1. General Transcription Initiation Factors

2. Assay for accurate initiation by bacterial RNA polymerase Promoter E. coli RNA pol holoenzyme + initiation elongation Nascent RNA Run-off transcript, Discrete size ++ RNA Pol core

3. Random transcription by bacterial RNA polymerase core Promoter E. coli RNA pol core + Random initiation elongation Transcripts of random size

4. Random transcription by eukaryotic RNA polymerase Promoter Eukaryotic RNA pol II + Random initiation elongation Transcripts of random size

5. Accurate initiation by Euk RNA polymerase II plus factors in the nucleus Promoter + initiation elongation Nascent RNA Run-off transcript, Discrete size ++ Eukaryotic RNA pol II + Nuclear extract, S-100 General Transcription Initiation Factors

6. General transcription factors = GTFs Proteins other than RNA polymerase involved in transcription –Initiation, Elongation, Termination –Not subunits of purified RNA polymerase. GTFs for RNA polymerase II are called TFIIx, where x = A, B, D, … –Can have multiple subunits General transcription initiation factors (GTIFs) –Proteins required for specific transcription from a minimal promoter (core) –Required for RNA polymerase to bind avidly and specifically to promoters.

7. Fractionation of nuclear extracts to find GTFs Phosphocellulose column: ion exchange Template: Adenovirus late promoter Plus Purified Pol II Run-off Transcript: Accurate Initiation at promoter Polyacrylamide gel separating products of in vitro transcription

8. Fractionation of nuclear extracts to find GTFs Fractionation scheme, DEAE cellulose Matsui, Segall, Weil, Roeder (1980) JBC 255:11992 Plus Purified Pol II Run-off Transcript Accurate Initiation at promoter

9. GTIFs for RNA polymerase II TFIID TBP } TAFs IIB IIA IIE IIF IIH helicase protein kinase TBP Inr IIB IIA Pol IIa IIF IIE IIH CTD of large subunit of Pol II Recognize core promoter Targets Pol II to promoter Modulates helicase Helicase CTD protein kinase Many GTIFs are possible targets for activators of transcription.

10. TATA Binding Protein = TBP TBP binds in the narrow groove of DNA at the TATA box found about 20-25 bp 5’ to the start site for transcription of many (but not all) genes transcribed by RNA polymerase II. TBP bends the DNA about 90 degrees. TBP alone or with TBP-associated proteins (TAFs) plays an important role in recognizing the core promoter and recruiting RNA polymerase II to the promoter.

11. TBP bound to DNA Image from crystal structures, provided by Dr. T. Nixon.

12. RNA Pol II bound to DNA and general transcription initiation factors

13. Movie of TBP, then binding to TATA DNA (and bending it), and then TFIIA binding to TBP-TATA Thanks to Drs. Song Tan and Tracy Nixon for movies

14. Sequential Binding Model for assembly of preinitiation complex -30+1 TATA Inr Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA. IIB Eukaryotic RNA polymerase II TFIID } TBP TAFs IIB IIE CTD of large subunit of Pol II Pol IIa or TBP IIA IIF helicase protein kinase IIH TATA Inr IIA Pol IIa IIF IIE preinitiation complex TATA Inr IIA IIB Pol IIa IIF IIE ATP hydrolysis initiation complex, DNA melted at Inr IIH = PIC Activated PIC

15. Direct Binding of Holoenzyme model for assembly of the preinitiation complex = PIC Activated PIC

16. Other proteins involved in transcription and regulation In addition to RNA polymerase II and GTIFs: Proteins required for regulation, e.g. –Gal11: regulation of the GAL operon –Rgr1: resistance to glucose repression Srb proteins –Yeast strains with truncations in the CTD of the large subunit of RNA polymerase B are cold-sensitive –SRB genes: when mutated, suppress the phenotype of CTD deletions –Extragenic suppressors: implicated in RNA polymerase function

17. RNA polymerase II Holoenzyme and Mediator Holoenzyme –RNA polymerase II + (TFIIB, E, F, H )+ (Srb2, 4, 5, 6) + (Rgr1, Gal11, others) –Correct initiation in presence of TBP (TFIID) –Responds to transcriptional activators Mediator –Complex needed for a response to transcriptional activators by purified RNA Pol II plus GTFs –Yeast Mediator has 20 subunits, including Srb2, 4, 5, 6; Srb7, Rgr1, Gal11, Med 1, 2, 6, 7, Pgd1, Nut 1, 2, and others RNA Pol II + Mediator (+ some GTIFs?) = Holoenzyme

18. Expanding the functions of RNA polymerase

19. Stages in Initiation of Transcription Bacterial transcription Closed complex: holoenzyme+promoter Open complex (DNA melting, not need ATP ) Abortive transcripton Productive initiation –Transcribe past +9 to +12 –Sigma dissociates Elongation Eukaryotic transcription Preinitiation complex (PIC) assembly PIC activation (DNA melting, needs ATP) Abortive transcription Productive initiation –CTD phosphorylated –Promoter clearance Elongation

20. Parallels between initiation pathway in prokaryotes and eukaryotes From Eick et al. (1994) Trends in Genetics 10: 292-296

21. GTFs for RNA polymerase I

22. GTFs for RNA polymerase III

23. TBP is used by all 3 RNA polymerases TBP is a subunit of an important GTIF for each of the 3 RNA polymerases: –TBP or TFIID for Pol II –SL1 for Pol I –TFIIIB for Pol III It does NOT always bind to TATA boxes; promoters for RNA Pol I and Pol III (and even some for Pol II) do not have TATA boxes, but TBP is still used. The GTIFs that contain TBP may serve as positioning factors for their respective polymerases.