modified tRNAs are presented below.…”

Section: Trnamentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Experiments With Arninmll$ Adenyhtatesmentioning

confidence: 92%

Section: Anda-containing Trivas and Fatpmentioning

confidence: 99%

Section: Trnamentioning

confidence: 99%

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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 derivatives of yeast tRNAPhe have been shown to be active in a number of assays including the ribosome systems from E. coli [13] and reticulocytes [14], indicating that their structure is not very different from that of native tRNAPhe. Both proflavine-labeled and ethidium-labeled tRNAPh" have been successfully used in a fluorescence spectroscopic study of synthetase-tRNA interaction [15]. For the present study of tRNA-ribosome interaction, we have used the ethidium label because it has a rather long fluorescence life time, making it suitable for polarization measuremets, and because its fluorescence properties are sensitive to conformational changes of the nucleic acid [16] (and M. Ehrenberg, R. Rigler, and W. Wintermeyer, unpublished results).…”

mentioning

confidence: 99%

Interactions of Yeast tRNA^Phe with Ribosomes from Yeast and Escherichia coli

Robertson

Kahan

Wintermeyer

et al. 1977

European Journal of Biochemistry

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The interaction of ethidium-labeled tRNAPhe from yeast with ribosomes from yeast and Escherichiu coli was studied by steady-state measurements of fluorescence intensity and polarization. The ethidium label was covalently inserted into either the anticodon or the dihydrouridine loop of the tRNA. The codon-independent formation of a tRNA.ribosome complex led to only a moderate increase of the observed fluorescence polarization indicating a considerable internal mobility of the labeled parts of the tRNA molecule in the ribosome complex. When the ribosome complex was formed in the presence of poly(U), the probes both in the dihydrouridine loop and in the anticodon loop were strongly immobilized, the latter exhibiting a substantial increase in fluorescence intensity. A smaller intensity change was observed when E. coli ribosomes were used, although the extent of immobilization was found to be similar in this case. Competition experiments with non-labeled tRNAPhe showed that the labeled tRNA!& was readily released from the complex with yeast ribosomes when poly(U) was absent, whereas in the presence of poly(U) it was bound practically irreversibly. The finding that the mobility of a probe in the dihydrouridine loop is affected by the codon-anticodon interaction on the ribosome suggests a conformational change of the ribosome-bound tRNA which may involve opening of the tertiary structure interactions between the dihydrouridine and the TYC loop.In the past few years a rather detailed picture of the functional aspects of the tRNA-ribosome interaction has been elaborated [l -51. However, the physical basis of the high precision of protein synthesis and the mechanism of the individual steps have not yet been understood. Sensitive physicochemical methods, such as fluorescence, have to be applied in order to obtain more structural and kinetic information. In other systems fluorescence techniques have been widely used because they offer high sensitivity and a wealth of information [6,7]. A pre- Cohn. EtdBr, 2,7-diarnino-lO-ethyl-9-phenyl-phenanthridinium bromideorethidium bromide; t R N A h , a derivative of tRNAPhe in which wyebutine is replaced by ethidium; tRNA~~:Sl6;,,, tRNA"h' in which dihydrourdcil is replaced by ethidium; tRNAP_h;Wye, tRNAPhe lacking wyebutine.

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Aminoacyl‐tRNA Synthetases: Some Recent Results and Achievements

Kisselev¹,

Фаворова²

1974

Advances in Enzymology - And Related Areas of Molecular Biology

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On the Specificity of Interactions between Transfer Ribonucleic Acids and Aminoacyl‐tRNA Synthetases

Cited by 37 publications

References 22 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

Interactions of Yeast tRNA^Phe with Ribosomes from Yeast and Escherichia coli

Aminoacyl‐tRNA Synthetases: Some Recent Results and Achievements

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On the Specificity of Interactions between Transfer Ribonucleic Acids and Aminoacyl‐tRNA Synthetases

Cited by 37 publications

References 22 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

Interactions of Yeast tRNAPhe with Ribosomes from Yeast and Escherichia coli

Aminoacyl‐tRNA Synthetases: Some Recent Results and Achievements

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Interactions of Yeast tRNA^Phe with Ribosomes from Yeast and Escherichia coli