1. Chapt. 10 Eukaryotic RNA Polymerases and their Promoters Student learning outcomes : Explain composition of 3 different nuclear RNAPs; emphasis on pol II Explain which genes each pol transcribes Describe nature of 3 classes of promoters Generally appreciate how structural information of pol II has informed mechanistic understanding Appreciate that mitochondria and chloroplasts also have their own RNA polymerases (not discussed) Appreciate that Archaeal RNA polymerases share features with eukaryotes (not discussed) 10-1

2. Important Figures: 1*, 2, 3, 4, 6*, 9*, 12, 14*, 19*, 20*, 22, 23, 24, 26; Tables: 1, 2, 3 Review problems: 1, 4, 5, 6, 7, 8, 11, 15, 16, 17, 18, 21, 23, 26, 28; AQ problems: 1, 2, 3, 4 10-2 Roger Kornberg: crystal structure of Pol II  4/7 with DNA/RNA active site Nobel Prize

3. 10-3 10.1 Multiple Forms of nuclear RNAP Nuclei contain 3 RNAP s eparated by ion-exchange chromatography Pol I in nucleolus: rRNA genes Pol II and pol III in nucleoplasm Fig. 1

4. 10-4 Roles of 3 RNA nuclear Polymerases Pol I large rRNA precursor Pol II mRNA –Heterogeneous nuclear RNA (hnRNA) –Small nuclear RNA (snRNA) Pol III tRNAs, 5S rRNA, other small RNA

5. 10-5 Human and Yeast RNAP Subunit Structures Note orthologs to bacterial subunits; CTD = C-terminal domain

6. RNA polymerases I, II, III distinguished by sensitivity to inhibitor  -amanitin 10-6 Figs. 3 Poisonous Amanita mushroom “death cap” Fig. 4 pol II is most sensitive

7. 10-7 Epitope tagging, purification, helps distinguish true subunits from associated proteins Add extra domain to one subunit; others normal (e.g., His-tag) (RNAP labeled by growing cells in labeled amino acids) Purify complex with antibody to epitope Denature, separate on SDS-PAG gel TAP tag common technique ( tandem affinity purification) Fig. 6 Pol II Structure

8. Pol II Original 10 subunits: Core – related in structure and function to bacterial core subunits Common – found in all 3 nuclear RNAP Nonessential subunits – conditionally dispensable for enzymatic activity Numbered by size; later subunits 11 and 12 Fig. 7; 32 P shows phosphorylated subunits A, Rick Young; b. Roger Kornberg

9. 10-9 Core Subunits Rpb1, Rpb2, Rpb3 absolutely required for enzyme activity: deletion mutants are dead; some ts mutants –Homologous to  ’-,  -, and  -subunits –Both Rpb1 and  ’-subunit bind DNA –Both Rpb2 and  -subunit are at or near the nucleotide- joining active site –Rpb3 does not really resemble  -subunit, but some aa similarity, 2 subunits per holoenzyme Common Subunits Found in all 3 nuclear polymerases: –Rpb5, Rpb6, Rpb8, Rpb10, Rpb12 All are essential genes (deletion mutants are dead)

10. 10-10 Subunits Nonessential for Elongation Rpb4 and Rpb7 –Dissociate fairly easily from polymerase –substoichiometric quantities –Rpb4 may help anchor Rpb7 to pol II –Mutants without Rpb4 and Rpb7 transcribe well, but cannot initiate at real promoter Rpb7 is essential subunit, so must not be completely absent in the rpb  4 mutant Assist binding of transcription factors

11. 10-11 Review Pol II subunits

12. Heterogeneity of Rpb1 (Subunit II) Subunit IIa is primary product in yeast –Converted to IIb by proteolytic removal of carboxyl-terminal domain (CTD) which is 7-peptide (YSPTSPS) repeated 26 times (heptad repeat) –Kinase converts IIa to IIo by phosphorylation of Ser2 in heptads –RNAP with IIa binds promoter –RNAP IIo involved in elongation –Phosphatase converts IIo back to IIa Fig. 8 C = Rpb2

13. 10-13 3-D Structure of RNA Pol II - Kornberg Nobel prize Yeast pol II (pol II  4/7) at 2.8 A: Deep cleft accepts linear DNA template Catalytic center at bottom of cleft contains Mg 2+ ion Second Mg 2+ ion present in low concentrations Geometry of linear DNA permits: –TFIID to bind at TATA box of promoter –TFIIB link pol II to TFIID –Places polymerase to –initiate transcription Fig. 10

