Nucleotides and nucleic acids IBiochemistry 302Central Dogma of Molecular Biology(Cell as a factory analogy)Complexity of Cell “Factories”Basic chemical structure of DNA and RNA (heteropolymers of nucleotides)Major purine and pyrimidine bases found in DNA and/or RNARibonucleotide nomenclatureChemical properties of nucleotidesChemical stability of polynucleotidesDNA: an historical perspectiveElucidation of DNA structureFranklin and Wilkins 1953; King’s CollegeWatson and Crick 1953; Cambridge Univ.Important properties of nucleotide bases ? 3D structure of nucleic acidWatson and Crick 1953Intuition: H-bonding between certain bases on opposite strands stabilizes the helixH-bonding pattern in W-C base pairs and numbering conventionOther features of Watson-Crick modelOther views of the Watson-Crick model for the structure of DNAWere Watson and Crick right?Secondary structural variants (deduced from fiber diffraction and crystal structures)Properties of the three forms of DNAStructural variation in DNA ? nucleotide conformationStructural variation in DNA and RNA? ?-furanose or sugar puckerWhat drives B-DNA into an A-DNA conformation?Nucleotides and nucleic acids IBiochemistry 302Bob KelmJanuary 19, 2005Central 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. 4.23Complexity of Cell “Factories”Fig. 1.11 Fig. 1.10Basic 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 chainsFig. 4.1Major purine and pyrimidine bases found in DNA and/or RNA• DNA: A, G, C, T• RNA: A, G, C, U• N-β-glycosyl bond: 1′ carbon of ribose and N9 (A, G) or N1(C, T, U)• Base + ribose = nucleosideFig. 4.2Ribonucleotide nomenclatureAdenylateGuanylateCytidylateUridylate**Fig. 4.3Chemical properties of nucleotides• Phosphate group– Strong acid– pKa of ~1 for primaryionization, ~6 for secondary• Bases (but weak)– pKa ~2.4-9.5– Tautomeric – Isomers differing in position of H atoms & double bond. Less stable imino & enol forms found in special base interactions• Absorb light– Near UV region of spectrum– Conjugated double bond systems (Amax~260 nm)Fig. 4.4Chemical stability of polynucleotides• 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 and exo, specific and non-specific) promote rapid hydrolysis of PDE bonds in DNA and RNA in vivo.• Dehydration-resistant (e.g DNA in fossils).Lehninger Principles of Biochemistry, 4th ed., Ch 8DNA: an 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 NTs vs~20 AAs → DNA merely a structural material present in the cell nucleus.• 1944 to 1952 – DNA transfer & labeling studiespoint to DNA as the repository of genetic info.• 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 helixImportant properties 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) • Highly conjugated → resonance among atoms– Pyrimidines (planar)– Purines (slight pucker)• Functional groups (H-bonding)– ring nitrogens– carbonyl groups– exocyclic amino groups• Hydrophobic character– base stacking interactions – van der Waals interactionsWatson and Crick 1953Intuition: H-bonding between certainbaseson opposite strands stabilizes the helixStabilizing Features:• H-bonding between A=T, G≡C base pairs → distance between C-1′ Cs the same → constant helical diameter• pentose ring in C-2′ endo conformation • van der Waals interaction between stacked bases• deoxyribose-phosphate backbone exposed to waterantiparallel 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 conventionG ≡ C(O6,N1,N2) ≡ (N4,N3,O2)A = T (N6,N1) = (O4,N3)Lehninger Principles of Biochemistry, 4th ed., Ch 8Other features of Watson-Crick model• Right handedness• Antiparallel strands• Major/minor grooves– 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 8Were 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