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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

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Molecular Microbiology: DNA supercoiling IMC

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International Systems Biology Conference 2004 in Heidelberg

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

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

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

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Molecular Microbiology: DNA supercoiling zTV >>> transistors; zCell >>> molecules; zOrganism >> cells; zEcosystem >> organisms Important properties emerge from the interactions/integration How do molecules and cells do this ? The subject of System Biology The subject of System Biology

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Molecular Microbiology: DNA supercoiling How scientific conferences differ from (some) lectures.. zTalk by scientist A zScientist B listens zAsks a question and makes A think and respond zA and B go home zDo a new experiment and make a new discovery zNext time the talks of both A and B go further zThe essence: zA influences A through a dynamic action through B zIt is important that both A and B are dynamically responsive

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

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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

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Molecular Microbiology: DNA supercoiling Both system and molecules are different

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Molecular Microbiology: DNA supercoiling Look at the system

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

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

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Molecular Microbiology: DNA supercoiling The primary and secondary structure of B-DNA zHydrophobic interior zHydrophilic exterior zBase pairing zMajor and minor groove zRight-handed helix zPitch: 3.4 nm; 10.4 bps z1 bp: 0.33 nm

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Molecular Microbiology: DNA supercoiling Static? Or? zHydrophobic interior zHydrophilic exterior zBase pairing zMajor and minor groove zRight-handed helix zPitch: 3.4 nm; 10.4 bps z1 bp: 0.33 nm

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Molecular Microbiology: DNA supercoiling The twist zTw= number of times the two strands rotate around each other y(when one follows the ds DNA through space) zB-form DNA: 1/10.4 basepairs zE. coli 4.6 million basepairs, I.e. zTw = 0.44 million

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

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Molecular Microbiology: DNA supercoiling A, B and Z DNA; differences zZ: left-handed zB: minor and major groove zPitch (10, 10.4 and 12 bps) zTwist

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Molecular Microbiology: DNA supercoiling The twist zB-form DNA: 1/10.4 basepairs zE. coli 4.6 million basepairs, I.e. zTw = 0.44 million zA-form DNA: 1/10.0 basepairs zTw=0.44 million zZ-form DNA: -1/12 basepairs: zTw=-0.38 million

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Molecular Microbiology: DNA supercoiling Special primary structure: circular DNA zSome DNAs are circular zThe 5 and 3 ends of each strand are covalently closed zBecause of the twist of the strands, zthis causes the strands to be linked

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Molecular Microbiology: DNA supercoiling Linking number zThe number of times the one strand has been caught by the other) zHow many times does one need to cut, strand pass and reseal to liberate the two strands zFlat circle: Lk = Tw zSupercoiled circle Lk Tw 1 3 4 5 6 7 8 9 2

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Molecular Microbiology: DNA supercoiling For a flat circle Linking and twisting are not independent zLk = Tw zFlat pBR322 circle: Lk = Tw = 419 zNow: increase the Lk by 1 zLk=420 zNow Tw also 420 if circle remains flat

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Molecular Microbiology: DNA supercoiling Changing the topology

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Molecular Microbiology: DNA supercoiling 1 3 4 5 6 7 8 9 2 4 1 3 2 6 7 89 5 10 9 2 3 4 1 7 8 1 6 5 Link +1 Let go Change Lk, Tw and Wr

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Molecular Microbiology: DNA supercoiling 1 3 4 5 6 7 8 9 2 4 1 3 2 6 7 89 5 10 9 2 3 4 1 7 8 1 6 5 Link +1 Let go Lk=10; Tw=10; Wr=0 Lk=9; Tw=9; Wr=0 Lk=10; Tw=9; Wr=1 Note: Lk Tw+Wr

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

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

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Molecular Microbiology: DNA supercoiling How can the cell change supercoiling zWr: nucleosomes, DNA binding proteins zTw: intercalating agents, ionic strength, RNA polymerase zLk: nicking then closing: yTopoisomerase I: single strand nick yTopisomerase II; double strand nick yTopoisomerases and transcription zNote: yTw, Lk and Wr: always 2 change together ychange in Lk can be measured and therefore this is usually called change in supercoiling

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Molecular Microbiology: DNA supercoiling How can the cell change supercoiling zLk: nicking then closing: yTopoisomerase I: single strand nick

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Molecular Microbiology: DNA supercoiling How can the cell change supercoiling zLk: nicking then closing: yTopoisomerase II: double strand nick, passage of double stranded DNA, closure

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Molecular Microbiology: DNA supercoiling How does the cell bring about supercoiling zBacterial Topoisomerase II (gyrase): reduces Lk zLk=Tw+Wr zCauses negative Wr zSupercoiling is negative: zLk deficit: Lk