1. Introduction to Genetics Winter semester 2014 / 2015 Seminar room 00.005 INF230 Thursdays 18:15 - 19:45

2. 14 lectures Molecular biology of genes and genomes. Key technologies: Next generation sequencing. Bioinformatics analysis of sequencing data. Background. Molecular Biology of the Cell (Alberts, 5th Edition) Genes X (Lewin) Background reading (guided by lectures). Talks (pdfs or powerpoints of talks posted on “Moodle” e- learning platform after lectures).

3. Introduction to Genetics: timetable 2014 October 23 Thomas DickmeisIntroduction / Basic transcription mechanisms I(Lecture 1) October 30 Thomas DickmeisBasic transcription mechanisms II (Lecture 2) November 6 Clemens GrabherControl of transcription in eukaryotes (Lecture 3) November 13 Felix LoosliDNA replication, recombination and repair I (Lecture 4) November 20 Felix LoosliDNA replication, recombination and repair II (Lecture 5) November 27 Felix LoosliRNA machines and translation I (Lecture 6) December 4 Clemens GrabherRNA machines and translation II (Lecture 7) December 11 Harald KoenigmRNA splicing and processing (Lecture 8) December 18 David Ibberson Next generation sequencing (Lecture 9) 2015 January 8 Juan MateoData analysis of Next generation sequencing (Lecture 10) January 15 Clemens GrabherTransposable elements, recombination, hypermutation: immune system. (Lecture 11) January 22 Rüdiger RudolfEpigenetic control of gene expression (Lecture 12) January 29 Rüdiger RudolfViruses (Lecture 13) February 5th Jochen WittbrodtGenome structure, function, evolution (Lecture 14) Date to be announced. Nicholas S. Foulkes Final Examination Contacts: Nick Foulkes: nicholas.foulkes@kit.edu Jochen Wittbrodt: jochen.wittbrodt@cos.uni-heidelberg.de Thomas Dickmeis: thomas.dickmeis@kit.edu Clemens Grabher: clemens.grabher@kit.edu Rüdiger Rudolf: ruediger.rudolf@kit.edu Felix Loosli: felix.loosli@kit.edu Harald Koenig: h.koenig@kit.edu David Ibberson: david.ibberson@bioquant.uni-heidelberg.de Juan Mateo: juan.mateo@cos.uni-heidelberg.de

4. Examination: Mid-February Short questions / answers 34 questions, 2.5 hours. Resit, new set of questions (Andrea Wolk). Announcement of date, place and time closer to the date (January) Questions based on information content of lectures Course contact: Nicholas S. Foulkes nicholas.foulkes@kit.edu

5. Basic transcription mechanisms Thomas Dickmeis Institut für Toxikologie und Genetik, KIT, Karlsruhe thomas.dickmeis@kit.edu 5

6. Transcription:Definitions I (DNA dependent) RNA polymerase Transcription: 5′ to 3′ on a DNA template strand that is 3′ to 5′ non-template strand of the DNA = coding strand 6

7. Transcription:Definitions II promoter – region of DNA where RNA polymerase binds to initiate transcription transcription startpoint or start site (TSS) - position on DNA corresponding to the first base incorporated into RNA terminator – a sequence of DNA that causes RNA polymerase to terminate transcription 7

8. Transcription:Definitions III upstream – sequences in the opposite direction from transcription downstream – sequences in the direction of transcription 8

9. Transcription: Definitions IV transcription unit – the sequence between sites of initiation and termination by RNA polymerase primary transcript – the original unmodified RNA product corresponding to a transcription unit (a transcription unit may contain several genes, e.g. in bacteria) 9

10. typical cartoon of a transcription unit promoter TSS coding region 5‘ UTR 3‘ UTR AUG Stop (UnTranslated Region) 10

11. The transcription „bubble“ 11

12. Reaction catalyzed by RNA polymerase RNA (n residues) + ribonucleotide triphosphate (NTP) ↔ RNA (n+1 residues) + PP i PP i + H 2 O ↔ 2 P i Stryer 2002 12

13. Transcription bubble of bacterial RNA polymerase Bubble size: 12-14 bp (DNA-RNA-hybrid within the bubble: 8-9 bp) Speed: 40-50 nucleotides/second (DNA replication: 800 bp/second) 13

14. The stages of transcription 14

15. Prokaryotic Transcription 15

16. Bacterial RNA polymerase consists of multiple subunits Core polymerase: α 2 ββ′  catalyzes transcription Holoenzyme: α 2 ββ′  and σ (sigma factor) core enzyme and σ factor together competent for initiation 16

