1. Three RNA polymerases in eukaryotes

2. RNA polymerase III Hundreds of promoters - 40% of a cell transcriptional activity -Moderately sensitive to  -amanitin (Ki=1mM) -Transcribe gene encoding tRNAs, 5S rRNA and some stable RNAs (U6 and others) -Promoter may be internal to the transcribed region RNA polymerase II Thousands of promoters - 10% of a cell transcriptional activity -Highly sensitive to  -amanitin (Ki=10 nM) Transcribe gene encoding mRNAs and some stable non coding RNAs (U1, U2, U4, U5 and others) RNA polymerase I 1 promoter - 50% of a cell transcriptional activity - low sensitivity to  -amanitin Transcribe gene encoding the 45S precursor of rRNAs (18S, 5.8S, 28/26S rRNAs) 18S 28S 18S 5.8S 45S precursor Non-transcribed spacer 45S precursor

3. TAT A Inr TAT A Inr TAT A Inr TAT A Inr TAT A Inr + TFIID + TFIIA + TFIIB + TFIIF - Pol.II + TFIIE + TFIIH Pre Initiation Complex (PIC) IIA TFIID (TBP + TAFs) TFIID TFIIB IIB TFIIF RNA Polymerase II Polymerase II TFIIF TFIIE TFIIH Assembly of the RNA polymerase II machinery onto Eukaryotic Promoters General Transcription Factors: used to assist RNA Polymerase binding to most promoters: TFIIA TFIIB TFIID = TBP +TAFs TFIIE TFIIH

4. TAT A Inr ATP hydrolysis (helicase in TFIIH) Also used in Nucleotide Excision Repair Pre Initiation Complex (PIC) Open Complex IIA TFIID IIB Polymerase II TFIIF TFIIE TFIIH TAT A Inr IIA TFIID IIB Pol. II TFIIF TFIIE TFIIH TAT A Inr IIA TFIID IIB Pol. II TFIIF TFIIE TFIIH P P P P Pol. II CTD Phosphorylation by CAK (TFIIH) Phosphorylation at S5 (initiation) of YSPTSPS repeats of the CTD

5. TAT A Inr IIA TFIID IIB Pol. II TFIIF TFIIE TFIIH P P P P TAT A Inr IIA TFIID IIB Pol. II TFIIF TFIIE TFIIH P P P P Capping Enzyme SR Proteins CPSF CstF Phosphorylated Pol. II Recruitment of RNA processing Factors Capping enzyme,SRs (splicing) CPSF, CstF (3’end) Transcription Initiation TAT A Inr IIA TFIID Pol. II TFIIF TFIIE TFIIH TFIIB Capping Enzyme SR Proteins CPSF P P P P 5’ 3’ Phosphorylation at S2 (elongation) of YSPTSPS repeats of the CTD

6. TATA Binding Protein (TBP) - binds and recognize the TATA box TFIID TBP-associated Factors (TAFs) TAF 250,60, 110, 95, 78, 38, 28 - binds and recognize the Inr (TAF250) -provide binding sites for gene specific transcription factors TFIIH XPB ATP-dependent DNA helicase p62 p52 p44 P34 XPD ATP-dependent DNA helicase Cyclin-activated kinase: Phosphorylates the CTD of Pol. II Structure of TBP bound to the TATA element (coding strand shown in green) Composition and Function of two GTFs – TFIID and TFIIH PDB ID = 1CDW

7. Transcriptional Control in eukaryotes: a few things specific to eukaryotes ------------------------------------------------------------------------------------------- Gene-specific transcription factors (as opposed to sigma factors) Coactivators of transcription Transcriptional control and chromatin modifications (“epigenetics”) (control of transcription by controlling RNA Pol.II binding) ------------------------------------------------------------------------------------------- Pausing of the RNA polymerase near promoters (control of transcription by controlling RNA Pol.II elongation)

8. TATA box Inr Activator Sequence (sometimes called enhancer) +1 -30 GTFs + Pol. II Gene-specific transcription factor binds here Promoter Why Are activator sequences necessary ? Transcriptional Activation in Eukaryotes: Genes-specific transcription factors 1) Assembly of GTFs and Pol. II is inefficient The binding of gene-specific TFs facilitate assembly of GTFs and Pol. II 2) Transcription is cell- or time-specific The presence of a combination of gene specific activators in a particular cell type at a particular stage of differentiation ensures the transcription of the proper set of genes.

