Fluorimetric Study of the Complex between Yeast Phenylalanyl‐tRNA Synthetase and tRNA^Phe

Abstract

The interaction between yeast tRNAPhe and phenylalanyl-tRNA synthetase was studied by monitoring different emission properties of the wybutine residue. The following results were established: (a) the isolated tRNAPh" exists in solution under at least two forms in a magnesiumdependent equilibrium; (b) in the complex with the cognate synthetase, the tRNA still has two different conformations, slightly different from the above conformers. Mg2 + ions appear to play a crucial role in the adaptation of both macromolecules one to another: for concentrations of the order of 1 mM, magnesium ions trigger a conformational change of the anticodon loop of tRNA, resulting in the expulsion of the wybutine from a stacked region. Furthermore, experimental evidence suggests that the anticodon region lies in a groove of the protein.This local conformation change, occurring in the anticodon loop, can be correlated to structural modifications of the enzyme as shown by the modification of circular dichroism and tryptophan fluorescence.During these different conformation changes, an energy transfer from tryptophan residues to wybutine is triggered in the critical magnesium concentration range.The specific fluorescence of wybutine (YWye), located in the anticodon loop of yeast tRNAPh", has been used in numerous studies of the conformation of the tRNA in solution, as well as to monitor the interaction of this molecule with its partners, in the different biological processes involving the tRNA. The early studies [l-31 showed that the YWye fluorescence is a very sensitive probe for the local conformation of the anticodon loop and that it strongly depends upon external conditions, especially the Mg2+ concentration and pH. Studies of the YWye fluorescence intensity in barley tRNAPhe [4,5] and in yeast tRNAPhe [6], as a function of divalent cation concentration (respectively Mg2 + and Mn2 +) and monovalent cations, afforded an insight into the role of cations in the structure of tRNAs.Abbreviations. The modified base at position 37 of tRNAPhe from S. cerevisiae (formerly called Y base) is called wybutine, abbreviated YWye, as proposed by W. E. Cohn; CD, circular dichroism.Enzymes. Phenylalanyl-tRNA synthetase (EC 6.1 . I .20); aspartyl-tRNA synthetase (EC 6.1.1.12); valyl-tRNA synthetase (EC 6.1.1.9).Krauss et al. [7] showed that YWye fluorescence was quenched upon interaction with the cognate aminoacyl-tRNA synthetase, in the presence of 10 mM Mg2+ : this property was used to measure the stoichiometry of the tRNA-enzyme complex [S]. Other studies have been devoted to the interaction of yeast tRNAPh" with the elongation factor Tu [9], and with poly-(uridylic acid) [lo, 1 I] or with oligonucleotides the sequence of which was complementary to the anticodon [lo].Finaly the YWye fluorescence was used as a builtin probe to study conformation modifications occurring either during aminoacylation [9] or upon thermal denaturation [12,13]. Quite recently Urbanke and Maass [I41 reported a kinetic investigation of the conformational changes occurring i...

Comparison of the Hydrolysis Patterns of Several tRNAs by Cobra Venom Ribonuclease in Different Steps of the Aminoacylation Reaction

“…Finally, despite many common features, each tRNA seems to have its own way of interacting with its cognate aminoacyl-tRNA synthetase. This may be the origin of the aminoacylation specificity, based on a multistep dynamic recognition process (possibly involving small ligands [2,3,34, leading to a discrimination of the cognate versus non-cognate tRNA through the rate of aminoacylation [40,41].…”

“…Such a mechanism has indeed been postulated in studies with model compounds [ l l ] and could explain the critical role of Mg2+ ions reported in the mutual adaptation of yeast phenylalanyl-tRNA synthetase and tRNAPhe [2,3].…”

“…The binding of ATP can result either in a different protection afforded to the t R N h by the aminoacyl-tRNA synthetase, or even to a structural modification of the tRNA molecule itself. It is well known that an aminoacyl-tRNA synthetase may trigger conformational changes in the complexed tRNA: in the phenylalanine system, structure modifications have becn shown to occur in the anticodon region [2], in the 3'-terminal region [4] or in the corner between acceptor stem and D stem [32]. Furthermore, both aminoacyl-tRNA synthetase and tRNA most likely undergo conccrtcd structure modifications upon complex formation, since the circular dichroism spectra of glutamyl-tRNA synthetase and phenylalanyltRNA synthetase were reported to be modified in the tRNAenzyme complex [2,33].…”

“…One of the most interesting questions is to know whether the tRNA molecules undergo conformational changes in the course of the aminoacylation process. Indeed, recent results showed such structural modifications, upon binding to the cognate aminoacyl-tRNA synthetase, in the yeast phenylalanyl system [2,3]. These changes may be responsible for the appearance of the catalytic activity or even for the specificity of aminoacylation [3].…”

Structural variability of tRNA: small-angle x-ray scattering of the yeast tRNAphe-Escherichia coli tRNAGlu2 complex.

“…This idea is supported by experimental observations of concomitant conformational transitions in anticodon loop and acceptor end of tRNAPhe on binding to the cognate synthetase (38,39) and by relaxation kinetic data suggesting that a conformational transition of tRNAPhe, with a relaxation time of 0.2-0.5 s, might be the rate-limiting step ofthe acylation reaction (36). A close spatial relation between the anticodon and aminoacyl sites on the tRNA has been connected to the unique assignment of the amino acid to the anticodon by primitive synthetases (40,41).…”