Regulation of Gene Expression

1. Regulation of Gene Expression Instructor: Kirst King-Jones

2. Contact info: Kirst King-Jones Room G-504, Biological Sciences Building Phone: 492-8605 Email: kirst.king-jones@ualberta.ca Office hours: Wednesdays,15:30-16:30 Ran Zhuo Room G-502, Biological Sciences Building Phone: 492-7534 rzhuo@ualberta.ca Office hours: Wednesdays 15:00-17:00, otherwise email

3. Mark breakdown • Three in class problems (quizzes): 3 x 6% Nov. 2, 16 & 30 (Wednesdays), each 20 min • Final exam: 32% (topics of the second half) • Date of the final: Tuesday, December 17. • Where & when: CCIS 1-160, 14:00 - 16:00

4. Reading Material • No textbook • However, recommended chapters in some common textbooks are: - Essential Genes III (Lewin): chapters 10, 19-21, 28-30. - Genes X (Lewin): chapters 10,19-21, 26, 28-30. - Molecular Biology of the Gene (6th edition, Watson et al.): chapters 16-19 - Molecular Biology of the Cell (Alberts et al.): chapter 4 & chapters 6-8 • Articles will be on GENET304 webpage • Lectures will be will be uploaded the night before.

5. http://www.biology.ualberta.ca/courses/genet304/

6. Password: chromatin Username: histone

7. The central Dogma DNA RNA Protein

8. Gene Regulation occurs mainly at the Level of Transcription DNA Regulation of gene expression RNA Protein

9. Prokaryotes Eukaryotes

10. So, what makes eukaryotes so complex? DNA DNA DNA DNA NOT cell-specific RNA RNA RNA RNA cell-specific Protein Protein Protein Protein cell-specific cell type 1 (brain) cell type 2 (kidney) cell type 3 (liver) cell type 4 (hindgut)

12. So, what is so different about eukaryotes? brain heart gut liver DNA DNA DNA DNA NOT cell-specific RNA RNA RNA RNA cell-specific Protein Protein Protein Protein cell-specific (cell type 1) (cell type 2) (cell type 3) (cell type 4)

13. Key differences at different regulatory levels multiple tissues, same DNA highly compact chromatin (Histones) DNA complex intron/exon structure bigger genomes, more genes DNA packaged into nucleus alternative splicing & RNA editing microRNAs RNA RNA interference three RNA polymerases transcription separated from translation Protein more protein families Signaling molecules for cell-cell communication

14. What is different in Eukaryotes? Important questions Distance: Regulatory sequences are far away from transcript start site How is this distance overcome and how is specificity maintained? 3D Structure: Histones/Nucleosomes restrict access to DNA How is chromatin made accessible for transcription factors? Complexity: Genes have exons and introns Why are certain splice forms made while others are not? Specificity: Eukaryotes have many cell types and complex development Why are genes turned on in specific tissues or at specific times?

15. The complexity of eukaryotic gene regulation • ~10% of all genes are involved in gene regulation ….most of them encode transcription factors (Proteins other than RNA Polymerase participating in transcription). • Many transcription factors bind short DNA sequences (~5 to 20 bp) …which are also called cis-regulatory elements • cis-regulatory elementsare often “bundled” into cis-regulatory modules • Examples forcis-regulatory modules are enhancers, silencers and insulators

16. What we will cover in this course • core promoter function: bacteria and eukaryotes compared • Chromatin remodeling & histone modification • Methods to study gene expression • RNA processing (splicing & editing) • Translational control of gene expression (miRNA & iron) • Signaling pathways

17. Genes, core promoters and Polymerases • Gene & mRNA structure: Features and terminology • RNA polymerases • Core promoters

18. Anatomy of a eukaryotic gene Proximal Promoter Enhancer Transcription Start Site Core Promoter A gene consists of DNA that encompasses regulatory DNA sequences as well as the transcribed region (= transcription unit), both regions may overlap.

19. Transcription occurs in a DNA bubble (true for both bacteria and eukaryotes) • DNA is melted • RNA synthesis occurs in bubble • RNA chain extends and dissociates from DNA • DNA reanneals

20. Transcription: Sequence of events (true for both bacteria and eukaryotes) • Factors required for initiation, elongation and termination not shown

21. There are three RNA polymerases in eukaryotes (and only one in bacteria)

22. Roles of the 3 RNA polymerases RNA polymerase I: transcribes rRNA genes (to make ribosomal RNA) RNA polymerase II: transcribes protein-coding and microRNA genes RNA polymerase III: transcribes tRNA and other small RNA genes

23. How are eukaryotic RNA polymerases recruited to the core promoter? In contrast to bacteria, all eukaryotic RNA Polymerases require pre-bound “general transcription factors” (“GTFs”) . general transcription factors for POL II: TFIId = TBP + TAFs TBP : TATA-binding protein TAF: TBP-Associated Factor

24. The Start Point for RNA Polymerase II • RNA polymerase II requires general transcription factors (GTF’s) to initiate transcription. • Most RNA polymerase II promoters have a short conserved sequence called the initiator element (InR) at the start point • TATA-Binding Protein (TBP) binds to the TATA box, but not necessarily in the correct orientation. • Correct directionality for the polymerase is provided by a second promoter element, which is contacted by other GTF’s

25. DNA elements found in core promoters ~25% ~33% ~69% ~40%

26. Species-specific comparison of eukaryotic core promoter elements

27. Promoter contacts made by the general transcription machinery

28. TBP Binds DNA in an Unusual Way • TBP binds to the TATA box in the minor groove of DNA using • an uncommon DNA-binding domain composed of 8 b-sheets • Compared to the major groove, binding to the minor groove offers less chemical contacts for a DNA-binding protein. As a result, binding specificity is reduced, which is why most DNA-binding proteins form contacts in the major groove. • It forms a saddle around the DNA and bends it by ~80°.

29. TATA-binding protein (TBP) bends DNA

30. The Basal Apparatus Assembles at the Promoter • Binding of TFIID to the TATA box is the first step in initiation. • Other transcription factors bind to the complex in a defined order • TFIIH is required to melt DNA and allows polymerase movement via CTD tail phosphorylation (ATPase and Kinase functions) • The tail also serves as a binding site • for capping, splicing and polyA • factors.

31. Eukaryotic genes have complex regulatory regions

32. RNA polymerase II requires TFIId for binding to DNA

33. TFIId requires activators for binding to DNA Step 1 Step 3 Step 2