Nucleolytic processing of a tRNAArg-tRNAAsp dimeric precursor by a homologous component from Saccharomyces cerevisiae

A subcellular extract from Saccharomyces cerevisiae has been used to transcribe cloned yeast tRNA genes in vitro and to process the primary transcripts at the 5' and 3' termini. Chromatographic fractionation of the extract has separated the transcription components from two distinct nucleolytic activities: an endonuclease that cleaves the precursors to produce mature 5' termini; and a 3'-5' exonuclease. These fractions have been used to elaborate a processing pathway for the dimeric primary transcript of the yeast tRNAArg-tRNAAsp gene pair. Under optimal conditions in vitro this gene is expressed at a rate of 200 transcripts/gene/hour, initiating at position -10 with respect to the mature 5' terminus of tRNAArg and terminating near position +160. The primary transcripts are cleaved by an endonuclease to give tRNAAsp with a mature 5' terminus, and a pre-tRNAArg monomer with a 5' leader and 3' trailer sequences. A second endonuclease cleavage of pre-tRNAArg generates the mature 5' terminus of tRNAArg. The endonuclease cleavages are not ordered. Exonuclease activity(ies) remove the spacer sequences from the 5' mature tRNAArg, and trim the 3' trailer portion from tRNAAsp. Exonucleolytic removal of the 3' trailer does not require prior endonuclease action, but removal of the spacer sequences from pre-tRNAArg is incomplete without prior removal of the 5' leader sequences.