NUCLEOLYTIC PROCESSING OF A TRANSFER RNAARG-TRANSFER RNAASP DIMERIC PRECURSOR BY A HOMOLOGOUS COMPONENT FROM SACCHAROMYCES-CEREVISIAE

Abstract

A subcellular extract from S. cerevisiae was 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 separated the transcription components from 2 distinct nucleolytic activities: an endonuclease that cleaves the precursors to produce mature 5'' termini; and a 3''-5'' exonuclease. These fractions were 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 per h, 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 sequence. A 2nd endonuclease cleavage of pre-tRNAArg generates the mature 5'' terminus of tRNAArg. The endonuclease cleaves are not ordered. Exonuclease activity(ies) remove the spacer sequence 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.