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DNA: Structure, Dynamics and Recognition

DNA: Structure, Dynamics and Recognition. L2: Introductory DNA biophysics and biology. Les Houches 2004. STRUCTURE DETERMINATION. X-ray l ≈ 1 Å ≈ atomic separation requires crystals phase problem (homologous structures, or heavy atom doping). X-RAY DIFFRACTION. 1.2 Å. 2 Å. 3 Å.

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DNA: Structure, Dynamics and Recognition

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  1. DNA: Structure, Dynamics and Recognition L2: Introductory DNA biophysics and biology Les Houches 2004

  2. STRUCTURE DETERMINATION

  3. X-ray l≈ 1 Å ≈ atomic separation • requires crystals • phase problem (homologous structures, or heavy atom doping) X-RAY DIFFRACTION

  4. 1.2 Å 2 Å 3 Å - Resolution limit = l/2.Sin qmax - R-factor = S [|Fobs| - |Fcal|]/|Fobs| (0.15-0.25 implies good agreement) Crystallographic resolution

  5. Doucet et al. Nature 337, 1989, 190 Crystal packing effects

  6. DiGabriele et al. PNAS 86, 1989, 1816 Crystallographic curvature

  7. NMR SPECTROSCOPY • Can excite atoms with nuclear spins, 1H, 13C, 15N, 31P • Relaxation leads to RF emissions which depend on the local environment • 1D spectra of macromolecules suffer from overlapping signals

  8. 2D NMR SPECTRA COSY (COrrelation SpectroscopY) - covalently coupled atoms NOESY (Nuclear Overhauser Effect) - through space coupling

  9. Sequential Resonance Assignments “Biomolecular NMR Spectroscopy” J.N.S. Evans (1995).

  10. STRUCTURE FROM NMR DATA • identify residues in contact (>5 Å) • model structure using distance + torsional constraints and known valence geometry • check quality by reconstructing NMR spectrum • a range of structures generally fit the data (accounting for flexibility) • not easy to define resolution • problems of crystallisation are replaced with problems of solubility and size • may need isotopic labelling

  11. OTHER SPECTROSCOPIC TECHNIQUES

  12. Simple inexpensive technique • Optical density of sample compared to buffer solution • IR - molecular vibrations, • UV - electronic transitions • Macromolecules give broad spectra formed of many overlapping transitions Absorption Spectroscopy

  13. UV • More disorder more absorption (e.g. diamonds) • ds DNA  ss DNA more absorption Absorption Spectroscopy

  14. IR • Raman scattering gives acces to vibrations without water peak • can identify percentages of sugar puckers, glycosidic conformations, ... Absorption Spectroscopy

  15. Circular dichroism (CD) • Measures the difference in absorption between left- and right-handed circularly polarized light (ellipticity) • Sensitive to molecular chirality • ms resolution • simple experiments poly(dG-dC).poly(dG-dC) 0.2 M NaCl 3.0 M NaCl Pohl & Jovin J. Mol. Biol. 67, 1972, 675

  16. Neutron scattering spectroscopy DNA/D2O • Access to dynamics in psns timescale • Vibrational density of states • Needs a lot of material and a reactor • H/D exchange for selective studies Slow relaxation in solvent > 210 K Sokolov et al. J. Biol. Phys. 27, 2001, 313

  17. FRET - fluorescent resonance energy transfer • varies as r -6 • detection ≈ 5-10 Å

  18. S S HN3 imino proton Still to come .... Hydrogen exchange Single molecule experiments

  19. STABILIZATION OF THE DOUBLE HELIX

  20. Chemical bonds C-H 105 kcal.mol-1 C=C 172     Ionic hydration Na+ -93 Ca2+ -373    Hydrogen bonds O…H -5 (in vacuum)     Protein folding ~ 2-10 (in solution)    Protein-DNA binding ~ 5-20 (~200 Å2 contact) Biological energy scale

  21. Helix  Coil

  22. UV melting curve for a bacterial DNA sample Tm= T at which 50% of DNA is melted

  23. Tm increases with GC content

  24. Stabilising factors : Base pairing (hydrogen bonds) Base stacking (hydrophobic) Ion binding (electrostatics) Solvation entropy Destabilising factors : Phosphate repulsion (electrostatics) Solvation enthalpy (electrostatics/ LJ) DNA strand entropy DNA energetics - I

  25. Pairing in vacuum : Yanson, et. al. 18 (1979) 1149 Bases DH CG -21.0 AU -14.5 Pairing in chloroform : Kyoguku et al.BBA 179 (1969) 10 Bases DH CG -10.0 -11.5 AU -6.2 AA -4.0 Stacking in water (stronger than pairing) :T’so 1974 Bases DH AA -6.5 UU -2.7 TT -2.4 Base pairing and stacking

