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Nucleic Acids

Nucleic Acids. CH339K. Monomers: Nucleotides. Component 1: 5-Carbon Sugar. Ribose vs. Deoxyribose. Difference in components of DNA and RNA Extra hydroxyl makes RNAs much more reactive. Sugar Pucker. Furanoses are not planar Can pucker out of the plane of the ring at C2 or C3

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Nucleic Acids

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  1. Nucleic Acids CH339K

  2. Monomers: Nucleotides

  3. Component 1: 5-Carbon Sugar

  4. Ribose vs. Deoxyribose • Difference in components of DNA and RNA • Extra hydroxyl makes RNAs much more reactive

  5. Sugar Pucker • Furanoses are not planar • Can pucker out of the plane of the ring at C2 or C3 • Pucker effects higher order structures (or vice-versa)

  6. Component 2: Nitrogenous Base

  7. Purines

  8. Pyrimidines • Cytosine and Thymine in DNA • Cytosine and Uracil in RNA

  9. Component 3: Phosphate

  10. Nomenclature

  11. Syn and Anti Conformations

  12. Syn / anti energetics From: Neidle, S. (2008) Principle of Nucleic Acid Structure Elsevier, London, pg. 33

  13. Condensation – Polymer Formation

  14. Phosphodiester Linkages

  15. Simple Condensation is Energetically Unfavorable DGo‘≈ +25 kJ/mol Keq=4.15*10-5

  16. Synthesis is from the triphosphate Energetics: Phosphodiester formation - +25 kJ/mol nTP cleavage – -31 kJ/mol Pyrophosphate cleavage - -19 kJ/mol Keq = 24100

  17. Tautomeric Forms of Bases -NHx groups can be in the amino or imino conformation =O groups can be in the keto or enol conformation The predominant form for the free base is not necessarily the predominant form in the nucleotide Lack of basic O-Chem knowledge caused problems for Watson and Crick when they were trying to figure out the structure of DNA Keto Enol

  18. Base Pairing keto amino amino keto Cytosine Guanine

  19. Animation

  20. Secondary Structure of Nucleic Acids • Helical • Result of base pairing • Defined by • Pitch • Rise • In turn governed by structure of the monomers

  21. B Helix Typical DNA

  22. Determination of helix parameters Rosalind Franklin’s Diffraction Photo of B-DNA

  23. A Helix RNA, DNA/RNA hybrids, dehydrated DNA

  24. Z Helix Alternating Purine-Pyrimidine

  25. Z DNA Function? • Z DNA is antigenic • Antibodies are found in autoimmune disorders like systemic lupus erythematosus • Antibodies bind to puffs in Drosophila polytene chromosomes • Also bind macronuclei of ciliates • Z DNA-prone sequences found in transcription start sites • May act as spacer between RNA polymerases • Z DNA binding proteins required for pathogenicity by vaccinia and smallpox

  26. Helix Parameters Summarized

  27. Major and Minor Grooves

  28. Grooves provide access to base sequence • Telomere binding protein • a-helix fits into major groove • Side chains can recognize bases

  29. Another Example cro Repressor protein of bacteriophage l. Small (66 amino acids) Forms dimers Binds to specific sites on DNA that activate / deactivate genes Expression of cro results in the phage entering the lytic cycle

  30. Absorption of UV Light

  31. UV Absorption Spectrophotometry

  32. Beer-Lambert Law c = concentration l = path length e = extinction coefficient An Absorbance = 2 means that only 1% of the incident beam is getting through.

  33. Transmittance and Absorbance Absorbance vs. Concentration Transmittance vs. Concentration

  34. Physical Properties - Absorbance

  35. Physical Properties - Hypochromicity • Stacked bases in nucleic acids absorb less ultraviolet light than do unstacked bases, an effect called hypochromism • Rules of thumb: • e280dsDNA: 20 • e280ssDNA/RNA: 37.5 • e280 small oligonucleotides: 50 Calculated spectrum of equivalent mixture of free nucleotides Double stranded RNA (38% G+C) Single stranded RNA (38% G+C) From Cox, R. A. (1970) Conformation of Nucleic Acids and the Analysis of the Hypochromic Effect,Biochem. J. (1970) 120, 539-547

  36. Denaturation: “Melting” • Heat, alkali cause the double helix to unwind • As H-bonds break, they form “bubbles” in the helix • As the equilibrium shifts towards H-bonds breaking, the bubbles coalesce • The strands come apart

  37. As temperature increases, local denatured regions coalesce

  38. Effect of G+C content on Tm

  39. ss Bubbles Coalesce until Strands Separate

  40. Effects of changing DHo’ and DSo’ Artificially generated curves

  41. DNA Sequencing – Sanger Method

  42. DNA Sequencing - Sequencers

  43. Polymerase Chain Reaction(aka DNA Amplification)

  44. Internal Structure Palindromes and inverted repeats tend to be sites for recognition by proteins Palindromes: Kay, a red nude, peeped under a yak Some men interpret nine memos Campus Motto: Bottoms up, Mac

  45. Internal Structure (cont.)

  46. Replication Origin of Duck Hepatitis B

  47. Nonstandard Base Pairs

  48. Triplex DNA Structure • Duplex DNA Structure • Triplex DNA with 3rd Strand in Major Groove • Bissler, John J. (2007) Triplex DNA and human disease, Front. Biosci. 12: 4536-4546.

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