Die Temperaturabhängigkeit der Aminoacylierungsgeschwindigkeit von Phenylalanin- und Serin-spezifischer Transfer-Ribonucleinsäure aus Brauerhefe

Abstract

It is known that several physical properties in tR N A * are changed between 35 °C and 45 °C. These changes have been attributed to unfolding of tertiary structure. In this paper the correlation between these changes and the rate of aminoacylation is studied. W ith purified tR N A yILt and tRNAyeast and purified aminoacyl synthetases, aminoacylation reaches a plateau within 20 min. (Fig. 2). The rate of charging was determined in the linear re gion (5 min.) at varying temperatures. W ith tRN A ylLst there is a sharp drop in the rate of charging at 39° (Fig. 3 a) corresponding to the first melting of tRNAyJLt (Fig. 6 a). By changing the Mg2® concentration, rate of aminoacylation and first melting are shifted in the same direction. W ith tRNAyeast no stepwise decrease of aminoacylation with temperature is observed (Fig. 3 b). At the same time this molecule does not exhibit a two step melting profile ( Fig. 6 b ) . The effects ob served cannot be due to a change or irreversible inactivation of the enzyme since the thermal in activation of the enzyme follows a normal pattern above 43 °C and 38.5 °C respectively (Figs. 4 a and 4 b ) . Likewise the amino acid activation reaction is not responsible for these effects. This reac tion has been measured by the pyrophosphate formation to have a normal temperature profile up to 50 °C. Only at this temperature the enzyme begins to become denatured (Fig. 5). The results indicate that the observed changes in aminoacylation are caused by a change in physical properties of the tRNA. This change yields a molecule having a conformation that can no longer be aminoacylated. Since at the higher temperature still the same plateau is reached, however, at a slower rate, an equilibrium between the two forms must exist. This equilibrium could be of the following manner: Completely Ordered Structure _____^ Partially Unfolded Structure (Tertiary Structure, chargeable) ^ (Secondary Structure, not chargeable) Die Temperaturabhängigkeit physikalischer, phy sikalisch-chemischer und chemischer Eigenschaften von Transfer-Ribonucleinsäure weist auf struktu relle Änderungen des Moleküls im Bereich von etwa 40 °C hin. Dies kann unter anderem geschlossen werden aus Messungen der O R D 1, der Röntgen-Kleinwinkelstreuung2 und des Schmelzverhaltens (Hyperchromie) 3' 4, aus hydrodynamischen Daten 5 und Befunden der Adenosin-TV-Oxydation4 sowie kinetischen Messungen des enzymatischen Abbaus 6 von tRNS. Die vorliegende Arbeit untersucht, in welcher Weise strukturelle Änderungen im Temperatur bereich um 40 °C die Aminoacylierung der tRNS * Abkürzungen: O RD = Optische Rotationsdispersion, tRNS= T ransf er-Ribonucleinsäure, ATP = Adenosin-5'-triphosphat, CTP = Cytidin-5'-triphosphat, PEP = Phosphoenolpyruvat, Aminoacyl-tRNS-Synthetasen (E.C.6.1.1.).1 P. S. S a r i n , P. C. Z a m e c n ik , P. L . B e r g q u i s t u.

Hydrolytic action of aminoacyl-tRNA synthetases from Baker's yeast: "chemical proofreading" preventing acylation of tRNAIle with misactivated valine

“…After drying the spots were marked under a uv lamp. Nucle-otide containing spots were cut out and prepared for liquid scintillation counting as described (Schlimme et al, 1969).…”

Phenylalanyl-tRNA Synthetase from Baker's Yeast. Salt Dependence of Steady-State Kinetics Indicates Two Molecular Forms of the Enzyme

“…All assays were run at 37 "C. Liquid scintillation counting was done as described [4,7]. Throughout the paper velocities, u, are expressed as radioactivity bound per unit time; 1000 counts/min correspond to 17 pmol ['4C]phenylalanine.…”

Isoleucyl‐tRNA synthetase from baker's yeast: The 3′‐hydroxyl group of the 3′‐terminal ribose is essential for preventing misacylation of tRNAIle‐C‐C‐A with misactivated valine

“…Isoleucylation and valylation were performed according to [7]. The assay mixture contained 150 mM TrisHCl buffer pH 7.6,150 mM KCl, 10 mM MgSO*, 0.06 mM ['4C]isoleucine or [*4C]valine and 0.003 mM tRNAUe-C-C-N.…”