TRANSCRIPTION OF A CLONED BOMBYX-MORI TRANSFER RNA-2ALA GENE - NUCLEOTIDE-SEQUENCE OF THE TRANSFER-RNA PRECURSOR AND ITS PROCESSING INVITRO

The transcription of a cloned silkworm (B. mori) .**GRAPHIC**. gene in germinal vesicle extracts of X. laevis (frog) oocytes was analyzed. The primary transcript was sequenced; it is 98 nucleotides long, beginning with a 5'' triphosphate nucleotide and ending in a 3'' oligouridine stretch. After transcription for long periods of time, enzymes in the frog extract also process the .**GRAPHIC**. precursor to remove extra 5'' and 3'' nucleotides and to add a CCA end. The 22 extra nucleotides at the 3'' end of this precursor are recovered as an intact fragment, implicating a new site of endoribonuclease cleavage in eukaryotic tRNA processing. This enzyme activity was also demonstrated by reincubation of isolated .**GRAPHIC**. with a germinal vesicle extract. The products of in vitro cleavage are the same as those seen in the transcription reactions. The .**GRAPHIC**. precursor molecules are made faithfully in the system with as few as 6 bp of B. mori DNA upstream of the transcription initiation site of the .**GRAPHIC**. gene. This result narrows down the minimal amount of DNA adjacent to the 5'' end of a eukaryotic tRNA gene needed to support proper initiation by RNA polymerase III.