Slide 1Lecture OutlineSlide 3Slide 4Conventions in writing DNA/RNA sequencesSlide 6Slide 7Summary of B-DNA Structural FeaturesWhat forces are responsible for DNA structure?Slide 10Slide 11Clicker QuestionSlide 13Sequence-specific DNA binding proteinsSlide 15DNA Structure is Not Uniform Along the Helix and Can VarySequence Dependent Bends in DNAProtein Binding can Bend DNAPropeller TwistSlide 20Slide 21Chemical and Physical Properties of DNASchematic of a UV-Vis SpectrophotometerSpectrophotometry of DNAUV Absorption Spectrum of DNASlide 26Absorption Spectra of Aromatic Amino AcidsSlide 28Denaturation of DNASlide 30DNA Melting CurvesSlide 32DNA Renaturation or Annealing Denaturation is reversible!Sequences do not have to be identical to renatureSlide 35MCB 250 Lecture 4More about DNALecture Outline•DNA structure (conclusion)•Denaturation/renaturation of DNA–UV spectrum of DNA–Use of spectrophotometry to quantitate DNA and study denaturation/renaturation•DNA hybridizationA=T and G=C because DNA is a double helix and A in one strand pairs with T in the other strand via H-bonds. The same thing goes for G and C.Note that the strands are antiparallel. One runs 5’-3’, the other 3’-5’.Figure 4-3Hydrogen Bonding Defines the Specificity of Base Pairing•Excellent relative alignment of hydrogen bond donors and acceptors•Non-complementary bases do not align and cannot form hydrogen bonds or even fit in to the DNA helix. Figs 4-6 4-7Conventions in writing DNA/RNA sequences•5’ (left) 3’ (right): ATGTCG means 5’A, 3’G. It’s complement is CGACAT.•You can always indicate the polarity explicitly: the complement of the sequence above can be written 3’TACAGC5’•Usually if you write a duplex sequence the top strand is 5’-3’ left to right. (5’) ATGTCG TACAGC (5’)B-form DNAA right-handed helix1Å = 1 x 10-10 meter = 0.1 nanometer (nm)Right HandedLeft HandedThe Double HelixB-DNASummary of B-DNA Structural Features•Right handed helix •Strands are antiparallel•Hydrogen bonded bases lie in the same plane•Plane is perpendicular to the helix axis•20Å in diameter•34Å per turn•10.4 base pairs per turn•Bases rotated 36° with respect to each other•Major groove (12Å), Minor groove (6Å)What forces are responsible for DNA structure?•Base Pairing - H-bonds•Base Stacking - stacking interactions between aromatic rings•Hydrophobic interactions of bases•Repulsion of negatively charged phosphate groups – cations are required to neutralize10Stacked Base PairsH2O moleculeIn addition to van der Waals (stacking) forces the nonpolar surfaces of the stacked aromatic rings undergo hydrophobic interactions, thus excluding H2O from the interior region of the helix.In contrast, the negatively charged phosphate groups are repelling each other and are, therefore, on the outside of the helix.Chemical information in the major and minor groovesEdges of base pairs are differentiated by the pattern and presence of-H bond acceptors (A)-H bond donors (D)-Nonpolar Hydrogens (H)-Methyl groups (M)Fig. 4-10Clicker Question•Some proteins specifically bind to DNA of a given sequence. In other words, the protein can “read” the sequence. •If you were designing a DNA binding protein the binds to a specific sequence, how would you make it work? A. Form ionic bonds with the phosphates in the backboneB. Form hydrogen bonds with the “spare” groups in the Minor grooveC. Form hydrophobic interactions with the bases in the Minor grooveD. Form hydrogen bonds with “spare” groups in the Major grooveE. Form hydrophobic interactions with the bases in the Major groove( Hint – a common secondary structural element of protein, the alpha-helix, is ~12 A in diameter).AADHDAADAMADAMAMHere's what a protein sees inside a major groove...3'-ACAA-5'5'-TGTT-3'12Å6ÅSequence-specific DNA binding proteins •Interactions with H-bond donors and acceptors and van der Waals surfaces in the grooves allow proteins to recognize specific sequences without disrupting the double helix.•The major groove contains more information. E.g., C-G and G-C look different in the major groove but not in the minor groove.The Double HelixB-DNADNA Structure is Not Uniform Along the Helix and Can Vary•Most DNA in cells is believed to be more or less in the B form•Different base sequences can induce structural variations–Intrinsic bends–“Propeller twist”•Binding of proteins to DNA can also induce bending•DNA is a dynamic molecule–Some regions may be transiently single stranded (melted).–Loops and cruciforms may form transiently.–Water and ions are continuously coming and going. Look at Youtube: http://www.youtube.com/watch?v=JjDyWgAqTkE&feature=channel_video_title–Some regions may form alternative structures transiently (e.g., Z-DNA).[GCTCGAAAA]4Sequence Dependent Bends in DNARuns of A-T base pairs spaced about 10 bps apart cause bending.Protein Binding can Bend DNAHistone binding bends the double helix.Propeller TwistNormal B-form base pairsTwisted base pairs Fig. 4-12Sequences Dependent StructuresCruciformG QuadruplexFigure 4-11Different DNA Structures ExistB DNA A DNA Z DNAChemical and Physical Properties of DNA•Bases absorb UV light with a maximum at 260nm (remember they’re aromatic).•Absorption is dependent on the environment of the chromophore (the chromophores are the bases).•The environment is different in single stranded and double stranded DNA so they absorb differently.Schematic of a UV-Vis SpectrophotometerEntrance SlitDispersion DeviceExit SlitSampleDetectorMonochromatorSourceSpectrophotometry of DNAThe Hyperchromic Effect Wavelength (nm)AbsorbanceNativeDenatured200 250 260 3001.51.00.50UV Absorption Spectrum of DNA220 240 260 280 300AbsorbanceAl=elclBeer’s LawFor DNA:A260 e260clA280 e280cl=A260 e260A280 e280==1.8DNAPeak Absorbance at ~260 nmWavelength (l; nm)A=Absorbancee=Extinction Coefficientc=Concentrationl=Path lengthSpectrophotometry of DNAMeasuring the UV absorption of a sample allows…• Quantitation of [DNA] -At 50mg/ml, dsDNA A260 = 1.00 ssDNA A260 = 1.37 free bases A260 = 1.60• Purity check of a preparation - A260/A280 = 1.8 for highly pure dsDNAAbsorption Spectra of Aromatic Amino AcidsTrpTyrPheAl=elclBeer’s Lawe280>>e280>>>e280Trp Tyr Phe\Trp predominates in absorption at 280 nmfor any given protein230 250 270 290 310Wavelength (l;