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CUP1 gene can still be induced to 60% of the WT level after preinactivating Srb4 (Fig. 3a). The CUP1 gene is fully induced after inactivating Srb6 (Fig. 3b). To assess the effect of mediator depletion on SSA4 gene activation, we generated a copper-inducible Srb4-knockout strain (Fig. 3c). After depleting Srb4, the SSA4 gene is activated to a similar level ( 30% of WT) as thatseen in the Kin28-knockout strain (Fig. 3c). Therefore, a strong correlation exists between Kin28-independent and mediator-independent transcription. We have found a set of genes that require neither TFIIH kinase nor normally critical components of the Pol II mediator complex for their activated transcription. From these findings and previous in vitro7,8 and in vivo5 data, we conclude that there areat least two distinct transcriptional activation mechanisms in vivo: one requires functional TFIIH kinase and mediator complex, and the other does not. Both of the activation mechanisms can be explained by the simple recruitment models described above. Interestingly, a second activity of TFIIH, the helicase activity, is required by both mechanisms. We have not addressed whether other mechanismsof transcriptional activation, such as those found in higher eukaryotes27, may also exist in yeast. Nevertheless, our findings with TFIIH kinase and the mediator complex, with studies of promoter-specific TATAbinding protein (TBP)28 and TBP-associated factors29, show that transcription from various Pol II associated promoters in vivo is accomplished by different mechanisms, using different coretranscriptional components.
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