On the conformation of transfer RNA.

The determination of base sequences of several transfer ribonucleic acids has led to proposals for their secondary structure. The most popular structural model has had the form of a cloverleaf.' There have also been some indications of an additional fixation of the arms of the model in a tertiary structure. So far there is no direct experimental proof for any of these structures, although investigations have included studies of sedimentation, hyperchromicity, viscosity, and kinetics of enzymatic degradation.2 Recently, methods of nuclear magnetic resonance (NMR),8 4circular dichroism (CD),5 and optical rotatory dispersion (ORD) have been applied.6-8 Attempts have been made to draw conclusions about the secondary and/or tertiary structure from chemical modifications of tRNA. The specific cyanoethylation of pseudouridine9 in the Gp-Tp-ip-Cp-Gp loop occurs only at low ionic strength. Water-soluble carbodiimide reacts preferentially with certain regions in tRNA fl, st, an indication that these are exposed.'0 At elevated temperatures, additional regions become exposed and available to the reagent. Selective N-oxidation of adenosine to adenosine-l-N-oxide in polynucleotides is possible with monoperphthalic acid and can be followed easily by changes in ultraviolet absorption. The reaction takes place only at the non-hydrogenbonded N-i-nitrogen of adenosine. Therefore, since this method discriminates between base-paired and non-base-paired adenosines, it can be used to determine the structure of tRNA. The validity of this method has been proved in several investigations." We now wish to propose a general structure for the conformation of tRNA molecules. This structure is compatible with physicochemical, chemical, and biochemical evidence.'2 Materials and Methods.-tRNAP"8t and tRNA" t were obtained from crude tRNA (Boehringer: Mannheim, Germany) by combining extraction' with chromatography on benzoylated 0-diethylaminoethyl (DEAE) cellulose'4 and DEAE-Sephadex. 1" 16 Brewers' yeast and bakers' yeast were used. Chargeability was 82%o with both tRNAPe6e 58% with tRNAbr. yeast, and 89% with tRNAba. yeast. The tRNA's were pure with respect to 15 other amino acids tested (C14 L amino acid, kit B: Schwarz Bio-Research, Orangeburg, N.J.). tRNAb"' oi was a product of CalBiochem, Los Angeles, Calif. The following enzymes were used: pancreatic RNase, snake venom phosphodiesterase, and alkaline phosphomonesterase from E. coli (Boehringer: Mannheim, Germany); TjRNase (Sankyo: Tokyo, Japan). Spleen phosphodiand phosphomonoesterase were prepared in our laboratory by Dr. H. Sternbach. Aminoacyl-tRNA synthetases (E.C.6.1.1.): These were prepared from bakers' yeast (Langemeye.r: Mettingen, Germany) by a modification of the procedure of Makman and Cantoni.'7 Seryland phenylalanyl-tRNA synthetases were both purified 350-fold.1' Charging experiments were carried out as described elsewhere.24 Hyperchromicity: The measurements were taken on a Gilford type 2000 recording spectrophotometer with a temperature controlled cell-compartment.