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PMID:9887099

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Citation

Ranish, JA, Yudkovsky, N and Hahn, S (1999) Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes Dev. 13:49-63

Abstract

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.

Links

PubMed PMC316368

Keywords

Fungal Proteins/genetics; Mutation/genetics; Promoter Regions, Genetic/genetics; Protein Binding/genetics; RNA Polymerase II/genetics; TATA Box/genetics; Trans-Activators/genetics; Transcription Factor TFIIA; Transcription Factor TFIIB; Transcription Factor TFIID; Transcription Factors/genetics; Transcription Factors, TFII/genetics; Transcription, Genetic/genetics; Transcriptional Activation/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:TF2B

involved_in

GO:0051123: RNA polymerase II preinitiation complex assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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