Abstract
RNA polymerase (RNAP) III synthesizes tRNAs and other transcripts, and mutations to its subunits cause human disorders. The RNAP III subunit-heterodimer C37/53 functions in initiation, elongation and in termination-associated reinitiation with subunit C11. C37/53 is related to heterodimers associated with RNAPs I and II, and C11 is related to TFIIS and Rpa12.2, the active site RNA 3' cleavage factors for RNAPs II and I. Critical to termination is stability of the RNA:DNA hybrid bound in the active center, which is loose for RNAP III relative to other RNAPs. Here, we examined RNAP III lacking C37/53/C11 and various reconstituted forms during termination. First, we established a minimal terminator as 5T and 3A on the non-template and template DNA strands, respectively. We demonstrate that C11 stimulates termination, and does so independently of its RNA cleavage activity. We found that C37/53 sensitizes RNAP III termination to RNA:DNA hybrid strength and promotes RNA 3' end pairing/annealing with the template. The latter counteracts C11-insensitive arrest in the proximal part of the oligo(T)-tract, promoting oligo(rU:dA) extension toward greater hybrid instability and RNA release. The data also indicate that RNA 3' end engagement with the active site is a determinant of termination. Broader implications are also discussed.