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We have isolated a precursor of yeast tRNATyrand shown that it contains an intervening sequence identical to that found in the gene for tRNATyr. The conformation of pre-tRNATyris similar to that of mature tRNATyrexcept for the anticodon loop. The loop is sensitive to endonucleolytic cleavage by S1nuclease near to the ends of the intervening sequence. This pre-tRNA is functionally inactive as it cannot be aminoacylated and the anticodon is not accessible for hydrogen bonding. A crude nuclear extract from yeast contains an excision-ligase activity which will process pre-tRNATyrinto mature tRNATyr.