Abstract
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.
Highlights
From their experiments with a ternary complex having UCUC at the 3Ј end, these authors concluded that the hydrolytic activity cuts primarily in dinucleotide increments. They noticed that mononucleotides were produced but at a lower frequency. To examine this process in detail, we studied cleavage reactions in polymerase III (Pol III) transcription complexes stalled within many different sequence contexts
RNA Polymerase III Nuclease complexes were formed in a promoter-dependent system that required Pol III factors for initiation
The ribonuclease activities associated with E. coli RNA polymerase and with eukaryotic Pol II both degrade nascent RNA sequentially from the 3Ј terminus, releasing, in many instances, mono, di, and trinucleotide split products [1, 4, 5, 28]
Summary
In this paper we have extended the study of pol III nuclease activity to elongation complexes stalled within a number of different sequence contexts For several of these we characterized both products of nuclease cutting, the released 3Ј oligonucleotides as well as the shortened 5Ј transcript. Because the bond between a 3Ј-terminal uridylate and the penultimate nucleotide is not efficiently cleaved, we infer that in growing RNAs having an oligouridine stretch at the 3Ј end, the last residue is not base paired to the DNA template This suggests that at arrest sites, the 3Ј transcript end becomes detached from the template due to the exceptional instability of (rU1⁄7dA) hybrids [17]. This line of reasoning suggests that Pol III nuclease may have a proofreading function similar to the well known 3Ј-5Ј exonuclease of many DNA polymerases [18]
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