Conformation and binding of lysyl-tRNA to poly-A-ribosome complexes.

The role of tRNA' in accepting and transferring amino acids during protein synthesis has been well documented.2 However, the specific physicochemical mechanisms involved in these reactions have not been completely elucidated. Peterkofsky3 and Carbon4 have shown that physiological or chemical alteration of tRNA affects its capacity to accept amino acids. Recently, Lipsett5 and Carbon4 observed the presence of thiopyrimidines in tRNA of E. coli. Similar results were obtained for tRNA of B. subtilis.6 The reversible oxidative inactivation of amino acid acceptor ability reported by Carbon can also be demonstrated with certain tRNA species of B. subtilis. Although this effect is tenatatively linked to the occurrence of thiobases in tRNA, no direct evidence has been obtained as yet. Evidence is presented in this paper for reversible changes in conformation and binding efficiency of lysyl-tRNA following mild oxidation treatment. This suggests another possible role of thiobases in tRNA, and demonstrates the requirement for a specific conformation of tRNA in the binding process. Methods and Materials.-Organisme and medium: B. subtilis W23 was used as the source of tRNA and aminoacyl synthetase. Penassay medium (Difco) was used for growing cells. Log phase cells were harvested at a density of 1-2 X 108 cells per ml. E. coli Q13, kindly provided by Dr. L. Overby, was used for the preparation of ribosomes. These cells were grown to mid-log phase in Penassay medium (DifCo) at 37°C. Preparation of tRNA: The tRNA was extracted from cells as described by von Ehrenstein and Lipmann,7 except for three additional phenol extractions. The tRNA was treated with 0.5 M Tris-HCl buffer, pH 8.8, for 60 min at 350C to remove bound amino acids. The solution was made 1.0 M in NaCl, and the tRNA was precipitated by the addition of 2 vol of ethanol. The precipitate was dissolved in 0.01 M Tris-HCl buffer, pH 8.0, and dialyzed against the same buffer overnight. After concentrating by lyophilization, the tRNA was stored in the frozen state. Preparation of aminoacyl-tRNA synthetase: The synthetase was prepared generally as described by Zubay.8 Preparation of aminoacyl-tRNA: The reaction mixture of 0.5 ml contained in ,umoles: TrisHCl buffer, pH 7.3, 40; ATP, 1; KCl, 5; MgCl2, 5; phosphoenolpyruvate, 5; 1 jMmole each of 19 unlabeled amino acids; and 0.1-0.15 mg of enzyme; 0.5-1 mg tRNA; 10 Ag pyruvate kinase; and 1 gc of lysine-C'4. For larger batches, the reaction mixture was scaled up tenfold. The mixture was incubated at 370C for 10 min with gentle shaking. The reaction was stopped by adding an equal volume of water-saturated phenol to the mixture in an icebath and extracted by thc method of Gierer and Schramm.9 The final ethanol precipitate was dissolved in 0.05 M citratephosphate buffer, pH 5.5, and kept at -20°C. The method of Carbon et al.10 was used for oxidation and reactivation of the lysyl-C'4-tRNA. Preparation of MAK column: The method is essentially that of Sueoka and Yamane,11 and the assay method was described in detail previously.12 Assay of lysyl-C14-tRNA binding to poly-A-ribosome complex: The method of Nirenberg and Leder13 was used for the binding experiments. The only exception was the omission of mercaptoethanol during preparation of the ribosomes. Optical rotatory dispersion analysis was performed as described by Sarin and Zamecnik.14 Materials: Reagents were obtained from the following sources: Schwarz BioResearch, Inc., L-lysine-C14 (240 mc/mmole); Calbiochem, 2-phosphoenolpyruvate, pyruvate kinase (rabbit muscle), ATP, CTP; and Sigma Chemical Co., poly-A.