We investigated the timing of the recruitment of Spn1 and its partner, Spt6, to the CYC1 gene. Like TATA binding protein and RNA polymerase II (RNAPII), Spn1 is constitutively recruited to the CYC1 promoter, although levels of transcription from this gene, which is regulated postrecruitment of RNAPII, are low. In contrast, Spt6 appears only after growth in conditions in which the gene is highly transcribed. Spn1 recruitment is via interaction with RNAPII, since an spn1 mutant defective for interaction with RNAPII is not targeted to the promoter, and Spn1 is necessary for Spt6 recruitment. Through a targeted genetic screen, strong and specific antagonizing interactions between SPN1 and genes encoding Swi/Snf subunits were identified. Like Spt6, Swi/Snf appears at CYC1 only after activation of the gene. However, Spt6 significantly precedes Swi/Snf occupancy at the promoter. In the absence of Spn1 recruitment, Swi/Snf is constitutively found at the promoter. These observations support a model whereby Spn1 negatively regulates RNAPII transcriptional activity by inhibiting recruitment of Swi/Snf to the CYC1 promoter, and this inhibition is abrogated by the Spn1-Spt6 interaction. These findings link Spn1 functions to the transition from an inactive to an actively transcribing RNAPII complex at a postrecruitment-regulated promoter.For a large number of well-characterized genes, the ratelimiting step in the transcription process is the formation of the preinitiation complex at the promoter. At these genes, the recruitment of TATA binding protein (TBP) and RNA polymerase II (RNAPII) to the promoter correlates strongly with transcriptional output (35,41,67). Indeed, delivery of TBP and RNAPII appears to be sufficient for gene activation in many contexts (for reviews, see references 63 and 64). However, there are a growing number of genes that have been found to be regulated at a step after the recruitment of RNAPII. Such genes include the yeast CYC1 gene, the Drosophila heat shock genes, and mammalian human immunodeficiency virus type 1 and the c-Myc proto-oncogene (3,39,41,51,77). Indeed, whole-genome studies suggest that a significant portion of the human genome may be regulated postrecruitment of RNAPII (26,35). As such, these mechanisms have the potential to impact the expression of thousands of human genes. Our understanding of these mechanisms and the rate-limiting steps involved is incomplete, but these observations suggest that functions critical for a high level of transcription are either inhibited or absent under noninducing conditions. To determine the nature of these functions, further characterization of genes regulated after the assembly of the general transcription machinery is imperative.The yeast CYC1 gene encodes iso-1-cytochrome c, a protein involved in the electron transport chain in the mitochondria (75). In the presence of a fermentable carbon source (such as dextrose), CYC1 gene expression is inhibited and transcriptional levels are extremely low (24,25). When cells are grown on a nonfermentable c...

A growing number of promoters have key components of the transcription machinery, such as TATAbinding 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.