1. Transcription Biology Review Bios 691 – Systems Biology January 2008

2. Outline Gene structure Chromatin structure & modifications Transcription apparatus Transcription factors and cofactors Elongation and termination RNA capping, splicing, and adenylation RNA processing and miRNA’s

3. Chromosome Organization Mammalian chromosomes tend to fill discrete regions within the nucleus An elaborate network of fibrils maintains these arrangements RNA ‘factories’ at distinct locations do most of the transcription work Nucleoli are factories for rRNA

4. Chromatin Structure Protein scaffolds anchor the DNA Within the scaffold there are loops Most transcription happens on the loops Much chromatin is wrapped in 30nm ‘heterochromatin’

5. Fine Structure of Chromatin Heterochromatin – inaccessible –Bound with many proteins –Centromeres; telomeres; some other areas Euchromatin – accessible –Still needs to be opened Telomeric Heterochromatin and SirtuinsEuchromatin: 30 nm & open

6. DNA Packaging & Nucleosomes

7. Gene Structure – Exons & Introns Exon Size distribution

8. Gene Structure – Initiation Sites Most (~2/3) genes have multiple promoters Most promoters are either ‘sharp’: –Very narrow range –Usually TATA + Inr –Often tissue specific or ‘broad’: –Typically 70 bp range –Rarely TATA / Inr –Often widespread

9. Histones and Modifications DNA contacts histones on their tails Histone tails can be modified Histones can stay loose or assemble tightly

10. Proteins Modify Histones

11. DNA Methylation Adding a Methyl to Cytosine Cytosine methylation is passed on to daughter cells

12. Controlling Transcription

13. DNA-Binding Proteins All proteins interact weakly with DNA Proteins with projecting amino acids interact with the DNA major groove Hydrogen bonds stabilize position of proteins on DNA Proteins that line up several amino acid contacts bind strongly to specific DNA sequences

14. Transcription Factor Families Several structures line up amino acids –Helix-turn-Helix (Homeodomain) –Helix-loop-helix –Zinc Finger Mostly dimers These families have proliferated because of their role in attracting transcription apparatus

15. Cofactors Frequently the effect of DNA-binding proteins depends on co-factors E.g. ER sits on the DNA but requires estrogen as a co-factor to function Myc requires Max as a co- factor to stimulate transcription If Max is coupled with Mad instead, the genes are repressed

16. Kick-starting Pol II & Elongation Mediator protein bridges TF proteins and RNA Pol II Contains kinase domains – may phosphorylate CTD of RNA Pol II

17. Initiating Transcription TBP on a TATA Box

18. RNA Polymerase II RNA (red) copied from DNA (blue) by RNA Polymerase II RNA Polymerase II Structure The cycle of adding nucleotides

19. Terminating Transcription

20. RNA Processing

21. RNA Processing Steps Nucleus –capped, –spliced, –cleaved, –polyadenylated Exported Cytoplasm –stored –translated –degraded

22. Capping mRNA The RNA factory

23. RNA Splicing

24. Poly-adenylating RNA Poly-A Polymerase adds ~100- 150 Adenines to 3’ end After export to cytoplasm, nucleases chop off ~10-20 A’s at a bite Nucleases compete with ribosomes for mRNA’s When ~30 A’s left degradation speeds up

25. RNA Export RNA has to be passed through nuclear pores to show up in the cytoplasm (where we measure it)

26. Micro RNA’s

27. P-Bodies Loci where RNA accumulates and is degraded Have their own structural proteins

28. Implications for Systems Biology Levels of TF’s on a promoter may not predict levels of transcripts Rate of transcription may not predict level of mRNA in the cytoplasm Levels of mRNA in cytoplasm may not predict levels of protein