17. σ factor ensures promoter specific binding and is required for initiation cannot initiate able to initiate 17

18. How does RNA polymerase find promoter sequences? too fast for simple diffusion unspecific DNA binding, then „one-dimensional random walk“ proposed mechanisms: (wrong labels in the book) intersegment transfer „hopping“ direct transfer 18

19. Aligment of many promoters reveals stretches of conserved sequences -> functionally important What defines a promoter? cis-acting element: recognized and specifically bound by proteins consensus sequence Sequence logo: illustrates conservation and frequency of bases in each position consensus sequence: the most conserved bases in each position adapted from Stryer 2007 19

20. The bacterial promoter consensus sequence TATAATTTGACA main elements: -35 box and -10 box (or Pribnow box) additional elements (UP, Ext, Dis...) can affect promoter efficiency individual promoters usually differ from the consensus also: not all elements have to be present: modularity distance between -35 and -10 boxes: 16-18 bp in 90% of promoters -> Important! (Why?) 20

21. The bacterial promoter consensus sequence TATAATTTGACA several regions of  factor and the  subunit C-Terminal-Domains bind at the consensus elements seen in crystal structure of the bacterial holoenzyme in bound to promoter DNA 21

22. X ray crystallography in a nutshell Stryer 2002 electron density map atomic model ribbon diagram 22

23. Crystal structure model of holoenzyme-DNA complex Illustration adapted from D. G. Vassylyev, et al., Nature 417 (2002): 712-719  (Detailed view – but you cannot always determine a crystal structure each time you want to map protein- DNA interactions) Sigma factor is extended, with short alpha-helical domains connected by flexible linkers 23

24. footprinting Stryer 2007 (How do you get labelling just at one end?) 24

25. Preparing a footprinting probe I putative binding site 1 putative binding site 2 PCR-amplification 5‘ 3‘ genomic DNA 25

26. putative binding site 1 putative binding site 2 (blunt) (5‘ overhang) Nature Protocols 3, 900 - 914 (2008) Preparing a footprinting probe II „asymmetric“ digestion Klenow enzyme can add radioactive nucleotide (*) at this end („Klenow fill-in reaction“) (What if no suitable restriction sites in the sequence?) (How must the nucleotide be labelled?) 26

27. Real world examples Increasing protein concentration Nature Protocols 3, 900 - 914 (2008) sequencing reaction footprint experiment Footprinting achieves single nucleotide resolution! Nucl. Acids Res. (2000) 28 (18): 3551-3557. 27

28. (Sanger Sequencing – original method) 28

29. (Sanger Sequencing – the standard today) Now even faster methods with higher throughput become available – „next generation sequencing“ – see lecture by David Ibberson 29

30. (another new method to map protein binding down to single bp resolution: ChIP-exo) ChIP = Chromatin Immuno Precipitation Rhee et al., Cell Volume 147, Issue 6, 9 December 2011, Pages 1408–1419 30

31. Footprinting reveals polymerase shape changes during the stages of transcription  factor core enzyme  factor Knippers 1997 closed binary complex open complex ternary complex general elongation complex Initiation Elongation 31

32. Detection of unwinding  factor core enzyme  factor Knippers 1997 Initiation Elongation Unwound bases become accesible for reagents that cannot reach them within the double helix e.g. KMnO 4 32

33. Functional promoter analysis by mutation „down“ mutations – decrease in efficiency „up“ mutations – increase (e.g. mutation towards consensus) not all promoters match the consensus – the „perfect“ example above doesn‘t exist in nature! „maximal“ activity not necessarily „optimal“ activity „down“ in -35: closed complex formation rate ↓ open complex conversion ↔ „down“ in -10: either closed complex formation rate ↓ or open complex conversion ↓ or both AT-rich sequence around -10 helps melting – why? 33

34. Summary promoter 1.Modular 2.Consensus sequence 3.Most important: -35 and -10 box 4.Mutations may affect:  factor and polymerase binding DNA unwinding 34

35. The stages of initiation  factor core enzyme  factor Knippers 1997 closed binary complex open complex ternary complex general elongation complex Initiation Elongation 35

36. crystal structure models of initiation - snapshots of a molecular machine closed binary complex most contacts on non-template strand Nature Reviews Microbiology 6, 507-519 (July 2008) 36