9. Modular Structure of Gene-Specific Transcription Factors N C DNA-binding Domain Activator Domain Examples of Activator Domains: - Acidic - Glutamine-rich - Proline-rich Examples of DNA-binding Domains: - Helix Turn Helix - Zn Finger - leucine Zippers/bZip - bHLH Flexible Linker TATA box Inr +1 GTFs + Pol. II binding site 1 Activator Domain-2 Activator Domain-1 DNA Binding Domain-2 DNA Binding Domain-1 Enhancer 2 How do long distance (enhancer-promoter) relationships work ? Local Curvature Of the DNA region => PIC Stabilization

10. Helix-turn-Helix DNA binding domains most frequent DBD in prokaryotes (e.g. Lac Repressor, etc..) Also found in euk. for example in Homeodomain proteins = Transcription Factors that govern Development Engrailed, Bithorax etc… Courey Plate 4.4 PDB ID = 1HDD

11. 3 Zn Fingers complexed to DNA 2 Zn Fingers The Zn Finger motif 1 Zn Finger 3D structure The multiplicity of Zn Fingers on the same protein allows recognition of complex DNA sequences PDB ID = 1ZAA

12. Direct base readout by  -helices of DNA binding domains bZip domains Helix-loop-Helix domains PDB ID: 1FOS PDB ID: 1HDD Courey Plate 4.4

13. TATA box Coactivator Inr Gene-Specific Transcription Factor GTFs + Pol.II TATA box Inr Gene-Specific Transcription Factor GTFs + Pol.II Examples of Coactivators: -CBP/p300 -Mediator Complex Direct vs. Indirect Activation by Gene-Specific Transcription Factors Direct Activation: The Gene-Specific Transcription Factor interacts directly with the GTFs and/or RNA Polymerase II Indirect Activation: The Gene-Specific Transcription Factor does not interact directly with the GTFs and/or RNA Polymerase II and needs a Coactivator

14. - The Mediator complex stimulates transcription of genes containing activator sequences. - The action of the Mediator complex is dependent on the presence of proteins binding to the activator sequences. TATA Inr TATA Inr Enhancer mediator From Boyer et al., Nature 1999 May 20;399(6733):276-9 RNA produced by “basal” transcription RNA produced by “activated” transcription Conclusion: The presence of the mediator complex only affects RNA produced by “activated” transcription, not by “basal” transcription One Example of Coactivator complex:The Mediator In vitro transcription with two different DNA templates, RNA Pol.II, and increasing amounts of mediator complex

15. Binding of histones to DNA through electrostatic interactions: Histones are + charged, DNA is - charged -Modulation of interactions of histones with DNA by covalent modifications: Histones acetylation, deacetylation, methylation, ubiquitination on the N-terminal tails of histones Structure of the nucleosome of eukaryotic cells The problem of Chromatin in Eukaryotic Cells How to access genes in this context ??

16. Post-translational modifications of histones modulate chromatin accessibility and transcription Current Opinion in Plant Biology Volume 5, Issue 5, 1 October 2002, Pages 437-443 NH 3 Ac-CoA CoA Lys side chain in a histone tail HAT HAT = histone acetyl transferase HDAC = histone deacetylase CH 3 H N O H2OH2O CH 3 COO - HDAC

17. Histone modifications modulate chromatin accessibility and transcriptional status Regulated chromatin modifications,allow access of the transcription machinery and activation/inactivation of genes Methyl Transferase overall Histone H3 acetylation/ methylation status controls transcription levels

18. -> Enhancer Sequences -> Promoters -> Transcribed Regions Hum. Mol. Genet. (2009) 18 (R2): R195-R201. Specific Histone modifications mark gene regions in eukaryotes “Chromatin Signature”

19. Chromatin immunoprecipitation of RNA pol.II across genomes reveal the enrichment of the polymerase nearby promoters (“poised”) Transcriptional Control by Controling RNA Polymerase II elongation

20. Promoter proximal pausing is controlled by the elongation factor NELF and helps control eukaryotic gene expression by rapid switch from poised state to elongation