Abstract
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.
Highlights
RNA polymerase (Pol)3 III synthesizes tRNA, 5 S rRNA, and a variety of other types of small nuclear and cytoplasmic RNAs
Mutational Analysis of the SNR52 Promoter—The S. cerevisiae SNR52 gene has recently been identified as a novel class III transcription unit in which a snoRNA coding region is preceded by a leader region containing putative box A and box B control regions, as well as a TATA element located ϳ60-bp upstream of the box A [10]
The A box and B box are both required for SNR52 transcript accumulation in vivo, whereas in vitro the box B appears to be dispensable for transcription
Summary
RNA polymerase (Pol) III synthesizes tRNA, 5 S rRNA, and a variety of other types of small nuclear and cytoplasmic RNAs. An indication of the constraints imposed on ICR by their overlapping structural and functional roles comes from the variability of promoter organizations displayed by the minority of class III genes not coding for tRNA and 5 S rRNA In some of these genes, the TFIIIC-interacting control regions (box A and box B) have been maintained within the transcribed region, and adapted to the structure of the small RNA without losing the transcriptional function. The upstream regions of ICR-containing class III genes are characterized by the presence of a TATA box, starting at around Ϫ30, which strongly contributes to transcription efficiency as an essential component of the promoter [25, 26]. This finding prompted us to re-evaluate the contribution of TFIIIC-box B interaction to promoter strength and its actual requirement for tRNA gene transcription
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