Nucleotides and Nucleic AcidsJM

Nucleotides and Nucleic AcidsJMNucleic AcidsObjectives

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Nucleotide

BaseSugar (Base + Sugar = Nucleoside)Phosphate (Nucleoside + phosphate = Nucleotide)ElementsNucleic Acids contain all the CHNOPS elements except sulfur (S).6CCarbon12.01077NNitrogen14.00678OOxygen15.99941HHydrogen1.0079415PPhosphorus30.973765Nucleotide Structure - 1SugarsOHOCH251432OHOCH2OHOHOHOHOCH2OHHOHRiboseDeoxyriboseGeneric Ribose StructureN.B. Carbons are given numberings as a prime

Important PyrimidinesPyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA.

HNNHOOUracil

HNNHOOCH3ThymineHNNHNH2OCytosinePyrimidinesNN561234ThymineCytosineNHNOTOHH3CCNNNH2OHPyrimidinesNN561234UracilNHNOUOHThymine is found ONLY in DNA.In RNA, thymine is replaced by uracilUracil and Thymine are structurally similarImportant PurinesAdenine and guanine are the principal purines of both DNA and RNA.Adenine

NNNH2NNHGuanine

OHNNHNNH2NPurine = adenine and guanine Pure silver Pure = AG10PurinesNNNN123456789AdenineGuanineAGNNNNHNH2NNNNHOHNH2Chargaff's Rules1950's: Erwin Chargaff studies heterocyclic base ratios in DNA from various organismsChargaff's Rules: In DNA of all organisms...(G+A)/(C+T) = purines/pyrimidines ratio ~1:1A/T ratio ~1:1G/C ratio ~1:1 A+G=T+C.A/T and G/C ratios random in RNASpeciesGACT(G+A)/(C+T)A/TG/CS. aureus21.030.819.029.21.111.051.11E. coli24.926.025.223.91.081.090.99Wheat germ22.727.322.827.11.001.011.00Bovine thymus21.528.222.527.80.961.010.96Human thymus19.930.919.829.41.011.051.01Human liver19.530.319.930.30.981.000.98The Problem Solved1953:}Not compatible with single helixJames Watson and Francis Crick combine...Franklin's x-ray dataChargaff's rulesExamination of molecular models}DNA is a base-paired double helixRosalind Franklin: x-ray studies of DNA show helical structureDiameter = 20 Length = 34 per 360o turnCalculated density

Franklin's Photo 51. The X pattern is characteristic of a helical structureWatson and Crick made extensive use of models to study molecular structure. Follow their example!

DNA replicationIt has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.James WatsonFrancis Crick195314The greatest understatement in biology!Base PairingWatson and Crick proposed that A and T were equal because of complementary hydrogen bonding.2-deoxyribose

2-deoxyriboseATBase PairingLikewise, the amounts of G and C were equal because of complementary hydrogen bonding.

2-deoxyribose2-deoxyriboseGCThe DNA DuplexWatson and Crick proposed a double-stranded structure for DNA in which a purine or pyrimidine base in one chain is hydrogen bonded to its complement in the other.

Watson-Crick Base PairsHeterocyclic bases associate via two or three hydrogen bondsBase pairs similar size and shape efficient packing into double helixGuanine-Cytosine

Adenine-Thymine

Two antiparallel strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases.

Based-Paired Double Helix

DNA strands are antiparallelSpace-filling model: atoms represented at their van der Waals radii (electron cloud volumes)

Aromatic stacking5' end 3' end3' end 5' endStrongHydrogen bondseasily disassembledHelical structure of DNA. The purine and pyrimidine bases are on the inside, sugars and phosphates on the outside.

The DNA Space ProblemHuman genome = 3 x 109 base pairs (bp)(3 x 109 bp) x (34 per 10 bp) x (10-10 m per ) = ~1 meter in lengthSolution: DNA tertiary structure = supercoiling

NucleosidesThe classical structural definition is that a nucleoside is a pyrimidine or purine N-glycoside of D-ribofuranose or 2-deoxy-D-ribofuranose.

Informal use has extended this definition to apply to purine or pyrimidine N-glycosides of almost any carbohydrate.

The purine or pyrimidine part of a nucleoside is referred to as a purine or pyrimidine base.Uridine and AdenosineUridine and adenosine are pyrimidine and purine nucleosides respectively of D-ribofuranose.Uridine(a pyrimidine nucleoside)Adenosine(a purine nucleoside)

ONHOCH2HNOOHHOO

NNNNHOCH2OOHHONH2NucleotidesNucleotides are phosphoric acid esters of nucleosides.