37. crystal structure models of initiation - snapshots of a molecular machine closed binary complexopen complex Conformational changes: DNA bends opens between -11 and +3 moves into the enzyme („jaws close“) Nature Reviews Microbiology 6, 507-519 (July 2008) 37

38. crystal structure models of initiation - snapshots of a molecular machine closed binary complexopen complexternary complex „ternary“ – RNA polymerase, DNA and first RNA nucleotides abortive initiation: short RNAs formed and released RNA polymerase stays on promoter „DNA scrunching“ Nature Reviews Microbiology 6, 507-519 (July 2008) 38

39. Transition to elongation – promoter escape Two problems: 1)Initiation requires tight binding to specific sequences Elongation requires binding to all sequences encountered 2)  occupies exit channel for the RNA:  mediates specific binding and blocks RNA exit → get rid of it! → TEC = Transcription Elongation Complex Nature Reviews Microbiology 6, 507-519 (July 2008) 39

40. the sigma factor cycle 40

41. The elongation complex  factor core enzyme  factor Knippers 1997 closed binary complex open complex ternary complex general elongation complex Initiation Elongation 41

42. The catalytic mechanism Groove lined with positively charged amino acid residues, why? 42

43. The catalytic mechanism 43

44. The catalytic mechanism 44

45. The catalytic mechanism Mg 2+ facilitates attack of 3‘ OH stabilizes negative charges of transition state Nature Reviews Microbiology 6, 507-519 (July 2008) and Stryer 2007 45

46. The mechanism of elongation template DNA non-template DNA RNA „trigger loop“ Volume 19, Issue 6, December 2009 Pages 708-714 „bridge helix“ nucleotide in catalytic site 46

47. Brownian ratchet model Nat Struct Mol Biol. 2008 August ; 15(8): 777–779 Mg 2+ 47

48. Direct observation of base-pair stepping of single RNA polymerase molecules „optical tweezers“: small beads can be trapped in highly focused laser beams, position of the beads can be monitored with high precision: = ~ 1 bp RNA polymerase laser beam bead Nature 426:684–87 (2003) Nature. 2005 November 24; 438(7067): 460–465 48

49. Summary initiation and elongation  factor core enzyme  factor Knippers 1997 Initiation Elongation Closed binary complex: promoter recognition Open complex: melting of DNA „jaws close“ Ternary complex: RNAP, DNA, RNA abortive transcription Elongation complex:  factor off catalysis: transition state stabilized elongation movement: Brownian ratchet model 49

50. What happens if transcription is blocked? Transcription can be transiently blocked e.g. by hairpin structures in the RNA or misincorporation of NTPs Annu. Rev. Biochem. 2008. 77:149–76 (transitory) RNA polymerase can cleave the RNA to generate new 3´-OH end (cleavage activity intrinsic to RNA Pol, stimulated by accessory factors) 50

51. Transcriptional termination Two classes of terminators: 1)intrinsic terminators (no other factors required) 2)rho (  dependent terminators Annu. Rev. Biochem. 2008. 77:149–76 Often difficult to find the termination site: 1)in vivo, the primary transcript gets cleaved or partially degraded 2)in vitro, experimental conditions influence termination capacity -> if both approaches find the same, probably the true site... 51

52. Intrinsic termination Interaction of hairpin with RNA Pol. or forces created by its formation lead to misalignment of 3‘ end of the mRNA with the active centre -> destabilisation Why? 52

53. Rho termination rut = rho utilisation recognition site and effect site of rho are different pausing gives time for the other necessary events to occur What else binds to the nascent mRNA? 53

54. Summary termination 1. transient pausing – backtracking, hairpins, misincorporation 2. RNA can be cleaved by polymerase to give new free 3‘ -OH 3. termination: intrinsic (e.g. hairpin) or extrinsic (rho factor) 54

55. Polycistronic transcripts promoter TSS coding region 5‘ UTR 3‘ UTR AUG Stop the general cartoon a polycistronic transcript 55

56. Transcription and translation (in prokaryotes!) 56

57. The cycle of bacterial mRNA 57

58. Transcription in prokaryotes vs. eukaryotes Stryer 2002 58

59. three important differences Chromatin is the template (bacteria: „naked“ DNA) Polymerase needs general transcription factors (GTFs) for promoter binding and initiation (bacteria: holoenzyme binds directly) three polymerases (bacteria: one): – RNA pol I: 18S/28S rRNA – RNA pol II: mRNA, few small RNAs – RNA pol III: tRNA, 5S rRNA, other small RNAs 59