A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

Abstract

The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position ؊30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele of SNR6 with decreased spacing between the A and B blocks, snr6-⌬42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-⌬42 is much more sensitive to mutations in a (dT-dA) 7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-⌬42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIICindependent transcription of SNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA) 7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.Initiation of transcription by an RNA polymerase at the start site of a gene involves multiple protein-DNA interactions, but one protein-DNA interaction often has a dominant role in specifying the site and efficiency of transcription complex assembly. For example, a central step in initiation by eukaryotic RNA polymerase II (Pol II) on many protein-coding genes is binding of transcription factor (TF) IID, via its TATA-binding protein (TBP) subunit, to an upstream TATA box promoter element. A key step in initiation by Pol III on tRNA genes is binding of TFIIIC to two intragenic promoter elements, the A and B blocks. TFIIIC then places TFIIIB upstream of the transcription start site (reviewed in reference 9). These distinct promoter recognition steps in Pol II and Pol III transcription imply divergent mechanisms for initiation complex assembly. Yet further characterization of Pol II and Pol III transcription initiation complexes has revealed striking similarities. For example, like TFIID, TFIIIB contains the TATA-binding protein as a subunit. Although most Pol III transcription units lack a consensus TATA box in the TFIIIB-binding region, A/T-rich sequences 15 to 30 base pairs upstream of the start site are known to contribute to the efficiency of tRNA and 5S rRNA gene transcription in vitro (29, 38) and in vivo (23,40). In addition, an increasing number of Pol II transcription units have been found to contain TFIID-binding promote...

Requirement of Nhp6 Proteins for Transcription of a Subset of tRNA Genes and Heterochromatin Barrier Function in Saccharomyces cerevisiae

“…Studies of the role of Nhp6 in SNR6 transcription have led to the proposal that Nhp6 acts in this case by facilitating TFIIIC binding to the SNR6 promoter, perhaps by relieving the sterically unfavorable spacing of the SNR6 A-and B-block elements (44,48). In another study, however, Nhp6 was also found to stimulate TFIIIC-independent transcription of SNR6 in vitro, thus suggesting a direct effect of Nhp6 on TFIIIB-DNA complex formation (47).…”

“…In yeast, the HMG-like protein Nhp6 has previously been shown to cooperate with the Pol III machinery in the transcription of SNR6, a Pol III-transcribed gene coding for the U6 snRNA (44,47). Moreover, Nhp6 has been shown to promote transcription complex assembly on SNR6 by acting in concert with an upstream sequence element (48) and, more recently, to act as a transcriptional initiation fidelity factor for a subset of tRNA genes (39). We reasoned that this abundant, chromatin-associated protein might act as a transcriptional stimulator for at least some tRNA genes and that its ability to generate distorted DNA structures might also contribute to the positional roles of class III genes.…”

Quantitative Analysis of in Vivo Initiator Selection by Yeast RNA Polymerase II Supports a Scanning Model

“…Primer extension analysis of 5 g of total cellular RNA was carried out using 32 P-labeled oligonucleotide U4 -14B (complementary to nucleotides 140 -159 of yeast U4 RNA) or SCR1 (complementary to nucleotides 75-92 of yeast scR1 RNA) (31). Sequencing ladders were generated using the Sequitherm EXCEL II DNA sequencing kit (Epicentre).…”

Genetic Interactions Between Nhp6 and Gcn5 With Mot1 and the Ccr4–Not Complex That Regulate Binding of TATA-Binding Protein in Saccharomyces cerevisiae

“…However, at 30°, YEp-SNR6 or YEp-TBP strongly suppressed the growth defect, and YEp-TFIIB showed moderate suppression. nhp6ab mutants are defective in expressing SNR6, a pol III transcribed gene encoding the U6 splicing RNA (Lopez et al 2001;Martin et al 2001). YEp-SNR6 suppresses the temperature-sensitive growth defect seen in nhp6ab mutants, and it is suggested that decreased SNR6 RNA contributes to the poor growth at elevated temperatures (Kruppa et al 2001).…”

“…No phenotype is seen in nhp6a and nhp6b single mutants, while the nhp6a nhp6b double mutant (which we will describe as nhp6ab) is temperature sensitive for growth (Costigan et al 1994) and shows transcriptional defects (Paull et al 1996;Yu et al 2000;Fragiadakis et al 2004). Nhp6 also functions with Spt16 and Pob3, as part of the yeast FACT complex, to promote transcriptional elongation (Formosa et al 2001), and Nhp6 is important for expression of the SNR6 gene, transcribed by RNA polymerase III (Kruppa et al 2001;Lopez et al 2001;Martin et al 2001).…”