Phosphate GroupsPhosphate groups are what makes a nucleoside a nucleotidePhosphate groups are essential for nucleotide polymerizationPOOOOXBasic structure:Naming ConventionsNucleosides:Purine nucleosides end in -sine Adenosine, GuanosinePyrimidine nucleosides end in -dineThymidine, Cytidine, Uridine

Nucleotides:Start with the nucleoside name from above and add mono-, di-, or triphosphateAdenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine DiphosphateAdenosine 5'-Monophosphate (AMP)Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid.

NNNNOOHHONH2OCH2PHOOHOAdenosine 5'-Monophosphate (AMP)Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid.

NNNNOOHHONH2OCH2PHOOHO1'2'3'4'5'Adenosine Diphosphate (ADP)

NNNNOOHHONH2OCH2POOHOPOHOHOAdenosine Triphosphate (ATP)

NNNNOOHHONH2OCH2POOHOPOHOOPOHOHOMajor Compounds of LifeMacromoleculeSimple Molecule Structure4. Nucleic Acids nucleotides (DNA + RNA)(sugar + phosphate + N-base)NNNNNH2O HO - P - OOOOH OHadenine(N-base)

ribose(sugar)phosphoric acidNucleotide nomenclature

Nucleotide

BaseSugar (Base + Sugar = Nucleoside)Phosphate (Nucleoside + phosphate = Nucleotide)Nucleotides in nucleic acids

Bases attach to the C-1' of ribose or deoxyriboseThe pyrimidines attach to the pentose via the N-1 position of the pyrimidine ringThe purines attach through the N-9 positionSome minor bases may have different attachments.Roles of nucleotidesBuilding blocks of nucleic acids (RNA, DNA)Analogous to amino acid role in proteins

Energy currency in cellular metabolism (ATP: adenosine triphosphate)

Allosteric effectors

Structural components of many enzyme cofactors (NAD: nicotinamide adenine dinucleotide)Roles of nucleic acidsDNA contains genes, the information needed to synthesize functional proteins and RNAs

DNA contains segments that play a role in regulation of gene expression (promoters)

Ribosomal RNAs (rRNAs) are components of ribosomes, playing a role in protein synthesis

Messenger RNAs (mRNAs) carry genetic information from a gene to the ribosome

Transfer RNAs (tRNAs) translate information in mRNA into an amino acid sequence

RNAs have other functions, and can in some cases perform catalysisATPPerhaps the best known nucleotide is adenosine triphosphate (ATP), a nucleotide containing adenine, ribose, and a triphosphate group.

ATP is often mistakenly referred to as an energy-storage molecule, but it is more accurately termed an energy carrier or energy transfer agent. ATP is a nucleotide - energy currency

DG = -50 kJ/moltriphosphateBase (adenine)Ribose sugar ATP diffuses throughout the cell to provide energy for other cellular work, such as biosynthetic reactions, ion transport, and cell movement.

The chemical potential energy of ATP is made available when it transfers one (or two) of its phosphate groups to another molecule.

This process can be represented by the reverse of the preceding reaction, namely, the hydrolysis of ATP to ADP.NAD is an important enzyme cofactor

NADH is a hydride transfer agent, or a reducing agent.Derived from Niacin nicotinamide Nucleotides play roles in regulation

Nucleic AcidsNucleic acids are polymeric nucleotides (polynucleotides).

5' Oxygen of one nucleotide is linked to the 3' oxygen of another.

A section of a polynucleotide chain.Nucleic Acid StructurePolymerization

NucleotideSugar PhosphatebackboneTwo separate strands Antiparellel (53 direction)Complementary (sequence)Base pairing: hydrogen bonding that holds two strands togetherEssential for replicating DNA and transcribing RNA

5335 Sugar-phosphate backbones (negatively charged): outside Planner bases (stack one above the other): insideNucleic acids

Nucleotide monomerscan be linked together via a phosphodiester linkageformed between the 3' -OH of a nucleotide and the phosphate of the next nucleotide. Two ends of the resulting poly- or oligonucleotide are defined: The 5' end lacks a nucleotide at the 5' position, and the 3' end lacks a nucleotide at the 3' end position.

Helical turn: 10 base pairs/turn 34 Ao/turn

A-formB-formZ-formC1 Nucleic Acid Structure-6A, B and Z helicesNucleic acidsB form - The most common conformation for DNA.

A form - common for RNA because of different sugar pucker. Deeper minor groove, shallow major groove.

A form is favored in conditions of low water.

Z form - narrow, deep minor groove. Major groove hardly existent. Can form for some DNA sequences; requires alternating syn and anti base configurations.

