SUBSTRATE-SPECIFICITY OF THE RNASE ACTIVITY OF YEAST RNA-POLYMERASE-III

Using yeast RNA polymerase III ternary complexes stalled at various po sitions on the template, we have analyzed the cleavage products that a re retained and released by the transcription complexes. The retained 5' products result from cleavage at uridine residues during retraction , whereas the yield of mononucleotides and dinucleotides released indi cates that multiple cuts occur near the 3' end, Comparison of the clea vage patterns of uridine-containing and 5-bromouridine-containing tran scripts suggests that RNA within an RNA-DNA hybrid duplex is the subst rate for the 3'-5' exonuclease. During transcription of the SUP4 tRNA( Tyr) gene, RNA polymerase III produces not only full-length pre-tRNA(T yr) but also short oligonucleotides, indicating that exonuclease diges tion and transcription are concurrent processes. To explore the possib ility that these oligonucleotides are released by the action of the RN A polymerase III nuclease at previously observed uridine-rich pause si tes, we tested modified templates lacking the arrest sites present in the SUP4 tRNA(Tyr) gene, Comparative studies of cleavage during transc ription for these templates show a direct correlation between the numb er of natural pause sites and the yield of 3' products made, At the na tural arrest sites and the terminator, RNA polymerase III carries out multiple cleavage resynthesis steps, producing short Oligoribonucleoti des with uridine residues at the 3' terminus.