1. Activities of RNA Polymerase sequence specific DNA binding -promoters melts DNA to reveal the template strand selects ribonucleotide (not deoxynucleotides) that anneals to template strand polymerizes RNA strand translocates on DNA template, during which it must: -unwind DNA in front of polymerase -unwinds RNA:DNA hybrid -rewinds DNA behind polymerase recognize termination signals in the nascent transcript (or on the DNA template) In addition, the polymerase must be processive (have a high probability of reaching the end of the gene)

2. Modular Organization of Regulatory Information via Multiple Enhancers stripes of ftz and eve expression in a Drosophila embryo fragments of eve regulatory were inserted upstream of a  -Gal reporter and inserted into flies. different regulatory elements gave distinct patterns of  -Gal (dark staining) expression. Normal eve expression is shown in red. 1

3. Open complex formation Abortive initiation Promoter clearance Promoter binding Elongation Stages of Transcription [closed complex] [open complex]

4. Robert Roeder collecting sea urchin embryos 1968 today

5. Keys to Successful Protein Purification -an abundant source of material -a quantitative assay -must be able determine yield and purity -a strategy for separation -charge -size -hydrophobicity -stability -affinity reagents

6.  Roeder’s Assay, Incorporation of 32P- labeled UTP into RNA note that the  phosphate is incorporated into the RNA he could measure the amount of radioactivity incorporated into RNA (vs that that remained associated with the UTP) 32

7. Three Different RNA Polymerases in Eukaryotes (Roeder and Rutter, 1969) DEAE-Sephadex protein (measured by UV light absorbance in a spectrophotometer) polymerase activity ( 32 P incorporation into RNA) [salt] (50mM KCl -400 mM) functional group on column:

8.  -Amanitin (  g/ml) % Maximum activity RNA Polymerases I, II, and III Exhibit Different Sensitivities towards  -Amanitin Amanita phalloides

9. Enzymelocationrelative  -amanitin activitysensitivity Pol Inucleolus50-70%not inhibited Pol IInucleoplasm20-40%inhibited Pol IIInucleoplasm≈10%species-specific All three polymerase classes.... weigh >500,000 D contain 12-16 subunits - some conserved across evolution  ’ - like (~200,000 D)  - like (~140,000 D)  - like (~40,000 D) - some shared among all 3 polymerases - some unique Eukaryotic Nuclear RNA Polymerases

10. Three Classes of Transcription in Eukaryotes RNA polymerase I (pol I) ribosomal RNAs (5.8S, 18S, 28S rRNA) RNA polymerase II (pol II) mRNAs some small nuclear RNAs (snRNAs) non-coding RNAs (mostly of unknown function) RNA polymerase III (pol III) tRNAs 5S RNA some snRNAs small cytoplasmic RNAs (scRNAs)

11. RNA Polymerase II Underlies the Central Dogma of Molecular Biology Pol I Pol II Pol III 45S rRNA tRNA 5S rRNA Ribosome DNAmRNAProtein this explains our emphasis on the mechanisms of Pol II transcription

12. Roger Kornberg (1947) (With wife and sons, Stockholm Dec. 2006)

13. Important things to know about Pol II The general architecture of the polymerase, including the arrangement of nucleic acids in the active site That nucleotides likely enter through the “funnel” That the polymerase is a catalyst that specifically accelerates the rate at which the correctly paired ribonucleotide is added to a growing RNA chain (the exact details of the proposed reaction mechanism are not important to know) –Review, but don’t feel obligated to memorize the exact details of the role of the trigger loop in facilitating catalysis and substrate (I.e. nucleotide) selection That the polymerase must be able to translocate on the DNA template after it has added a nucleotide to the RNA

14. Crystal Structure of Yeast RNA Polymerase II at 2.8 Å Resolution (Cramer et al, 2001)

15. RNAP (T. aquaticus) RNA Pol II (S. cerevisiae)  ’’ Rpb2 Rpb1

16. What is Required for Promoter Function? cis (DNA sequences) vs trans (proteins) identify cis elements by, conservation, mutagenesis and assays of transcription identify trans factors by biochemical (mainly) and genetic (occasionally) approaches

17. Assays of Promoter Activity in vitro - use a specific promoter - mix with NTPs and extract or purified factors - measure RNA (directly, with 32 P-labeled NTPs or indirect assays, see Weaver pp 106-111) in vivo - introduce gene of interest into cells transformation (yeast and bacteria) transfection (cultured cells) - fuse promoter to a reporter gene that can be differentiated from normal genes in the recipient cells “reporter gene” - can collect and measure RNA from cells - can measure activity of reporter gene beta-Gal, selectable markers

18. Deletion mutations Linker scanning Point mutations Identifying Promoter Elements

19. Class II Promoters Several parts: –Core promoter –Upstream promoter elements –Enhancers, may be far from core promoters sequence elements found in many core promoters gene specific

20. Core Promoter Elements In addition to TATA box, core promoters are: –TFIIB recognition element (BRE) –Initiator (Inr) –Downstream promoter element (DPE) –note: the important thing to remember is the TATA box At least one of the four core elements is missing in most promoters TATA-less promoters tend to have DPEs Promoters for highly specialized genes tend to have TATA boxes Promoters for housekeeping genes tend to lack them

21. Upstream Elements Upstream promoter elements are usually found upstream of class II core promoters Differ from core promoters in binding to relatively gene-specific transcription factors. examples: –GC boxes bind transcription factor Sp1 –CCAAT boxes bind CTF (CCAAT-binding transcription factor) Enhancers, function in a position and orientation independent manner. (sometimes enhancers are considered to be a distinct type of element)

