Quantitative study of the ionic interactions between yeast tRNA<sup>Val</sup> and tRNA<sup>Phe</sup> and their cognate aminoacyl‐tRNA ligases

Bonnet, Jacques; Renaud, Michel; Jp, Raffin; Rémy, Pierre

doi:10.1016/0014-5793(75)80008-1

Cited by 29 publications

(9 citation statements)

References 22 publications

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“…The decrease in affinity upon excision of a quarter of the molecule is rather low, since the dissociation constant is only enhanced by a factor 7. This result is consistent with the values previously published [5]. A striking observation concerns the effect of the mere removal of the m7Gua base.…”

Section: Study Ofthe Interactions Between Yeast Phenylalanyl-trna Synsupporting

confidence: 93%

“…Furthermore, since the energy of interaction of a multisite molecule can be lower than the sum of the interaction energies of the isolated sites [29], a single site could, at the limit, be able to bind almost as efficiently as the entire molecule. This can explain why, upon excision of even a large part of the molecule, a high affinity is maintained [5,30,31].…”

Section: General Conclusionmentioning

confidence: 99%

“…Previous studies of our laboratory [5] have shown that a three-quarter fragment of tRNAPhe, tRNAPhe-(18 -76), still had a high affinity for the enzyme but could no longer be aminoacykdted. In the present paper we will report a more detailed analysis of the interaction of this three-quarter fragment of yeast tRNAPhe with phenylalanyl-tRNA synthetase, in the light of structural information obtained by thermal denaturation experiments according to Reiss et al [6].…”

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confidence: 94%

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Lack of Correlation between Affinity of the tRNA for the Aminoacyl-tRNA Synthetase and Aminoacylation Capacity as Studied with Modified tRNAPhe

et al. 1979

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The interactions of several modified yeast tRNAPh' [tRNAPhe lacking 7-methylguanine ; a fragment comprising about 3/4 of the whole molecule : tRNAPh' (1 8 -76) ; tRNAPhe (18 -76) lacking 7-methylguanine] with yeast phenylalanyl-tRNA synthetase were studied.Upon excision of the 5'-quarter of the tRNAPhe molecule, the residual fragment still tightly binds to the synthetase, but can no longer be aminoacylated. Surprisingly, upon removal of the 7-methylguanine base at position 46 in this fragment, although the affinity drops by a factor 10, a significant aminoacylation is restored. These results are discussed in terms of molecular flexibility and a model is proposed for tRNA-enzyme interaction, involving multisite recognition.Numerous studies have been performed with tRNA fragments in order to identify the interaction sites of the tRNAs with their cognate synthetases (for a review see [l-41). To draw information from this kind of experiment, both a quantitative study of the interaction of the tRNA fragment and the enzyme, and the knowledge of the structure of this fragment are necessary.Previous studies of our laboratory [5] have shown that a three-quarter fragment of tRNAPhe, tRNAPhe-(18 -76), still had a high affinity for the enzyme but could no longer be aminoacykdted. In the present paper we will report a more detailed analysis of the interaction of this three-quarter fragment of yeast tRNAPhe with phenylalanyl-tRNA synthetase, in the light of structural information obtained by thermal denaturation experiments according to Reiss et al. [6]. In this fragment the 7-methylguanine residue at position 46 appears to be of strategic importance, looking like a bolt in a zone which may be considered as a hinge between the two helical branches of the L-shaped tRNA molecule. Indeed, upon excision of m7Gua-46 in the three-quarter fragment new interesting properties arise, which will be analyzed. MATERIALS AND METHODSAll the chemicals used in this study were of the best available purity and were purchased from Merck (Darmstadt) and Prolabo (Paris). The radioactive chemicals were bought from the Commissariat a 1'Energie Atomique (Saclay). Purification of Yeast ~R N A~~" and Yeast Phenylalanyl-tR N A Syn thetasePure tRNAPhe was pepared from brewer's yeast tRNA (Boehringer, Mannheim) by counter-current distribution, according to Dirheimer and Ebel [7], followed by chromatography on benzoylated DEAEcellulose and elution with a combined gradient of NaCl (0.85 -1.2 M) and dimethylformamide (0 -4 x) in 5 mM sodium formate pH 4.0, 20 mM MgC12. The tRNAPh" obtained by this procedure accepted at least 1600 pmol amino acidlA260 unit.