36 base pairsBackbone - blue;Bases- gray

B-DNA

A, B and Z DNAA form favored by RNAB form Standard DNA double helix under physiological conditionsZ form laboratory anomaly, Left HandedRequires Alt. GCHigh Salt/ Charge neutralizationA, B & Z DNA Kinemages

DNA vs. RNA4 bases: guanine (G), cytosine (C), adenine (A), thymine (T)G-CA-Tdeoxyribose sugardouble strandednucleus4 bases: guanine (G), cytosine (C), adenine (A), uracil (U)G-CA-Uribose sugarsingle strandedrRNA, mRNA, tRNAsynthesized in nucleus (nucleoi for rRNA); function in cytosol5RNArRNARNA + protein form ribosomesmRNAcarries DNA code to cytosol for protein synthesis on the ribosomesread in triplets called codons (5 to 3)tRNAcarries amino acids to mRNA and matches up by base-pairing of triplet anti-codon with mRNA codon triplet8Stability of Nucleic AcidsHydrogen bonding Does not normally contribute the stability of nucleic acids

Contributes to DNA double helix, RNA secondary structure2. Stacking interaction/hydrophobic interaction between aromatic base pairs/bases contribute to the stability of nucleic acids. It is energetically favorable for the hydrophobic bases to exclude waters and stack on top of each other This stacking is maximized in double-stranded DNA Effect of AcidStrong acid + high temperature: completely hydrolyzed to bases, riboses/deoxyrobose, and phosphatepH 3-4 : apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ], can be generated by formic acidEffect of Alkali & Application

keto formenolate formketo formenolate formBase pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturationHigh pH (> 7-8) has subtle (small) effects on DNA structureHigh pH changes the tautomeric state of the bases RNA hydrolyzes at higher pH because of 2-OH groups in RNARNA is unstable at higher pH

OHfree 5-OH 2, 3-cyclic phosphodiesteralkaliChemical DenaturationUrea (H2NCONH2) : denaturing PAGE Formamide (HCONH2) : Northern blotDisrupting the hydrogen bonding of the bulk water solutionHydrophobic effect (aromatic bases) is reduced Denaturation of strands in double helical structureBuoyant density (DNA)1.7 g cm-3, a similar density to 8M CsClPurifications of DNA: equilibrium density gradient centrifugation

RNA pellets at the bottomProtein floatsUV absorption: nucleic acids absorb UV light due to the aromatic basesThe wavelength of maximum absorption by both DNA and RNA is 260 nm (lmax = 260 nm)Applications: detection, quantitation, assessment of purity (A260/A280)2. Hypochromicity: caused by the fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide C3 Spectroscopic and Thermal Properties of Nucleic Acids3. Quantitation of nucleic acidsExtinction coefficients: 1 mg/mL dsDNA has an A260 of 20ssDNA and RNA, 25The values for ssDNA and RNA are approximateThe values are the sum of absorbance contributed by the different bases (e : purines > pyrimidines)The absorbance values also depend on the amount of secondary structures due to hypochromicity. Purity of DNA A260/A280: dsDNA--1.8pure RNA--2.0protein--0.5

5. Thermal denaturation/melting: heating leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA.

RNA: the absorbance increases gradually and irregularlyDNA: the absorbance increases cooperatively.melting temperature (Tm): the temperature at which 40% increase in absorbance is achieved. 6. Renaturation:Rapid cooling: only allow the formation of local base paring. Absorbance is slightly decreasedSlow cooling: whole complementation of dsDNA. Absorbance decreases greatly and cooperatively. Annealing: base paring of short regions of complementarity within or between DNA strands. (example: annealing step in PCR reaction)Hybridization: renaturation of complementary sequences between different nucleic acid molecules. (examples: Northern or Southern hybridization)DNA SupercoilingClosed circular molecule

Supercoiling & energy

Topoisomer & topoisomerase

Almost all DNA molecules in cells can be considered as circular, and are on average negatively supercoiled.Counter helical turn2.Negative supercoiled DNA has a higher torsional energy than relaxed DNA, which facilitates the unwinding of the helix, such as during transcription initiation or replication Topoisomer: A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands.

Topoisomerases exist in cell to regulate the level of supercoiling of DNA molecules. Type I topoisomerase: breaks one strand and change the linking number in steps of 1.TypeII topoisomerase: breaks both strands and change the linking number in steps of 2.Gyrase: introduce the negative supercoiling (resolving the positive one and using the energy from ATP hydrolysis.

Ethidium bromide (intercalator): locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe.

Topoisomerases Type I: break one strand of the DNA , and change the linking number in steps of 1.

Type II: break both strands of the DNA , and change the linking number in steps of 2.