The Saccharomyces cerevisiae Ccr4-Not complex is a global regulator of transcription that is thought to regulate TATA binding protein (TBP) function at certain promoters specifically. In this paper, we show interactions between the essential domain of Not1p, which interacts with Not4p and Not5p, and the N-terminal domain of yTAF1. We isolated a temperature-sensitive nonsense allele of TAF1, taf1-4, which is synthetically lethal at the permissive temperature when combined with not4 and not5 mutants and which produces high levels of a C-terminally truncated yTAF1 derivative. Overexpression of C-terminally truncated yTAF1 is toxic in not4 or not5 mutants, whereas overexpression of full-length yTAF1 suppresses not4. Furthermore, mutations in the autoinhibitory N-terminal TAND domain of yTAF1 suppress not5, and the overexpression of similar mutants does not suppress not4. We find that, like Not5p, yTAF1 acts as a repressor of stress response element-dependent transcription. Finally, we have evidence for stress-regulated occupancy of promoter DNA by Not5p and for Not5p-dependent regulation of yTAF1 association with promoter DNA. Taken together with our finding that Not1p copurifies with glutathione S-transferase-yTaf1 in large complexes, these results provide the first molecular evidence that the Ccr4-Not complex might interact with yTAF1 to regulate its association at promoters, a function that might in turn regulate the autoinhibitory N-terminal domain of yTAF1.Transcription initiation of protein-coding genes by RNA polymerase II involves a large number of general transcription factors that position the polymerase specifically on the core promoter (for reviews, see references 32 and 47). The recruitment of the TATA binding protein (TBP) is the first step in the assembly of a functional preinitiation complex. TBP is a component of the TFIID multiprotein complex (for a review, see reference 6) that in the yeast Saccharomyces cerevisiae consists of 14 additional TBP-associated factors (TAF II s) (39,52,53). It specifically recognizes TATA elements present in the promoters of many genes transcribed by RNA polymerase II. For promoters that lack a canonical TATA sequence (referred to as TATA-less), TAF II s are likely to participate in the recognition of the promoter. First, certain TAF II s contact promoter elements such as the initiator (7) and the downstream promoter element (5), and it is possible that other contacts between the promoter and TAF II s are still to be discovered. Second, experiments with both mammalian systems (45, 58, 59) and yeast (54) have suggested the importance of TFIID rather than TBP in promoter recognition.TAF II s were first thought to be exclusively within the TFIID complex, but recent work has demonstrated the existence of multiple complexes that carry TAF II s (21,38,46,60). In the yeast Saccharomyces cerevisiae, the only two complexes identified so far are TFIID (49) and SAGA (21; for reviews, see references 22 and 55). In turn, the only well-characterized form of TBP recruited to promoter...

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The conserved multi-subunit Ccr4-Not complex regulates gene expression in diverse ways. In this work, we characterize the suppression of temperature sensitivity associated with a mutation in the gene encoding the scaffold subunit of the Ccr4-Not complex, NOT1, by the deletion of SPT3. We determine that the deletion of SPT3, but not the deletion of genes encoding other subunits of the SAGA complex, globally suppresses transcriptional defects of not1-2. We find that transcriptional activation in not1-2 is associated with increased binding of TFIID and SAGA at promoters of upregulated genes, and this is suppressed by the deletion of SPT3. Interestingly, Spt3p-dependent activation of transcription occurs in not1-2 even if the SAGA complex is disrupted by the deletion of SPT7 that encodes a subunit of SAGA required for its integrity. Consistent with a SAGA-independent function of Spt3p, the deletion of SPT3 displays synthetic phenotypes when combined with a deletion of SPT7. Taken together, our results provide a new view of the Spt3 protein by identifying a SAGA-independent function of this protein that is functionally linked to the Ccr4-Not complex.

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