The intermediates of the yeast tRNA splicing reaction have been characterized. The intervening sequence is excised as an unique linear molecule. It has 5'-hydroxyl and 3'-phosphate termini. Correspondingly, the half-tRNA molecules are shown to have a 3'-phosphate terminus on the 5' half and 5'-hydroxyl terminus on the 3' half. These isolated halves have been shown to be active in the ligation step of tRNA splicing. Removal of the 3'-phosphate from the 5' half eliminates the ability of the 5' half to participate in ligation.
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