22. A Typical RNA Pol II Promoter

23. Identifying the General Transcription Machinery establish robust in vitro assay with a strong core promoter –AdML adenovirus major late promoter purify proteins required for transcription goal is to identify a minimal set of purified proteins with which to reconstitute transcription

24. Fork loop 2

25. What is Required for Promoter Function? Observation: RNA polymerase alone is not capable of accurately initiated, gene-specific transcription. cis (DNA sequences) vs trans (proteins) identify cis elements by, conservation, mutagenesis and assays of transcription -core promoter (esp. TATA box) -upstream elements -enhancers identify trans factors by biochemical (mainly) and genetic (occasionally) approaches -general transcription factors (GTFs) -accessory factors required at all genes transcribed by a polymerase

27. Identifying the General Transcription Machinery establish robust in vitro assay with a strong core promoter –AdML adenovirus major late promoter purify proteins required for transcription goal is to identify a minimal set of purified proteins with which to reconstitute transcription

28. RNA Polymerase II Requires Additional Factors for Accurate Transcription Initiation at Promoters (Matsui et al, 1980) Phosphocellulose chromatography

29. Further Fractionation of S-100 Extract (Matsui et al, 1980)

30. Pol II 12 GTFs TFIID TFIIB 1 TFIIE 2 TFIIH 9 TFIIF 2 TFIIA 3 Mediator 22 RNA polymerase II Transcription Machinery Number of subunits TBP 1 TAFs 12 * *

31. Proposed Pathway of Initiation helicases in TFIIH catalyze open complex formation Pol II-TFIIF recruited by DNA-D/A/B complex initiation and escape

32. FactorRole TBP TFIIB TFIIE TFIIF TFIIH Promoter recognition; configures DNA to the pol II surface Promoter recognition; pol II recruitment; directs DNA path, stabilizes early transcribing complex; coupling of RNA synthesis to promoter clearance Recognizes closed complex, recruits TFIIH Captures nontemplate strand upon melting Untwisting of promoter DNA (helicase), CTD phosphorylation (kinase) Mechanism of Initiation of RNA Polymerase II Transcription H Pol TBP BCBC H Pol TBP BCBC FBNBN

33. Transcription Elongation...is slow compared to DNA replication 20-40 nucleotides / second Typical 1° transcript is ≈20,000 nts., corresponds to ≈10 minutes / transcript. Long transcripts can take hours to complete … is regulated TFIIF suppresses pausing TFIIS rescues arrested complexes others …. polymerases stalled at the 5’ ends of genes appear to be common...may involve proof-reading (observed in vitro)...is coupled to DNA repair

34. Activating domains can be replaced by randomly selected sequences (Ma and Ptashne, 1986) activator-gal+gal Gal41111895 Gal4DBD<1 none<1 B17415794 B42542756 B6429588 B9219.3 B159073 beta-gal activity Q. what are the essential features of activation domains? Approach: random e. coli sequences cloned downstream of Gal4 DBD and expressed in yeast containing a  -gal reporter with Gal4 sites in its promoter 16

35. Activating domains can be replaced by randomly selected sequences (Ma and Ptashne, 1986) 1.random e. coli sequences cloned downstream of the Gal4 DBD and expressed in a yeast strain containing a  -gal reporter with Gal4 sites in its promoter 2.~1% of all the clones activated transcription 3.activating sequences did not resemble known proteins, no catalytic domains etc. -activation domains unlikely to have enzymatic activity 4. negatively charged residues common 17

36. Overexpression of Gal4 from the strong ADH promoter inhibits promoters that lack Gal4 binding sites (Gill and Ptashne, 1988) core HIS3 +1 UAS H /core +12  -Gal UAS G = GAL enhancer (binds Gal4) UAS H = HIS3 enhancer UAS C = CYC1 enhancer decreased expression of reporter genes lacking Gal4 binding sites when Gal4 levels are high 18

37. TATA box activator A UAS Activator Interference or ‘Squelching’ activator B 19

38. TATA box activator A UAS Activator Interference or ‘Squelching’ activator B hypothesis? 20

39. 1.Eukaryotic activators do not bind to RNA pol II polymerase and therefore do not directly recruit polymerase to promoters. 2.Activators may, however, indirectly recruit RNA polymerase by recruiting factors (often called co-activators) that serve as a physical bridge between activator and polymerase. ‘TFIID hypothesis’ ‘Holoenzyme hypothesis’ What is the Limiting Target of Activators? 21

40. ChIP (Chromatin Immunoprecipitation) Formaldehyde crosslink Shear chromatin by sonication Immunoprecipitation Reverse crosslinks, PCR TBP input control: reverse crosslinks and analyze sample prior to IP 22

41. Extent of TBP binding correlates with promoter activity (Li et al, 1999) TBP crosslinks to active promoters 23

42. Pol II 12 GTFs TFIID TFIIB 1 TFIIE 2 TFIIH 9 TFIIF 2 TFIIA 3 Mediator 22 RNA Polymerase II Transcription Machinery Number of subunits TBP 1 TAFs 12 * * 24

43. The ‘TFIID Hypothesis’ in vitro assays suggest specific activator-TAF contacts predictions? 1. TAFs provide surfaces for the interaction of TFIID with activators. 2. TFIID recruits polymerase 25

44. Activated Transcription in the Absence of TAF II s western blot demonstrating depletion of TAF II s see p97 of Weaver or p769 of Watson for a description of the Western blot technique in vitro transcription shows that - transcription is abolished in the TFIID depleted extract - TBP is sufficient to restore activated transction - 4 different activators were tested Oelgeschlager et al., 1998 no transcription after depletion of TFIID and TAFs 26