Nucleic acids 2015.pptx

Nucleotides and Nucleic Acids

NucleobasesDerivatives of pyrimidine or purinePyrimidine is single-ringed Purine is double-ringedNitrogen-containing heteroaromatic moleculesPlanar ring structuresAbsorb UV light around 250-270 nm7

fig 8-1bBCH261 W 2015CHAPTER 8 Nucleotides and Nucleic AcidsBiological function of nucleotides and nucleic acidsStructures of common nucleotidesStructure of double stranded DNAStructures of ribonucleic acidsDenaturation and annealing of DNAChemistry of nucleic acids; mutagenesisKey topics: 2BCH261 W 2015MonomerOligomerPolymerAmino AcidsPeptidesPolypeptides(proteins)MonosaccharidesOligosaccharidesPolysaccharides(glycans)NucleotidesOligonucleotidesPolynucleotides(nucleic acids)Macromolecules in cells3So far..BCH261 W 2015Functions ofnucleotides and nucleic AcidsNucleotide FunctionsEnergy for metabolism (ATP)Enzyme cofactors (NAD+)Signal transduction (cAMP)Nucleic Acid FunctionsStorage of genetic info (DNA)Transmission of genetic info (mRNA)Protein synthesis (tRNA and rRNA)Processing of genetic information (ribozymes)4BCH261 W 20155Two major functions ofnucleotides and nucleic AcidsDeoxyribonucleic acid (DNA)Ribonucleic acid (RNA)Adenosine triphosphate (ATP)nucleotidenucleic acids

BCH261 W 2015The anatomy of a nucleotide

Phosphate6BCH261 W 2015

(DNA and RNA)Pyrimidine bases All are good H-bond donors and acceptorsNeutral at neutral pH8

(DNA only)(RNA only)BCH261 W 20158

9Purine basesBoth found in DNA and RNAAlso good H-bond donors and acceptors Neutral at neutral pH

BCH261 W 20159UV absorption of nucleobasesAbsorption of UV light at 250-270 nmThis is due to ____________________Excited states of common nucleobases decay rapidly via radiation-less transitionsEffective photoprotection of genetic materialNo fluorescence from nucleic acidsUsed to measure nucleic acid concentration

10Fig. 8-10, Lab 8BCH261 W 201511absorbance240250260270280290Wavelength (nm)NucleotidesAromatic amino acidsAbsorbance of nucleotides is slightly different that that of aromatic amino acidsBCH261 W 2015Pentose in nucleotides-D-ribofuranose in RNA-2-deoxy-D-ribofuranose in DNA12

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13Conformation of RiboseBCH261 W 201513

14Ribose rings are not planar: different puckered conformations of the sugar ring are possibleC-2 or C-3 are the carbons that undergo bendingBCH261 W 20151415Joining the base to the pentose

The N-Glycosidic BondBCH261 W 20151516

19The pentose ring is attached to the nucleobase via a N-glycosidic bond The anomeric carbon of the sugar is in b configuration

It forms a bond with position N1 in pyrimides and position N9 in purines

Free rotation in free nucleotidesStable, but hydrolyzable by acid b-N-Glycosidic bondBCH261 W 20151617Joining the phosphate to the pentose

The phosphoester bondBCH261 W 2015Phosphate group18

BCH261 W 2015No phosphate :

One phosphate:

Two phosphates:

Three phosphates:19

Nucleoside triphosphatesBCH261 W 2015

20NMP = NUCLEOSIDE MONOPHOSPHATENDP = NUCLEOSIDE DIPHOSPHATENTP = NUCLEOSIDE TRIPHOSPHATENucleotides and phosphate numberBCH261 W 201520Nomenclature

BCH261 W 20152121Nomenclature: Ribonucleotides

Recognize general structure, and the specific names and symbols (both one-letter (A) and three-letter (AMP) codes)22LEGEND 8.4BNomenclature: DeoxyribonucleotidesRecognize general structure, and the specific names and symbols (both one-letter (dA) and three-letter (dAMP) codes)

