Search

RNA polymerase (pol) III, assisted by the transcription factors TFIIIC and TFIIIB, transcribes small untranslated RNAs, such as tRNAs. In addition to known pol III-transcribed genes, the Saccharomyces cerevisiae genome contains loci (ZOD1, ETC1-8) associated to incomplete pol III transcription complexes (Moqtaderi, Z., and Struhl, K. (2004) Mol. Cell. Biol. 24, 4118-4127). We show that a short segment of the ZOD1 locus, containing box A and box B promoter elements and a termination signal between them, directs the pol III-dependent production of a small RNA both in vitro and in vivo. In yeast cells, the levels of both ZOD1- and ETC5-specific transcripts were dramatically enhanced upon nucleosome depletion. Remarkably, transcription factor and pol III occupancy at the corresponding loci did not change significantly upon derepression, thus suggesting that chromatin opening activates poised pol III to transcription. Comparative genomic analysis revealed that the ZOD1 promoter is the only surviving portion of a tDNA(Ile) ancestor, whose transcription capacity has been preserved throughout evolution independently from the encoded RNA product. Similarly, another TFIIIC/TFIIIB-associated locus, close to the YGR033c open reading frame, was found to be the strictly conserved remnant of an ancient tDNA(Arg). The maintenance, by eukaryotic genomes, of chromatin-repressed, non-coding transcription units has implications for both genome expression and organization.

The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA(Asn)(GTT) gene family, but not for two tRNA(Pro)(AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA(Asn) genes, but only in the presence of TFIIIB reconstituted with a crude B'' fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA(Asn) genes was strongly influenced by their 5'-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIIC-interacting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription.

We have shown previously that a T(10) peptide nucleic acid (PNA) bound to the transcriptional terminator of a Saccharomyces cerevisiae tDNA(Ile)(TAT) gene arrests elongating yeast RNA polymerase (pol) III at a position that precedes by 20 bp the upstream end of the PNA roadblock (Dieci, G., Corradini, R., Sforza, S., Marchelli, R., and Ottonello, S. (2001) J. Biol. Chem. 276, 5720-5725). Here, a PNA-binding cassette was placed at various distances downstream of a functional tDNA(Ile) transcriptional terminator (T(6)) that is not bound by the T(10) PNA, and the effect of the PNA roadblock on RNA 3'-end formation, transcript release, and transcription reinitiation was examined. With a PNA roadblock placed as close as 5 bp downstream of the T(6) terminator, pol III could still reach the termination site and complete pre-tRNA synthesis, implying that the catalytic site-to-front edge (C-F) distance of the polymerase can shorten by >10 bp upon recognition of the terminator element. In addition, transcripts synthesized by a PNA-roadblocked terminating pol III were found to be released from transcription complexes. Interestingly, however, the same roadblock dramatically reduced the rate of transcription reinitiation. Also, when placed 5 bp downstream of a mutationally inactivated terminator element (T(3)GT(2)), the PNA roadblock restored transcription termination, thus indicating that the inactivated terminator is compromised in its ability to cause pol III pausing, but can still induce C-F distance shortening and transcript release. The latter two activities were found to be further impaired in variants of the inactivated terminator bearing fewer than three consecutive T residues (T(2)G(2)T(2) and TG(2)TGT). The data indicate that RNA polymerase pausing, C-F distance shortening, and transcript release are functionally distinguishable features of the termination process and point to the RNA release propensity of pol III as a major determinant of its remarkably high termination efficiency.