  26. Stofer et al. J. Am. Chem. Soc. 121, 1999, 9503 Separating a GC basepair in water

  27. Breslauer empirical equation for ss  ds : (Biochemistry 83, 3748, 1986) DGp = (Dgi + Dgsym) + SkDgk Stack Dgk GG -3.1 AA -1.9 G G A A T T C C GA -1.6 C C T T A A G G CG -3.6 GC -3.1 DGp = (5.0 + 0.4) - 2 x 3.1 TG -1.9 - 2 x 1.9 - 2 x 1.6 - 1.5 AG -1.6 AT -1.5 GT -1.3 DGp = -9.3 Kcal/mol TA -0.9 DGexp = -9.4 Kcal/mol DNA energetics - II

  28. s1 : CGCATGAGTACGC Vesnaver and Breslauer PNAS 88, 3569, 1991 s2 : GCGTACTCATGCG ds ss(h) ss(r) Kcal/mol ds  ss(r) s1(hr) s2(hr) Sum DG 20.0 0.5 1.4 1.9 DH 117.0 29.1 27.2 56.3 TDS 97.0 28.6 25.8 54.4 DNA energetics -III

  29. ... and now for something completely different ...

  30. DNA TRANSCRIPTION

  31. Bond vibrations 1 fs (10-15 s) Sugar repuckering 1 ps (10-12 s) DNA bending 1 ns (10-9 s) Domain movement 1 s (10-6 s) Base pair opening 1 ms (10-3 s) Transcription 20 ms / nucleotide Replication 1 ms / nucleotide Protein synthesis 6.5 ms / amino acid Protein folding ~ 10 s Biological time scale

  32. RNA replicase DNA polymerase RNA polymerase DNA RNA Reverse Transcriptase PROTEIN CENTRAL DOGMA TRANSCRIPTION TRANSLATION

  33. RNA polymerase DNA mRNA NTPs snRNP • Regulation by transcription factor binding • Initiation (at a promoter site) • Formation of a transcription bubble • Elongation (3'5' on template strand,≈ 50 s-1) • Termination (at termination signal) • Many RNA polymerases can function on 1 gene (parallel processing) Splice out introns DNA Transcription

  34. Transcription Factors (TAFs) • Activators: specific DNA-binding proteins that activate transcription • Repressors: specific DNA-binding proteins that repress transcription • Some regulatory proteins can work as both activators and repressors for different genes • TAF sites are more difficult to locate than genes • Nucleosome positioning influences gene transcription

  35. s factor associates with -10 (TATA box) and -35 • RNA polymerase binds • Bubble forms at -103 Prokaryote transcription - initiation

  36. E.Coli. pol II, resolution ≈ 2.8Å RNA polymerase Cramer et al. Science 292, 2001, 1863

  37. 5' 3' 5' • form ≈ 10 bp RNA-DNA hybrid • 5'-end of RNA dissociates • s factor dissociates and recycles Prokaryote transcription - elongation

  38. inverted repeat preceding A-rich region • hairpin formation competes with RNA-DNA hybrid • RNA transcript dissociates • Can also involve RNA-binding protein Rho Prokaryote transcription - termination

  39. EukaryoteTranscriptosome

  40. DNA REPLICATION

  41. DNA Replication

  42. + Semiconservative • E.coli ≈ 1000 bp.s-1 • Replication is bidirectional • Prokaryotes have a single origin of replication (AT-rich repeats) DNA Replication

  43. DNA polymerase I requires NTPs , Mg2+ and primer • Works in the 5'3' direction • Leads to "Okazaki" fragments (10-1000 bp) • Initially these fragments are ≈10nt RNA primers • Fragments are finally joined together by a ligase DNA Replication

  44. Right hand: “palm”, “fingers”, “thumb” • Palm  phosphoryl transfer • Fingers  template and incoming nucleoside triphosphate • Thumb  DNA positioning, processivity and translocation • Some have 3'  5' exonuclease “proofreading” second domain DNA polymerases features

  45. Bacteriophage T7 T. gorgorianus DNA Polymerase variations

  46. Processivity is very variable (≈ 10  ≈ 105) • Fidelity ≈ 10-6-10-7 (primer plays an important role) • DNA polymerases can proofread (increases fidelity by ≈ 103) • Incorrect nucleotide stalls polymerase and leads to 3'5' exonuclease excision DNA Replication

  47. 3-component "ring"-type DNA polymerase

  48. b-subunit of E.Coli polymerase III

  49. Replication also requires: • DNA Helicase - hexameric, unwinds DNA, uses ATP • SSB - single-stranded DNA binding protein, stops ss re-annealing or behind degraded • Gyrase (Topo II) - relaxes +ve supercoiling ahead of replication fork • More complex in eukaryotes (telomeres, nucleosomes, ...) DNA Replication

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