The primary transcription product of a silkworm alanine tRNA gene: identification of in vitro sites of initiation, termination and processing

Abstract

A 13.5 Kb fragment of Bombyx mori DNA containing a single tRNA2Ala gene has been cloned, and transcribed in vitro with Xenopus germinal vesicle extracts. The primary transcription product of the tRNA2Ala gene has been isolated and shown to possess an unprocessed triphosphorylated 5' terminus. Products resulting from processing of this transcript have also been isolated and characterized. Complete nucleotide sequence analysis of this cloned alanine tRNA gene and its primary transcript shows that transcription initiates three nucleotides away from the mature tRNA2Ala 5' end and terminates in a U cluster 22 nucleotides beyond the last encoded 3' nucleotide of the mature species. Sequence determination of the products of in vitro maturation shows that in contrast to the tRNA processing mechanism characteristic of procaryotes, the extra 3'-nucleotides in this silkworm tRNA precursor are removed by a single endonucleolytic cleavage.