The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif.

Spn1/Iws1 plays essential roles in the regulation of gene expression by RNA polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP (TATA-binding protein), TFIIS (transcription factor IIS), and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here, we report the high-resolution (1.85 Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is composed of eight α-helices in a right-handed superhelical arrangement and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity.

Activation of a poised RNAPII-dependent promoter requires both SAGA and mediator.

A growing number of promoters have key components of the transcription machinery, such as TATA-binding protein (TBP) and RNA polymerase II (RNAPII), present at the promoter prior to activation of transcription. Thus, while transcriptional output undergoes a dramatic increase between uninduced and induced conditions, occupancy of a large portion of the transcription machinery does not. As such, activation of these poised promoters depends on rate-limiting steps after recruitment of TBP and RNAPII for regulated expression. Little is known about the transcription components required in these latter steps of transcription in vivo. To identify components with critical roles in transcription after recruitment of TBP in Saccharomyces cerevisiae, we screened for loss of gene expression activity from promoter-tethered TBP in >100 mutant strains deleted for a transcription-related gene. The assay revealed a dramatic enrichment for strains containing deletions in genes encoding subunits of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex and Mediator. Analysis of an authentic postrecruitment-regulated gene (CYC1) reveals that SAGA occupies the promoter under both uninduced and induced conditions. In contrast, Mediator is recruited only after transfer to inducing conditions and correlates with activation of the preloaded polymerase at CYC1. These studies indicate the critical functions of SAGA and Mediator in the mechanism of activation of genes with rate-limiting steps after recruitment of TBP.

Non-optimal TATA elements exhibit diverse mechanistic consequences.

To reveal mechanistic differences in transcription initiation between variant TATA elements, in vivo and in vitro assays of the functional activity of 14 different sequences were compared. Variant elements exhibited particular degrees of activation in vivo but universally were unable to support the -fold activation observed for an element consisting of TATAAA. Each element was classified by its functional activity for in vitro interaction with TATA-binding protein (TBP), TFIIA, and TFIIB. Certain off-consensus TATA elements form poor binding sites for TBP and this compromised interaction interferes with higher order complex formation with TFIIA and/or TFIIB. Other elements are only modestly decreased for TBP binding but dramatically affected for higher order complex formation. Another distinct category is comprised of two elements (CATAAA and TATAAG), which are not affected in the initial formation of the TBP, TFIIA-TBP, or TFIIB-TBP complexes. However, CATAAA and TATAAG are unable to form a stable TFIIA-TBP-DNA complex in vitro. Moreover, fusion of TFIIA to TBP specifically restores activity from these two elements in vivo. Taken together, these results indicate that the interplay between the sequence of the TATA element and the components of the general transcription machinery can lead to variations in the formation of functional complexes and/or the stability of these complexes. These differences offer distinct opportunities for an organism to exploit diverse steps in the regulation of gene expression depending on the precise TATA element sequence at a given gene.

SPN1, a conserved gene identified by suppression of a postrecruitment-defective yeast TATA-binding protein mutant.

Little is known about TATA-binding protein (TBP) functions after recruitment to the TATA element, although several TBP mutants display postrecruitment defects. Here we describe a genetic screen for suppressors of a postrecruitment-defective TBP allele. Suppression was achieved by a single point mutation in a previously uncharacterized Saccharomyces cerevisiae gene, SPN1 (suppresses postrecruitment functions gene number 1). SPN1 is an essential yeast gene that is highly conserved throughout evolution. The suppressing mutation in SPN1 substitutes an asparagine for an invariant lysine at position 192 (spn1(K192N)). The spn1(K192N) strain is able to suppress additional alleles of TBP that possess postrecruitment defects, but not a TBP allele that is postrecruitment competent. In addition, Spn1p does not stably associate with TFIID in vivo. Cells containing the spn1(K192N) allele exhibit a temperature-sensitive phenotype and some defects in activated transcription, whereas constitutive transcription appears relatively robust in the mutant background. Consistent with an important role in postrecruitment functions, transcription from the CYC1 promoter, which has been shown to be regulated by postrecruitment mechanisms, is enhanced in spn1(K192N) cells. Moreover, we find that SPN1 is a member of the SPT gene family, further supporting a functional requirement for the SPN1 gene product in transcriptional processes.