1. Biol/Chem 473 Schulze lecture 2: Eukaryotic gene structure

2. Prokaryotic vs. eukaryotic gene organization Eukaryotic genes are interrupted Polycistronic units are rare in eukaryotes

3. Eukaryotic genes have introns and exons

4. Eukaryotic transcription is complex

5. Eukaryotic RNA polymerases

6. All three eukaryotic RNA pols have 2 large sub units See some homology with bacterial RNA pol components …but also MANY additional subunits.

7. Eukaryotic RNA polymerases

9. RNA polymerase II

10. Has to transcribe a GREAT diversity of genes, that all have specific developmental and spatial expression profiles. RNA polymerase II has MANY subunits.

11. Using reporter genes to dissect regulatory sequences

14. Eukaryotic promoters consist of two components: 1: core promoter RNA pol II and the general transcription factors bind to the core elements in and around the transcription start site. This allows for a basal level of transcription.

15. Eukaryotic promoters consist of two components: 1: core promoter RNA pol II and the general transcription factors bind to the core elements in and around the transcription start site. This allows for a basal level of transcription.

16. Eukaryotic promoters consist of two components: 1: core promoter Not all promoters look alike in eukaryotes. Some genes have internal promoters. Some genes have no obvious promoters at all (even though they are expressed). Kutach and Kadonaga (2000) Mol. Cell Biol. 20(13): 4754

17. Eukaryotic transcription is complex Within 100-200bp from transcription start

18. Eukaryotic transcription is complex

19. The first enhancer discovered: SV40 Reticulocyte control fibroblasts

20. And they are promiscuous: they will influence any gene they have access to.

21. How does an enhancer function? The enhancer appears to function to bring proteins into the vicinity of the promoter

22. How does an enhancer function? The enhancer appears to function to bring proteins into the vicinity of the promoter

23. How does an enhancer function?

24. Specialized transcription factors

25. Transcription factors: an overview Can repress or activate transcription –Activators bind enhancers; repressors bind silencers Tend to be modularized Have distinctive DNA binding sites –Helix-turn-helix, Zn finger, Leucine zipper, homeodomain, Can multimerize Can work combinatorially

26. Transcription factors can work combinatorially

27. Complexity of eukaryotic transcription

29. Houston, we have a problem… Recall: this loop can be enormous

30. Houston, we have a problem… It almost certainly contains other genes

31. Houston, we have a problem… But recall that enhancers are promiscuous

32. And they are promiscuous: they will influence any gene they have access to.

33. If there is nothing blocking its way, an enhancer can activate any gene.

34. Problem: enhancers tend to be promiscuous! Enhancers located a great distance from their target genes have the potential to activate intervening non target genes How is the action of enhancers restricted to prevent promiscuous activation?

35. Solution: Insulators! DNA sequence elements that bind proteins with enhancer-blocking and/or chromatin barrier activity Insulator proteins have been extensively studied in flies (less so in humans)

36. Proteins that function with insulators Geyer and Clark 2002 flies mammals yeast A number of these proteins (most?) can self-associate

37. Sequences associated with insulator function Geyer and Clark 2002

38. Insulators as enhancer-blockers Gerasimova and Corces 2001 Insulators can disrupt enhancer-promoter communication only when placed between them Placement of insulators can create autonomous domains of gene activity

39. Insulators can organize the genome into transcriptionally autonomous domains Placement of insulators can create autonomous domains of gene activity

40. Insulators can organize the genome into transcriptionally autonomous domains

41. Overlay transcriptional and chromosomal territories Giant loop with active genes extending from chromosomal territory Active genes (white) move away from het (yellow), and back again when silent (black) Different chromatin densities within a chromosome territory Gene poor, late replicating domains within chromosome territories localize closer to the nuclear periphery Active genes (white) congregate where transcription factories (polymerases, splicing factors etc) are enriched Cremer & Cremer (2001) Nat. Rev. Genet. 2(292)