Two-dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyk3H]methionine in the presence of cycloheximide have revealed that the elongation factor la (EF-lcu) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.Elongation factor Iru Two-dimensional gel electrophoresis Methytation 3 T3B 3T3B/SV40
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.
The influence of the ion and water cloud shell on the structure of polyelectrolytes is very important especially for small angle studies. I n the present work the change in the radius of gyration of phenylalanine specific tRNA under the influence of various counterions was investigated. tRNAPhe(yeast) was studied in different solutions at pH 7.5, each containing only one certain kind of cation. As cations lithium, cesium, barium and spermidin were used. The influence of the ions on the radius of gyration of tRNAPhe (yeast) . As a rule it is not absolutely necessary t o use the coordinates of the single atoms [6] for the calculation and often the coordinates are assigned only t o the scattering centers of base, sugar and phosphate groups. This approximation has no decisive influence on the calculated value of the radius of gyration or on the inner portion of the scattering curve.The comparison of the experimental small angle X-ray data with those calculated for a particular model involves another problem. The experimental studies are carried out in solution and the influence of the solvent, the counterions and additional electrolytes on the radius of gyration is not well known.Since the problem of the structure of the water shell could not be completely clarified even with simple ions [7], the theoretical treatment of the water shell and hydration presents very great difficulties with a polyelectrolyte such as tRNA.Therefore the influence of the water and ion cloud shell is frequently ignored; some authors [3,4] assign one univalent ion to each phosphate group of the tRNA molecule for the calculation.Theoretically, polyions have discrete charged groups, and for many calculations it is assumed that counterions are bound only to these discrete groups. I n real polyions, however, a m u s e atmosphere of counterions in which the distribution of the counterions fluctuates must be assumed [7]. A theoretical treatment of this problem, based upon a geometric concept, was given by Luzzati et al. [S].I n order t o compare the experimental data with the data calculated for the various suggested models it seemed important to discover more about the influence of both hydration and the binding of counterions. Consequently we investigated tRNAPhe in solution in several cationic forms. Similar studies were effected by Luzzati et al.[S] and Bram [9] on DNA molecules; the authors determined the mass per unit length and the radius of gyration of the cross-section of DNA by using various counterions. The results obtained by the two groups are not in mutual agreement, as is discussed in detail by Eisenberg and Cohen [lo]. EXPERIMENTAL PROCEDURE MATERJAL.SPreparation of tRNAPhe( Yeast) with a Certain Cation 77 mg of tRNAPhe from yeast prepared as described earlier [l] was dissolved in 20 ml of double-distilled water. 5 ml aliquots were dialyzed once against 0.4 M sodium phosphate pH 7.0, twice
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