PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12 The polymeric structure of nucleic acidsSlide 14Slide 15Slide 16Slide 17Conventions in writing DNA/RNA sequencesChargaff’s Rules (1951-’52)Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33DNA Melting CurvesSlide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Lecture 21 (Ch. 33) Nucleotides and Nucleic Acids• Central Dogma review• Differences between DNA and RNA• Nitrogenous bases• Nucleotides• Nucleic acids (DNA) •Composition of DNA (Chargaff’s Rule)•The DNA double helix (Watson-Crick)•Secondary structure of DNA (A, B, Z DNA)•Denaturation and Renaturation of DNA•Higher order packing of DNA in cells (chromosomes and histones)• Nucleic acids (RNA) secondary and tertiary structures of RNA1Central Dogma2DNA and RNA differ in the sugar component and one of the bases BaseNucleoside = Base + sugar3BaseDNA and RNA differ in the sugar component and one of the bases 4BaseNucleotide = Base + sugar + phosphateBaseP PDNABackbones of DNA and RNAPhosphodiester bridgesRNA3’5’ 5Nitrogenous Bases: Pyrimidines-StructureFound in both RNA and DNA*Found in RNA only The pyrimidine ringFound in DNA only 6Nitrogenous Bases: Purines-StructureBoth are found in both RNA and DNAThe purine ring system7N-glycosidic bond links N1 of pyrimidines to C1 of deoxyribose" N9 of purines "The DeoxyribonucleotidesBases:Adenine Guanine Thymine Cytosine8N-glycosidic bond links N1 of pyrimidines to C1 of ribose" N9 of purines "The RibonucleotidesAdenine Guanine Uracyl Cytosine9Bases:Nucleotides“High-energy” phosphoanhydride bonds“Low-energy” phosphomonoester bond1011Cyclic Nucleotides- The phosphoric acid moiety is esterified to two of the available OH groups on the ribose ring12Cyclic AMP (cAMP)The polymeric structure of nucleic acidsRNA/DNA axis5’--OH--3’-OHSense StrandAntisense StrandSense StrandAntisense Strand13Chemical Properties of purines and pyramidines1. UV light absorbance: important for measuring DNA concentration2. Nucleotides are polyprotic acids14Purines and Pyrimidines- Absorption of UV lightPyrimidines and Purines typically strongly absorb at UV at 260 nm15At pH 7: A monomeric nucleotide (nucleoside monophosphate) has net charge of -2Nucleic acids (polymers of nucleotides) derive their name due to the acidic nature of the phosphate groups of their component nucleotidesNucleotides (ribo and 2ʹ-deoxyribo) are polyprotic acids 165′-P end 3′-OH endReading direction5’-ACGU- 3’5’-p-ACGU- 3’5’-pApCpGpU- 3’Nucleic acids have directionality17Conventions in writing DNA/RNA sequencesComplement to top strand (the bottom strand) can be written as:5’ - ATGTCG - 3’ 3’ - TACAGC - 5’Usually if you write a duplex sequence the top strand is 5’-3’ left to right.5’- CGACAT - 3’ or3’- TACAGC - 5’(forward)(backward)18Chargaff’s Rules (1951-’52)The % GC or AT varies between organisms but always A=T and G=C.(ds) [Purines] = [Pyrimidines]19Double HelixNote that the strands are antiparallel20Double Helix – B-form Double Helix – B-form ~ 10.4 nucleotides per turn21Axial view, looking down the helix axis2223The Major and Minor Grooves are lined sequence-specific hydrogen-bonding groupsGlycosidic bond = deoxyriboseGrooves arise because glycosidic bonds are not opposite each other Each groove is lined by potential hydrogen-bond donor and acceptor atoms24Major and Minor groove of the DNA double helix25Base Stacking Contribute to the Stability of the Double Helix26Right handed – A and B DNAPitch = 24.6 Å (2.46 nm)Base per turn = 11Rise per base = 2.3 ÅPitch = 34 Å (3.4 nm)Base per turn = 10.4Rise per base = 3.4 ÅA formB form27Z DNAB-DNA•Left-handed helix•Narrower and longer than B-DNA•Rise per base = 3.8 Å•12 bases per turn•45.6 Å pitch per turn28DNA replication is Semi-Conservative AntisenseSenseA SS AWhat would conservative replication look like+ nucleotides29A diagram of semiconservative replication+ nucleotides+ nucleotides(without blue nucleotides)(without blue nucleotides)(Only blue nucleotides)30The resolution of 14N dsDNA and 15N dsDNA by density-gradient centrifugation.UV-absorptionDensitometric tracingHeavy DNALight DNAds = double strandss = single strand31Meselson-Stahl experiment (1958)N15N15N14N1432Denaturation of DNA secondary structureDenaturing agents:1. Heat (DNA denaturation = DNA melting)2. Extremes of pH3. Strong H-bonding solutes33DNA Melting CurvesssDNAdsDNA34Denatured DNA can renature to re-form the double helixUpon cooling, returning pH to neutral or when denaturants are diluted out, denatured DNA will renature (reanneal) to re-form the duplexRenaturation is dependent on both DNA concentration and time35Circular DNA from MitochondriaRelaxed dsDNASupercoiled dsDNAMost DNA in cells is about5% underwound relative to B form.36Supercoiling in DNAlefUnderwinding Right-handed DNA (B-DNA) produces negative (-) supercoils. These supercoils are right-handed(260 bp)37Supercoils - DNA can adopt regular structures of higher complexity •Double stranded circular DNA form supercoils when the two strands are underwound (-ve supercoiled) or overwound (+ve supercoiled)•Topoisomerases are enzymes that can break one or both strands of the DNA, wind them tighter or looser and rejoin the ends•DNA Gyrase is a topoisimerase that introduces –ve supercoils into DNASupercoiling does not mean the DNA is more (or less) coiled than B form DNA.It means that there is a coiling super-imposed on the coiling of B form DNA.38DNA in cells occurs in highly folded compact structuresProkaryoticSingle dsDNA molecule length = 1.6 mmE. Coli cell = 0.002 mm lengthEukaryotic23 pairs of ds DNA(46 DNA molecules) = 3.6 m totalHuman cell = 20 μm diameterNucleus = 5 μm diameter39Nucleosomes are complexes of DNA and histonesWrapping DNA around histones introduces negative supercoils 40Eukaryotic DNA is wrapped around histones to form nucleosomesChromatin – DNA + histonenucleosomes (146 base pair of DNA around each nucleosome core)Histones are rich Lys and Arg41Net Result: Each DNA molecule has been packaged into a mitotic chromosome that is 50,000X shorter than the simple DNA helix.Still more compaction is required to form condensed mitotic chromosomes42Secondary Structures for Nucleic AcidsStem
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