STUDIES ON POLYNUCLEOTIDES, LXVIII * THE PRIMARY STRUCTURE OF YEAST PHENYLALANINE TRANSFER RNA

Following the pioneering work of Holley and co-workers' on yeast alanine tRNA,' nucleotide sequences of two similar serine tRNA's3 and of one tyrosine tRNA4 from yeast have been reported. The present report contains a brief account of the work leading to the elucidation of the nucleotide sequence of yeast phe-tRNA. Purification and Nucleotide Composition.-The method used for the purification of phe-tRNA was that of countercurrent distribution as described previously.5 This method gives a major and a minor peak; the two peaks differ most probably only in the nature of the 3'-end group, the major peak having lost the 3'-terminal adenosine residue.6 The structural work reported herein has all been on the major peak which contains cytidine at 3'-terminus. The purity of RNA used was estimated to be higher than 90 per cent. Phe-tRNA contains a total of 76 nucleotide units, including the terminal A required for amino acid acceptance. Fourteen nucleosides of minor bases, are present, 13 of which are as follows, their number/mole of tRNA being shown in parentheses: T(1), 4/(2), DiHU(2), MeC(2), 2'OMeC(1), MeA(1), 2M\eG(1), Di1\1eG(1), 2'ONIeG(1), and 71\1eG(1). The 14th nucleoside, which is still unidentified, is strongly fluorescent under ultraviolet light and is simply designated as Y. Of the identified minor nucleosides, 2'0\IeC and 7MeG have been isolated from a pure tRNA for the first time. Products of Digestion with Pancreatic and T1-RNases.-The structural work followed the general principles used by previous workers.".3 4 Thus, the first phase consisted of the characterization of the total products obtained on complete degradation with the two RNases, and the second phase involved the isolation and sequential analysis of larger fragments obtained by partial enzymic degradation until the overlaps permitted the derivation of a unique sequence. Methods used for the separation of the number of large oligonucleotide products formed involved column chromatography using diethylaminoethyl cellulose in the presence of 7 M urea.8 Table 1 lists the products of complete digestion of phe-tRNA with pancreatic RNase. Identification of the 5'-terminal sequence as pG-Csupports earlier conclusions arrived at using end-group labeling techniques.9 Analyses of most of the fragments were possible by the usual methods.'0 The octanucleotide G-G-G-A-G-A-G-Cwas characterized by the application of a technique developed for end-group labeling of 2',3'-diol residues in RNA.6, ' Sequence analysis of the hexanucleotide 2'OMeG-A-A-Y-A-4'was more difficult and involved the use of micrococcal nuclease and T2-RNase. Table 2 lists the products obtained by complete degradation with T1-RNase. As is seen, the only product not bearing a 3'-phosphate end group was the oligonucleotide C-A-C-COH, and this clearly represented the sequence at 3'-terminus of the RNA. This result is in agreement with conclusions drawn previously from end-