Fluorescence of tryptophanyl‐tRNA<sup>Trp</sup> from <i>E. coli</i>; An interaction between the indole and tRNA and its dependence on tRNA conformation

Buckingham, Richard H.; Danchin, Antoine

doi:10.1016/0014-5793(73)80659-3

Cited by 5 publications

(3 citation statements)

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“…Immediately after adenylate formation, the fluorescence intensity decrease of a reaction mixture containing stoichiometric amounts of dimeric enzyme and tryptophan is 40% of the starting value. A quenching of the order of 70% has been observed by Buckingham and Danchin [34] upon esterification of adenosine to the carboxyl group of tryptophan. Considering a similar quenching for tryptophan after adenylate formation and a contribution of 12% coming from tryptophan to the total fluorescence of the mixture, a fluorescence intensity decrease of 70 x 12/100 i. e. 8%, should be observed if the quenching was contributed by free tryptophan alone.…”

Section: Conformational Change Of Tryptophanyl-trna Synthetasementioning

confidence: 95%

Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase

Andrews

Trezeguet

Merle

et al. 1985

European Journal of Biochemistry

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When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATPMg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001 -1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-48791. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by highperformance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a K , of 1 mM for ammonia and a rate constant of 5.7 min-' at pH 8.3,25"C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.The aminoacylation of tRNA proceeds via the production of a high-energy intermediate formed between the carboxyl group of the amino acid and the phosphate group of AMP, derived from ATP by the release of pyrophosphate. The resulting mixed anhydride bond between the amino acid and AMP provides both species with a high chemical transfer potential, which can lead to several reactions deviating from the regular charging of tRNA. Depending on the synthetase studied, reactions of varying specificity and ...

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Section: Conformational Change Of Tryptophanyl-trna Synthetasementioning

confidence: 95%

Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase

Andrews

Trezeguet

Merle

et al. 1985

European Journal of Biochemistry

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“…However, the denatured wild-type tRNATrP shows no detectable interaction with tRNAPro. A preliminary account of this work was given recently (11).…”

Section: Introductionmentioning

confidence: 99%

“…tRNA tRNATrp (Su-) was prepared as previously described (11) from E. coli B tRNA (General Biochemicals). tRNA from the UGA suppressor strain CAJ64 was prepared as before (8) and tRNATrP (Su+) was purified by the same method as for the wildtype species, except for a preliminary chromatography on Sephadex A50 according to Nishimura et al (13).…”

Section: Introductionmentioning

confidence: 99%

Anticodon conformation and accessibility in wild-type and suppressor tryptophan. tRNA from E.coli.

Buckingham

1976

Nucleic Acids Research

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The association between Trp-tRNA and Pro-tRNA, which have complementary anticodon sequences, has been used as a probe of anticodon conformation. It is unaffected, however, by the base change in the D-stem present in UGA-suppressor Trp-tRNA. This does not support the hypothesis that UGA suppression depends upon a conformational change induced in the anticodon. The stable denatured form of wild-type Trp-tRNA no longer interacts with Pro-tRNA; the structure of the anticodon region must therefore be quite different in the denatured form.

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Structure of Viral Nucleic Acids in Situ

Tikchonenko

1975

Structure and Assembly

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Fluorescence of tryptophanyl‐tRNA^Trp from E. coli; An interaction between the indole and tRNA and its dependence on tRNA conformation

Cited by 5 publications

References 10 publications

Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase

Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase

Anticodon conformation and accessibility in wild-type and suppressor tryptophan. tRNA from E.coli.

Structure of Viral Nucleic Acids in Situ

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Fluorescence of tryptophanyl‐tRNATrp from E. coli; An interaction between the indole and tRNA and its dependence on tRNA conformation

Cited by 5 publications

References 10 publications

Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase

Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase

Anticodon conformation and accessibility in wild-type and suppressor tryptophan. tRNA from E.coli.

Structure of Viral Nucleic Acids in Situ

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Fluorescence of tryptophanyl‐tRNA^Trp from E. coli; An interaction between the indole and tRNA and its dependence on tRNA conformation