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UVM BIOC 302 - Nucleotides and nucleic acids

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Nucleotides and nucleic acids IBiochemistry 302January 18, 2006http://biochem.uvm.edu/courses/kelm/302User: studentPW: nucleicacidsCentral Dogma of Molecular Biology(Cell as a factory analogy)• DNA = permanent repository which stores master plans• RNA = temporary repository → “copy”of certain plans– Working RNAs (e.g. rRNA, snRNA).– Adapter RNAs (e.g. tRNA, miRNA)– Intermediary RNAs (e.g. mRNA).• Protein = working machineryFig. 4.23Basic chemical structure of DNA and RNA (heteropolymers of nucleotides)• Monomer composition (nucleotide)– heterocyclic pentose sugar – phosphate– nitrogenous base • RNA: polar ribose phosphate backbone• DNA: polar deoxyribose phosphate backbone (no 2′-hydroxyl)• Nucleotides joined by 3′,5′- phosphodiester linkages• Nitrogenous bases –side chainsLehninger Principles of Biochemistry, 4th ed., Ch 8Major nitrogenous bases found in DNA and/or RNA (purines & pyrimidines)• DNA: A, G, C, T• RNA: A, G, C, U• N-β-glycosyl bond: 1′carbon of ribose and N9 of Pur base (A, G) or N1 of Pyr base (C, T, U)• Pur or Pyr base + ribose = nucleosideFig. 4.2parent compoundsNucleotide NomenclatureDNARNAChemistry of nucleotide components• Phosphate group– Strong acid– pKa ~1 for primary ionization, ~6 for secondary• Purine/Pyrimidine (pKa ~2.4-9.5)– Weak tautomeric bases• Isomers differing in position of H atoms & double bond. • Less stable imino & enol forms found in special base interactions.– Conjugated double-bonds• Resonance among ring atoms• Absorb UV lightFig. 4.4Lehninger Principles of Biochemistry, 4th ed., Ch 8Chemical stability of polynucleotides(contribution of the ribose ring)• Hydrolysis of DNA and RNA is thermodynamically favorable but very slow.• Acid-labilebond (purine glycosidic linkage in DNA but not RNA• Base-labilebond (PDE bond in RNA but not DNA)• Nucleases(endo & exo, specific & non-specific) promote rapid hydrolysis of PDE bonds in DNA or RNA.• Dehydration-resistant(e.g. DNA in fossils) but water content (level of hydration) affects secondary structure Lehninger Principles of Biochemistry, 4th ed., Ch 8DNA and genetics: a historical perspective• ~1868 – Friedrich Miescher isolates phosphorus-containing substance “nuclein” from nuclei of leukocytes and salmon sperm, noted 2 portions… Acidic (DNA), Basic (Protein)• CW 1860s to 1940s – Genetic inheritance dictated by proteins → Nucleic acid too simple (4 nucleotides vs ~20 amino acids → DNA merely a structural material present in the cell nucleus.• 1944 to 1952 – DNA transfer & labeling studies point to DNA as the repository of genetic information.• Late 1940s – Chargaff’s rules of DNA composition A = T; G = C; A + G (purines) = C + T (pyrimidines) • 1953 – Watson & Crick propose structure of DNA.Hershey-Chase, 1952Avery, MacLeod, and McCarty, 1944T2 bacteriophage infectionViral T2 32P-DNA (not 35S-protein) transferred to and propagated in E. coliElucidation of DNA structureFranklin and Wilkins 1953; King’s CollegeWatson and Crick 1953; Cambridge Univ.• R. Franklin & M. Wilkins – X-ray diffraction pattern of wet DNA fibers consistent with regular, repetitive helical 3D structure w/ 2 distinct periodicities.– Primary repeat ( 3.4 Å)– Secondary repeat (34 Å)• J. Watson & F. Crick – Built best fit model based on X-ray data, Chargaff’s rules, DNA chemical composition, & clever deduction. – Ten residues/turn (34 Å)– Helical rise (3.4 Å, distance betw vertically stacked bases– Two DNA strands/helix (fiber density)R. E. Franklin and R. Gosling (1953) Nature 171:740Cross pattern typical of helixProperties of nucleotide bases → 3D structure of nucleic acid• pH-dependent tautomers– Adenine and Cytosine (amino form at pH 7) – Guanine and Thymine (keto form at pH 7)• Functional groups (H-bonding)– Ring nitrogens– Carbonyl groups– Exocyclic amino groups• Highly conjugated → resonance– Pyrimidines (planar)– Purines (nearly planar slight pucker)• Hydrophobic character– Hydrophobic stacking interactions – van der Waals interactions between uncharged atomsWatson and Crick 1953Intuition: H-bonding between certain baseson opposite strands stabilizes the helixGeometric Features:• H-bonding between A=T, G≡C base pairs → distance between C-1′ atoms the same → constant helical diameter• Bases “stacked” & slightly offset inside the double helix• Deoxyribose-phosphate backbone exposed to water• Pentose ring in C-2′ endoconformation (sugar pucker)antiparallel strandsbp stackingand rotation relative to long axisH-bonding (different # in A=T vs G≡C bps)~1.08 nm36°Rise = 0.34 nmFig. 4.10H-bonding pattern in W-C base pairs and numbering conventionA = T (N6,N1) = (O4,N3)Lehninger Principles of Biochemistry, 4th ed., Ch 8G ≡ C(O6,N1,N2) ≡ (N4,N3,O2)antiparallel strands(H-bond: two electronegative atoms, such as nitrogen and oxygen, interacting with the same hydrogen)Other features of Watson-Crick model• Right handedness (counterclockwise rotation)• Antiparallel strands• Major/minor grooves– Created by offset base pairing of 2 strands– Major groove allows direct access to bases– Minor groove faces ribose backbone• Base-pairing explains Chargaff’s rule → A/T or G/C ~1 in organisms with dsDNA genomes.van der Waals radius of atoms3′5′Fig. 4.11Other views of the Watson-Crick model for the structure of DNABecause B-DNA is really 10.5 bp/turn.Lehninger Principles of Biochemistry, 4th ed., Ch 8Ribose and phosphate oxygens are in blue. Phosphorus atoms are in yellow. Atoms comprising bases are in gray.Were Watson and Crick right?• Limitations of fiber diffraction studies– Fiber heterogeneity– Modeling intensive (idealized version)• Enhanced precision of crystallography– Atom positions specified – Structure of B-DNA more distorted than Watson-Crick model– Bending occurs wherever ≥ 4 adenosine residues appear in a row in one strandR.E. Dickerson et al. 1983DNA BendingFig. 4-16Secondary structural variants (deduced from fiber diffraction and crystal structures)• B-form – DNA fibers prepared under high humidity– Form found in cells• A-form (compact)– DNA fibers prepared under low humidity– RNA-RNA and RNA-DNA hybrids• Z-form (zigzag)– elongated left-handed DNA– alternating C (or 5-meC) & G residues in alternating anti and syn glycosyl bond


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