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The dynamic structure of the prokaryotic information

The dynamic structure of the prokaryotic information. Hans V. Westerhoff and friends Molecular Cell Physiology, Institute for Molecular Cell Biology and Swammerdam Institute for Life Sciences, BioCentrum Amsterdam. IMC. International Systems Biology Conference 2004 in Heidelberg.

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The dynamic structure of the prokaryotic information

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  1. The dynamic structure of the prokaryotic information Hans V. Westerhoff and friends Molecular Cell Physiology, Institute for Molecular Cell Biology and Swammerdam Institute for Life Sciences, BioCentrum Amsterdam

  2. IMC Molecular Microbiology: DNA supercoiling

  3. International Systems Biology Conference 2004 in Heidelberg Molecular Microbiology: DNA supercoiling

  4. Here we are ……………….. • Billons of bps, hundreds of transcriptomes, some proteomes, parts of the metabolome, even a phenome or two further …. and ……. ………… • Leena Peltonen: ‘hyperlipidemia: >10 factors involved; cannot be tracked down even with island populations’ • Svante Pääbo: ‘substantial expression divergence without functional consequences’ • Joseph Nadeau: ‘Different mouse strains show wide difference in homeostasis in response to folate depletion’ • Stefan Schreiber: ‘>600 genes differentially expressed between (Crohn) in inflamed mucosa; dissection does not allow to identify the multifactorial etiology’ • , i.e.: we still do not understand.. Molecular Microbiology: DNA supercoiling

  5. I will tell you what you know already …………….. But what we did not yet live up to…..

  6. Life arises not just in the isolated molecules but in their communication Jan Steen Molecular Microbiology: DNA supercoiling

  7. How scientific conferences differ from (some) lectures…….. • Talk by scientist A • Scientist B listens • Asks a question and makes A think and respond • A and B go home • Do a new experiment and make a new discovery • Next time the talks of both A and B go further • The essence: • A influences A through a dynamic action through B • It is important that both A and B are dynamically responsive Molecular Microbiology: DNA supercoiling

  8. What is System Biology? The properties that arise in interactions …….. Through the dynamic-dynamic modes

  9. The coin of System Biology has two sides The System differs from the sum of the molecules (without interactions) …….. The molecules behave differently in the system

  10. Both system and molecules are different Molecular Microbiology: DNA supercoiling

  11. Look at the system… Molecular Microbiology: DNA supercoiling

  12. … or look at the molecule…. in interaction Molecular Microbiology: DNA supercoiling

  13. DNA:Dynamic molecule in the system…. Really???? ? Molecular Microbiology: DNA supercoiling

  14. The primary and secondary structure of B-DNA • Hydrophobic interior • Hydrophilic exterior • Base pairing • Major and minor groove • Right-handed helix • Pitch: 3.4 nm; 10.4 bps • 1 bp: 0.33 nm Molecular Microbiology: DNA supercoiling

  15. Static? Or? • Hydrophobic interior • Hydrophilic exterior • Base pairing • Major and minor groove • Right-handed helix • Pitch: 3.4 nm; 10.4 bps • 1 bp: 0.33 nm Molecular Microbiology: DNA supercoiling

  16. The ‘twist’ • Tw= number of times the two strands rotate around each other • (when one follows the ds DNA through space) • B-form DNA: 1/10.4 basepairs • E. coli 4.6 million basepairs, I.e. • Tw = 0.44 million Molecular Microbiology: DNA supercoiling

  17. Three known secondary structures of DNA left-handed! Molecular Microbiology: DNA supercoiling

  18. Z: left-handed B: minor and major groove Pitch (10, 10.4 and 12 bps) Twist A, B and Z DNA; differences Molecular Microbiology: DNA supercoiling

  19. The ‘twist’ • B-form DNA: 1/10.4 basepairs • E. coli 4.6 million basepairs, I.e. • Tw = 0.44 million • A-form DNA: 1/10.0 basepairs • Tw=0.44 million • Z-form DNA: -1/12 basepairs: • Tw=-0.38 million Molecular Microbiology: DNA supercoiling

  20. Special primary structure:circular DNA • Some DNA’s are ‘circular’ • The 5’ and 3’ ends of each strand are covalently closed • Because of the twist of the strands, • this causes the strands to be linked Molecular Microbiology: DNA supercoiling

  21. 9 1 8 2 7 3 6 4 5 Linking number • The number of times the one strand ‘has been ‘caught by the other’) • ‘How many times does one need to cut, strand pass and reseal to liberate the two strands’ • Flat circle: Lk = Tw • Supercoiled circle Lk  Tw Molecular Microbiology: DNA supercoiling

  22. For a flat circle Linking and twisting are not independent • Lk = Tw • Flat pBR322 circle: Lk = Tw = 419 • Now: increase the Lk by 1 • Lk=420 • Now Tw also 420 if circle remains flat Molecular Microbiology: DNA supercoiling