14. 10-14 3-D Structure - Pol II in Elongation Complex pol II (  4/7) bound to DNA template, RNA product Clamp region of pol II closed over DNA and RNA –Closed clamp ensures transcription is processive – transcribe whole gene without falling off, early termination Fig. 13 DNA template strand in blue DNA nontemplate strand in green RNA is in red

15. 10-15 Position of Critical Elements in Transcription Bubble Loops of clamp extend into transcription bubble: –Lid: maintains DNA dissociation –Rudder: initiates DNA dissociation –Zipper: maintains dissociation of template DNA DNA template strand in blue DNA nontemplate strand in green RNA is in red Fig. 14b

16. Structure of 12-subunit pol II Initiating form of pol II; Clamp is shut Promoter must melt before template DNA inserts in active site Rpb4/7 extends dock region of pol II; makes binding of transcription factors easier, for initiation 10-16 Fig. 19

17. 10-17 10.2 Eukaryotic Promoters Three eukaryotic nuclear RNA polymerases have: –Different structures –Transcribe different classes of genes Expect the RNAPs recognize different promoters Emphasis on Pol II (mRNA)

18. 10-18 Class II Promoters Promoters recognized by pol II (class II promoters) are similar to prokaryotic promoters: Considered to have two parts: –Core promoter of 4 elements: TATAAA, TBP, BRE (IIB), –Upstream promoter element TATA box: on nontemplate strand (TATAAA consensus) Very similar to prokaryotic -10 box (TAtAaT consensus) Fig. 20

19. 10-19 Linker Scanning mutations clarify promoter elements Systematically substitute 10-bp linker for 10-bp sequences through promoter Found mutations within TATA box destroyed promoter activity Fig. 22

20. Fig. 10.23 Linker scanning mutations of TK promoter of herpes simplex virus defined critical elements mutations within TATA box destroyed promoter activity Injected plasmids into frog oocytes for transcription; control had pseudo-WT sequence

21. 10-21 Core Promoter Elements for pol II In addition to TATA box, core promoters have: –TFIIB recognition element (BRE) –Initiator (Inr) –Downstream promoter element (DPE) At least one core element 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 (constitutively active in all cells) tend to lack TATA boxes

22. 10-22 Upstream Elements Regulate pol II Differ from core promoters in binding to relatively gene-specific transcription factors (Chapt. 12) –GC boxes bind transcription factor Sp1 –CCAAT boxes (‘cat boxes’) bind CTF (CCAAT-binding transcription factor) Upstream promoter elements can be orientation- independent, yet relatively position-dependent Linker scanning mutations identified, clarified: –Delete upstream elements -> less expression –Ex. Mutation of GC boxes in SV40 early promoter

23. 10-23 Class I Promoters for rRNA - pol I Class I promoters are not well conserved sequence Usually two elements: –Core element surrounding transcription start site –Upstream promoter element (UPE) 100 bp farther –Spacing between elements is important Fig. 24; linker scanning mutations define sites

24. 10-24 Class III Promoters RNA pol III transcribes a set of short genes These have promoters that lie wholly within the genes There are 3 types of these promoters

25. 10-25 Class III Promoters of Pol III Genes are often within the gene Type I (5S rRNA) has 3 regions: Type II (tRNA) has 2 regions: Type III (nonclassical) resemble type II Fig. 26

26. 10-26 10.3 Enhancers and Silencers Position- and orientation-independent DNA elements stimulate or depress, respectively, transcription of associated genes Often tissue-specific - rely on tissue-specific DNA- binding proteins for activities Some DNA elements can act either as enhancer or silencer depending on what is bound to it: –Ex. SV40 Early genes 2 x 72-bp GC boxes Fig. 28 SV40

27. Enhancer in immunoglobulin genes Fig. 29 Deletion of enhancer reduces transcription; Fig. 30 enhancer works in either orientation, and position 10-27

28. Review questions 4. How many subunits does yeast pol II have? Which are core subunits? Which are common to all 3 nuclear RNA polymerases? 7. What is the structure of the CTD of RPB1? 17. Diagram a pol II promoter, showing all of the types of elements it could have. AQ3. You are investigating a new class II promoter. Design an experiment to locate promoter sequences 10-28