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Section: Study Ofthe Interactions Between Yeast Phenylalanyl-trna Synsupporting

confidence: 93%

Section: General Conclusionmentioning

confidence: 99%

mentioning

confidence: 94%

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Lack of Correlation between Affinity of the tRNA for the Aminoacyl-tRNA Synthetase and Aminoacylation Capacity as Studied with Modified tRNAPhe

et al. 1979

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“…It should be noticed that the number of phosphates protected by synthetases against alkylation substantially exceeds the number of electrostatic contacts calculated for interaction of tRNAs with synthetases [40]. This means that not all these phosphates are involved in electrostatic interaction, which is not surprising for the synthetases which are known to be acidic proteins [41].…”

Section: Discussionmentioning

confidence: 99%

Interaction of tRNA^Phe and tRNA^Val with Aminoacyl‐tRNA Synthetases

Vlassov

Kern

Romby

et al. 1983

European Journal of Biochemistry

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The alkylation by ethylnitrosourea of phosphodiester bonds in tRNAPh' from yeast and in tRNAVal from yeast and from rabbit liver and that by 4-(N-2-chloroethyl-N-methylamino)-benzylamine of N-7 atoms of guanosine residues in yeast tRNAVa' have been used to study the interaction of these tRNAs with aminoacyl-tRNA synthetases. The modifications occurring at low yield were carried out on 3' and/or 5'end-labelled tRNAs either free or in the presence of cognate or non-cognate synthetases. After splitting of the tRNAs at the alkylated positions, the position of the modification sites in the tRNA sequences were detected by acrylamide gel electrophoresis. It was found that the synthetases protect against alkylation certain phosphate or guanosine residues in their cognate tRNAs. Non-cognate synthetases failed to protect efficiently specific positions in tRNA against modification.In yeast tRNAPhe the cognate phenylalanyl-tRNA synthetase protects certain phosphates located in all four stems and in the anticodon and extra-loop of the tRNA. Particularly strong protections occur on phosphate 34 in the anticodon loop and on phosphates 23, 27, 28, 41 and 46 in the D and anticodon stems.In yeast tRNAVa' complexed with yeast valyl-tRNA synthetase the protected phosphates are essentially located in the corner between the amino-acid-accepting and D stems, in the D loop, anticodon stem and in the variable region of the tRNA. Three guanosine residues, located in the D stem, and another one in the 3' part of the anticodon stem were also found protected by the synthetase. In mammalian tRNAVa', complexed with the cognate but heterologous yeast valyl-tRNA synthetase, the protected phosphates lie in the anticodon stem, in the extra-loop and in the T $ arm.The location of the protected residues in the structure of three tRNAs suggests some common features in the binding of tRNAs to aminoacyl-tRNA synthetases. These results will be discussed in the light of informations on interaction sites obtained by nuclease digestion and ultraviolet cross-linking methods.

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“…Extremely high salt concentrations also stimulate the aminoacylation of yeast tRNA phe by the phenylalanyl-tRNA synthetase fromEscherichia coli [7], whereas generally the aminoacylation of non-cognate tRNAs is favored by low ionic strength [8]. Various explanations of the different salt effects were proposed, among them a saltdependent conformational change of the synthetase [4], stabilization of a certain tRNA conformation [2,9], or an interference by salt with the interaction between synthetase and tRNA [1,10].…”

Section: Introductionmentioning

confidence: 99%

The mechanism of salt‐induced stimulation of tRNA^Ser aminoacylation by yeast seryl‐tRNA synthetase

Dibbelt

Zachau

1981

FEBS Letters

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Quantitative study of the ionic interactions between yeast tRNA^Val and tRNA^Phe and their cognate aminoacyl‐tRNA ligases

Cited by 29 publications

References 22 publications

Lack of Correlation between Affinity of the tRNA for the Aminoacyl-tRNA Synthetase and Aminoacylation Capacity as Studied with Modified tRNAPhe

Lack of Correlation between Affinity of the tRNA for the Aminoacyl-tRNA Synthetase and Aminoacylation Capacity as Studied with Modified tRNAPhe

Interaction of tRNA^Phe and tRNA^Val with Aminoacyl‐tRNA Synthetases

The mechanism of salt‐induced stimulation of tRNA^Ser aminoacylation by yeast seryl‐tRNA synthetase

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Quantitative study of the ionic interactions between yeast tRNAVal and tRNAPhe and their cognate aminoacyl‐tRNA ligases

Cited by 29 publications

References 22 publications

Lack of Correlation between Affinity of the tRNA for the Aminoacyl-tRNA Synthetase and Aminoacylation Capacity as Studied with Modified tRNAPhe

Lack of Correlation between Affinity of the tRNA for the Aminoacyl-tRNA Synthetase and Aminoacylation Capacity as Studied with Modified tRNAPhe

Interaction of tRNAPhe and tRNAVal with Aminoacyl‐tRNA Synthetases

The mechanism of salt‐induced stimulation of tRNASer aminoacylation by yeast seryl‐tRNA synthetase

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Product

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Quantitative study of the ionic interactions between yeast tRNA^Val and tRNA^Phe and their cognate aminoacyl‐tRNA ligases

Interaction of tRNA^Phe and tRNA^Val with Aminoacyl‐tRNA Synthetases

The mechanism of salt‐induced stimulation of tRNA^Ser aminoacylation by yeast seryl‐tRNA synthetase