A Minimal Promoter for TFIIIC-dependent in Vitro Transcription of snoRNA and tRNA Genes by RNA Polymerase III

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 TFIIICinteracting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription.RNA polymerase (Pol) 3 III synthesizes tRNA, 5 S rRNA, and a variety of other types of small nuclear and cytoplasmic RNAs. In general, the transcription of class III genes is under the control of internal control regions (ICR) characterized by discontinuous structures, with essential boxes separated by non-essential nucleotides (1). In the case of tRNA and 5 S rRNA genes, the ICRs are highly conserved and comprise the binding sites for the general transcription factor TFIIIC (box A and box B) and for the 5 S-specific factor TFIIIA (box C). Once assembled, TFIIIC recruits TFIIIB upstream of the transcription start site (TSS); TFIIIB in turn recruits Pol III for transcription initiation. The strong conservation of the ICRs likely reflects their dual function as both nucleation sites for transcription complex assembly and key determinants of tRNA and 5 S rRNA structure. 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. For example, in the Saccharomyces cerevisiae SCR1 gene, coding for the 7SL RNA, box A and box B are both intragenic an...

Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

“…115 Importantly, it was also experimentally shown that the A-box of the SNR52 gene is sufficient for directing S. cerevisiae RNAP III transcription, supporting a functional specialization of submodules within TFIIIC into GTF (τA) and transcriptional activator (τB). 120 In accordance with this model, the B-Box element within the transcribed regions of genes with type 2 promoter elements may be considered as an enhancer element. Since the subunit composition of yeast and human TFIIIC has been conserved during evolution, 52 it can be assumed that this point of view may also hold true for human RNAP III transcription although this has not yet formally been shown.…”

RNA polymerase III transcription control elements: Themes and variations

“…Correspondingly, the A box has the characteristics of a core promoter element (proximity to TSS, role in PIC assembly) while the B box resembles an enhancer-like element. In support to this view, an A box has been shown to suffice for faithful and efficient in vitro transcription of mutated Pol III templates carrying no functional B box, thus sketching a scenario in which a minimal TFIIIC/TFIIIB assembly module, comprising an A box and possible upstream elements (such as a TATA and/or an Initiator-like element; see next section) surrounding the TSS, is reminiscent of the composite organization of RNA polymerase II core promoters (Guffanti et al, 2006a). As an integral part of such a core promoter module, the A box-interacting sub-complex of TFIIIC participates in TSS selection together with the TATA-box binding protein (TBP) which is part of TFIIIB (Joazeiro et al, 1996).…”

“…Interestingly such an effect (usually weakening the terminator, and becoming apparent only in the presence of T stretches of minimal length) can be exerted both by very short sequence stretches just downstream of the poly(dT) and by upstream sequences, suggesting at least two possible mechanisms of modulation, one involving contacts between the front end of polymerase and downstream DNA, the other possibly involving an allosteric action on polymerase of nascent RNA (Braglia et al, 2005). The strongest known termination-weakening context is the one occurring within the SNR52 transcription unit of S. cerevisiae, in which a T 6 stretch located between the A box and the B box of a tRNA-like leader sequence is largely read through by Pol III, a behavior which is determinant for the efficient production of the encoded snoRNA (Braglia et al, 2005;Guffanti et al, 2006a).…”

A differential sequencing-based analysis of the C. elegans noncoding transcriptome

“…UM2 snoRNA loci give rise to primary transcripts that resemble the dicistronic primary tRNA-snoRNA transcripts found in yeast and plants, which are cleaved by RNase Z, thereby generating the mature snoRNAs with 59 monophosphate termini (Kruszka et al 2003;Guffanti et al 2006). The processing mode of UM2 snoRNA primary transcripts is not known but is likely to generate mature snoRNAs with 59 monophosphate termini (since both endonuclease and exonuclease activity creates this type of 59 terminus).…”