Substrate specificity of the RNase activity of yeast RNA polymerase III.

Using yeast RNA polymerase III ternary complexes stalled at various positions on the template, we have analyzed the cleavage products that are 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 indicates that multiple cuts occur near the 3' end. Comparison of the cleavage patterns of uridine-containing and 5-bromouridine-containing transcripts suggests that RNA within an RNA-DNA hybrid duplex is the substrate for the 3'-5' exonuclease. During transcription of the SUP4 tRNATyr gene, RNA polymerase III produces not only full-length pre-tRNATyr but also short oligonucleotides, indicating that exonuclease digestion and transcription are concurrent processes. To explore the possibility that these oligonucleotides are released by the action of the RNA polymerase III nuclease at previously observed uridine-rich pause sites, we tested modified templates lacking the arrest sites present in the SUP4 tRNATyr gene. Comparative studies of cleavage during transcription for these templates show a direct correlation between the number of natural pause sites and the yield of 3' products made. At the natural arrest sites and the terminator, RNA polymerase III carries out multiple cleavage resynthesis steps, producing short oligoribonucleotides with uridine residues at the 3' terminus.