Three RNA polymerases in eukaryotes

1. Three RNA polymerases in eukaryotes

2. RNA polymerase I Non-transcribed spacer 1 promoter - 50% of a cell transcriptional activity - low sensitivity toa-amanitin Transcribe gene encoding the 45S precursor of rRNAs (18S, 5.8S, 28/26S rRNAs) 5.8S 5.8S 18S 18S 28S 28S 45S precursor 45S precursor Three RNA polymerases in eukaryotes RNA polymerase III • Hundreds of promoters • - 40% of a cell transcriptional activity • Moderately sensitive toa-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 a-amanitin • (Ki=10 nM) • Transcribe gene encoding mRNAs and • some stable non coding RNAs • (U1, U2, U4, U5 and others)

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

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

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

6. Composition and Function of two GTFs – TFIID and TFIIH TFIID TATA Binding Protein (TBP) - binds and recognize the TATA box • 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 Structure of TBP bound to the TATA element (coding strand shown in green) TFIIH PDB ID = 1CDW XPB ATP-dependent DNA helicase p62 p52 p44 P34 XPD ATP-dependent DNA helicase Cyclin-activated kinase: Phosphorylates the CTD of Pol. II

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. Transcriptional Activation in Eukaryotes: Genes-specific transcription factors Gene-specific transcription factor binds here GTFs + Pol. II -30 +1 Inr TATA box Activator Sequence(sometimes called enhancer) Promoter Why Are activator sequences necessary ? 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. How do long distance (enhancer-promoter) relationships work ? Enhancer 2 binding site 1 DNA Binding Domain-2 DNA Binding Domain-1 Activator Domain-2 Activator Domain-1 => PIC Stabilization GTFs + Pol. II Local Curvature Of the DNA region +1 Inr TATA box Modular Structure of Gene-Specific Transcription Factors DNA-binding Domain Activator Domain Flexible Linker C N Examples of DNA-binding Domains: - Helix Turn Helix - Zn Finger - leucine Zippers/bZip - bHLH Examples of Activator Domains: - Acidic - Glutamine-rich - Proline-rich

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… PDB ID = 1HDD Courey Plate 4.4

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

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

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

14. mediator TATA Inr Enhancer TATA Inr One Example of Coactivatorcomplex:TheMediator - 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. In vitro transcription with two different DNA templates, RNA Pol.II, and increasing amounts of mediator complex RNA produced by “basal” transcription RNA produced by “activated” transcription From Boyer et al., Nature 1999 May 20;399(6733):276-9 Conclusion: The presence of the mediator complex only affects RNA produced by “activated” transcription, not by “basal” transcription

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

16. NH 3 H O N CH 3 Post-translational modifications of histonesmodulate chromatin accessibility and transcription Lys side chain in a histone tail CH3COO- Ac-CoA HDAC HAT H2O CoA HAT = histone acetyl transferase HDAC = histonedeacetylase Current Opinion in Plant Biology Volume 5, Issue 5 , 1 October 2002, Pages 437-443

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

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

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

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