Mechanism of assembly of the RNA polymerase II preinitiation complex. Transcription factors delta and epsilon promote stable binding of the transcription apparatus to the initiator element.
Assembly of RNA polymerase II with the core region of TATA box-containing promoters requires the action of the TATA factor and four transcription factors designated alpha, beta gamma, delta, and epsilon, which have each been purified to near homogeneity from rat liver. Evidence from previous studies argues that alpha and beta gamma play a crucial role in delivering RNA polymerase II to the promoter (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6205-6209). Here we describe the interaction of transcription factor delta with preinitiation intermediates assembled in the presence of either recombinant yeast TFIID or the high molecular mass, endogenous TATA factor tau from rat liver (Conaway, J. W., Hanley, J. P., Garrett, K. P., and Conaway, R. C. (1991) J. Biol. Chem. 266, 7804-7811). Results of template challenge experiments argue that delta enters the preinitiation complex through interactions with multiple components of the transcription apparatus. We observe that, in the presence of recombinant TFIID, delta interacts stably with the preinitiation complex only in the presence of RNA polymerase II, alpha, and beta gamma, whereas, in the presence of tau, delta is capable of interacting stably with the Initial Complex independently of RNA polymerase II. Results of restriction site protection experiments reveal that delta and epsilon promote binding of the transcription apparatus to the Initiator element and support the model that RNA polymerase II assembles at the core promoter in at least two discrete steps, first "touching down" near the TATA element and finally extending its interaction downstream to encompass the cap site.
Conaway
J W
JW
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Bradsher
J N
JN
Conaway
R C
RC
D000818:Animals; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D005809:Genes, Regulator; D007700:Kinetics; D008099:Liver / Q000502:physiology*; D008297:Male; D008957:Models, Genetic; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016385:TATA Box; D013698:Templates, Genetic; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D011994:Recombinant Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D012319:RNA Polymerase II