  23. Changing the topology Molecular Microbiology: DNA supercoiling

  24. 9 1 8 10 9 8 1 2 7 2 7 3 6 3 6 4 5 5 8 7 4 9 1 3 5 1 2 6 Change Lk, Tw and Wr Link +1 4 Let go Molecular Microbiology: DNA supercoiling

  25. 9 1 8 10 9 8 1 2 7 2 7 3 6 3 6 4 5 5 8 7 4 9 1 3 5 1 2 6 Lk=9; Tw=9; Wr=0 Lk=10; Tw=10; Wr=0 Link +1 4 Let go Note: LkTw+Wr Lk=10; Tw=9; Wr=1 Molecular Microbiology: DNA supercoiling

  26. Strand cutting required to change Lk DNA molecule can itself exchange Tw with Wr DNA likes standard Tw Wr (supercoiling) tends to change when Lk is changed Wr: supercoiling, cruciforms Lk, not Wr can be measured by extracting the DNA Implications Molecular Microbiology: DNA supercoiling

  27. Same writhe; two conformations toroidal plectonemic Molecular Microbiology: DNA supercoiling

  28. How can the cell change ‘supercoiling’ • Wr: nucleosomes, DNA binding proteins • Tw: intercalating agents, ionic strength, RNA polymerase • Lk: nicking then closing: • Topoisomerase I: single strand nick • Topisomerase II; double strand nick • Topoisomerases and transcription • Note: • Tw, Lk and Wr: always 2 change together • change in Lk can be measured and therefore this is usually called ‘change in supercoiling’ Molecular Microbiology: DNA supercoiling

  29. How can the cell change ‘supercoiling’ • Lk: nicking then closing: • Topoisomerase I: single strand nick Molecular Microbiology: DNA supercoiling

  30. How can the cell change ‘supercoiling’ • Lk: nicking then closing: • Topoisomerase II: double strand nick, passage of double stranded DNA, closure Molecular Microbiology: DNA supercoiling

  31. How does the cell bring about ‘supercoiling’ • Bacterial Topoisomerase II (gyrase): reduces Lk • Lk=Tw+Wr • Causes negative Wr • Supercoiling is negative: • Lk deficit: Lk<Tw Molecular Microbiology: DNA supercoiling

  32. How can the cell change ‘supercoiling’ • Topoisomerases and transcription Molecular Microbiology: DNA supercoiling

  33. Puzzle: • Why are type II topoisomerases much better antitumor drug targets than type I topoisomerases? Can be trapped in double stranded DNA break state Molecular Microbiology: DNA supercoiling

  34. DNA supercoiling; a subtle change; can one detect it? Molecular Microbiology: DNA supercoiling

  35. Same molecule; different mobility Molecular Microbiology: DNA supercoiling

  36. Resolution of isomers Relaxed circle topoisomers Molecular Microbiology: DNA supercoiling

  37. The chloroquine trick • Difference large between and • Difference small between and • Lk=Tw+Wr • Increase Wr by decreasing Tw at constant Lk • Intercalating agent Molecular Microbiology: DNA supercoiling

  38. The chloroquine trick • Lk=Tw+Wr • Increase Wr by decreasing Tw at constant Lk • Tw=420; Lk=380; Wr=-40 • + intercalating agent • Tw=385; Lk=380: Wr=-5 Molecular Microbiology: DNA supercoiling

  39. Visualization of topoisomers on chloroquine gel

  40. Energy: topoisomers differ in elastic energy Concentrations should form a Gaussian distribution Molecular Microbiology: DNA supercoiling

  41. Gaussian distribution Molecular Microbiology: DNA supercoiling

  42. Energy: DNA gyrase Molecular Microbiology: DNA supercoiling

  43. In vivo supercoiling depends on [ATP]/[ADP] Molecular Microbiology: DNA supercoiling

  44. Hierarchical levels in the system Molecular Microbiology: DNA supercoiling

  45. Hierarchical levels in the system Molecular Microbiology: DNA supercoiling

  46. Topo I modulation and supercoiling Molecular Microbiology: DNA supercoiling

  47. Hierarchical levels in the system Molecular Microbiology: DNA supercoiling

  48. Gyrase modulation and supercoiling:Flux induced structure 0 -2 -4 -6 Supercoiling (aLk) -8 -10 -12 -14 -16 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 DNA gyrase (fraction of wild type) Molecular Microbiology: DNA supercoiling

  49. Function of DNA supercoiling • Compaction • Fit DNA into the cell • Nucleoid in absence of nuclear membrane Molecular Microbiology: DNA supercoiling

  50. Compaction: Is there a problem? 2000 • E. coli: …….. functions • E. coli genome ………. genes • 1 polypeptide chain ~…….. amino acid residues • E. coli chromosome: …………… bps • 1 bp ~0.33 nm • E. coli chromosome: …………. contour length • E. coli: 4000 300 4 million 1.3 mm 1 micron by 2 micron Molecular Microbiology: DNA supercoiling

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