Interplay of positive and negative regulators in transcription initiation by RNA polymerase II holoenzyme.

Activation of protein-encoding genes involves recruitment of an RNA polymerase II holoenzyme to promoters. Since the Srb4 subunit of the holoenzyme is essential for expression of most class II genes and is a target of at least one transcriptional activator, we reasoned that suppressors of a temperature-sensitive mutation in Srb4 would identify other factors generally involved in regulation of gene expression. We report here that MED6 and SRB6, both of which encode essential components of the holoenzyme, are among the dominant suppressors and that the products of these genes interact physically with Srb4. The recessive suppressors include NCB1 (BUR6), NCB2, NOT1, NOT3, NOT5, and CAF1, which encode subunits of NC2 and the Not complex. NC2 and Not proteins are general negative regulators which interact with TATA box binding protein (TBP). Taken together, these results suggest that transcription initiation involves a dynamic balance between activation mediated by specific components of the holoenzyme and repression by multiple TBP-associated regulators.

Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo.

Activation of eukaryotic class II gene expression involves the formation of a transcription initiation complex that includes RNA polymerase II, general transcription factors, and SRB components of the holoenzyme. Negative regulators of transcription have been described, but it is not clear whether any are general repressors of class II genes in vivo. We reasoned that defects in truly global negative regulators should compensate for deficiencies in SRB4 because SRB4 plays a positive role in holoenzyme function. Genetic experiments reveal that this is indeed the case: a defect in the yeast homologue of the human negative regulator NC2 (Dr1 x DRAP1) suppresses a mutation in SRB4. Global defects in mRNA synthesis caused by the defective yeast holoenzyme are alleviated by the NC2 suppressing mutation in vivo, indicating that yeast NC2 is a global negative regulator of class II transcription. These results imply that relief from repression at class II promoters is a general feature of gene activation in vivo.

A mammalian SRB protein associated with an RNA polymerase II holoenzyme.

A large multisubunit complex containing RNA polymerase II, general transcription factors and SRB regulatory proteins initiates transcription of class II genes in yeast cells. The SRB proteins are a hallmark of this RNA polymerase II holoenzyme as they are found only in this complex, where they contribute to the response to regulators. We have now isolated a human homologue of the yeast SRB7 gene and used antibodies against human SRB7 protein to purify and characterize a mammalian RNA polymerase II holoenzyme containing the general transcription factors TFIIE and TFIIH. This holoenzyme is more responsive to transcriptional activators than core RNA polymerase II when assayed in the presence of coactivators.