A transcription reinitiation intermediate that is stabilized by activator.

High levels of gene transcription by RNA polymerase II depend on high rates of transcription initiation and reinitiation. Initiation requires recruitment of the complete transcription machinery to a promoter, a process facilitated by activators and chromatin remodelling factors. Reinitiation probably occurs through a different pathway. After initiation, a subset of the transcription machinery remains at the promoter, forming a platform for assembly of a second transcription complex. Here we describe the isolation of a reinitiation intermediate that includes transcription factors TFIID, TFIIA, TFIIH, TFIIE and Mediator. This intermediate can act as a scaffold for formation of a functional reinitiation complex. Formation of this scaffold is dependent on ATP and TFIIH. The scaffold is stabilized in the presence of the activator Gal4-VP16, but not Gal4-AH, suggesting a new role for some activators and Mediator in promoting high levels of transcription.

Recruitment of the SWI/SNF chromatin remodeling complex by transcriptional activators.

SWI/SNF is a chromatin remodeling complex that facilitates expression of a number of yeast genes. Here we demonstrate that SWI/SNF can be recruited from yeast nuclear extracts by a transcriptional activator. Recruitment is dependent on an activation domain but not on promoter sequences, TBP, or RNA polymerase II holoenzyme. We also show that acidic activation domains can target SWI/SNF remodeling activity. These results demonstrate that SWI/SNF activity can be targeted by gene-specific activators and that this recruitment can occur independently of Pol II holoenzyme.

Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB.

Assembly and activity of yeast RNA polymerase II (Pol II) preinitiation complexes (PIC) was investigated with an immobilized promoter assay and extracts made from wild-type cells and from cells containing conditional mutations in components of the Pol II machinery. We describe the following findings: (1) In one step, TFIID and TFIIA assemble at the promoter independently of holoenzyme. In another step, holoenzyme is recruited to the promoter. Mutations in the CTD of Pol II, Srb2, Srb4, and Srb5, and two mutations in TFIIB disrupt recruitment of all holoenzyme components tested without affecting TFIID and TFIIA recruitment. These results indicate that the stepwise assembly pathway is blocked after TFIID/TFIIA binding. (2) Both the Gal4-AH and Gal4-VP16 activators stimulate formation of active PICs by increasing the extent of PIC formation. The Gal4-AH activator stimulated PIC formation by enhancing the binding of TFIID and TFIIA, whereas Gal4-VP16 could enhance the recruitment of TFIID, TFIIA, and holoenzyme. (3) Extracts deficient in TFIIA activity showed reduced assembly of all PIC components. These and other results suggest that TFIIA acts at an early step by enhancing the stable recruitment of TFIID. (4) An extract containing the TFIIB mutant E62G, had no defect in PIC formation, but had a severe defect in transcription. Similarly, mutation of the TATA box reduced PIC formation only two- to fourfold, but severely compromised transcription. These results demonstate an involvement of TFIIB and the TATA box in one or more steps after recruitment of factors to the promoter.