23LEGEND 8.4A24Joining nucleotides together: the phosphodiester bond

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25Nucleic acids are made by covalently bridging nucleosides with a phosphodiester bondPhosphate links the 3 end of one nucleoside to the 5 end of anotherPhosphodiester bonds are not fully extended with respect to bases and sugarsNucleic AcidsBCH261 W 20152526Formation of Phosphodiester bond is directional! A nucleotide is added to the 3 end of an existing chain

Formation of phosphodiester bonds results in release of pyrophosphateNucleic Acid SynthesisThe monomers used for nucleic acids are (d)NTPs!

No branchingPolynucleotide chain is directional: exposed 5 and 3 endsAlways read and write sequence from 5 to 3 end BCH261 W 201527

Two types of nucleic acidsFig. 8-7BCH261 W 2015Some properties of RNA and DNACovalent bonds formed via phosphodiester linkagesnegatively charged backboneLinear polymersNo branching or cross-linksDirectionality5 end is different from 3 endWe read the sequence from 5 to 3DNA backbone is fairly stableDNA from mammoths?Hydrolysis accelerated by enzymes (DNAse)RNA backbone is unstableIn water, RNA lasts for a few yearsIn cells, mRNA is degraded in few hoursDegraded by Alkali conditionsDNA is a helix and RNA is typically not28BCH261 W 201529

Watson-Crick model of B-DNA BCH261 W 20152930BCH261 W 2015

Overall DNA structure with GC and AT base pairs31What drives DNA HELIX formation?Phosphate and pentose are hydrophillic- exposed to water- forms a hydration shell

2. Hydrogen bonding between bases - between bases on opposite strands

3. Base stacking - bases are hydrophobic rings - bases are planar - adjacent bases in the same strand are able to stack very closely

BCH261 W 2015Hydrogen bonding interactions (1)Two bases can hydrogen bond to form a base pairIn double-stranded DNA, Watson-Crick base pairs predominate A pairs with TC pairs with GPurine pairs with pyrimidine32BCH261 W 2015BCH261 W 201533Stick model of a GC base pair showing H-bonds

BCH261 W 201534Stick model of a AT base pair showing H-bonds

Base stacking interactions35BCH261 W 2015

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Pi stacking (also called stacking) refers to attractive, noncovalent interactions between aromatic rings.

These interactions are important in nucleobase stacking within DNA and RNA molecules, protein folding, and molecular recognitionChemistry reminder: stacking interaction between aromatic rings37DNA can be visualized with fluorescent dyes: this takes advantage of the ring stacking interactions in DNA.Base 1Base 1Base 2Base 2Ethidium bromideExcited by UV, and fluorescent only when bound to DNA

BCH261 W 2015Complementarity of DNA strandsTwo chains run ________

Two chains are __________ together

Two chains are _______ in sequence (sequence is read from 5 to 3)

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BCH261 W 2015Discovery of DNA structureYou have or will learn about the discovery of the DNA helix in other courses: Genetics, Molecular BiologyElucidated by Watson and Crick

They used X-ray data obtained by Franklin and WilkinsOne of the most important discoveries in biology. Why?39BCH261 W 2015

40Replication of Genetic Code Strand separation occurs first Each strand serves as a template for the synthesis of a new strandSynthesis is catalyzed by enzymes known as DNA polymerases Newly made DNA molecule has one daughter strand and one parent strand.

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DNA can assume different conformationsThe B-form of DNA is the most common form,However Z-DNA is also very important.

Z-DNA is found in regions of DNA that are actively being transcribed, and we have proteins that specifically recognize Z-DNA in order to facilitate transcription.BCH261 W 20154141

42Rotation about the Phosphate and Phosphodiester bonds in the backboneRotation of the C-1-N-glycosyl bondConformation of deoxyribose (pucker of the 2 or 3 position)

What causes DNA to assume different conformations?

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Physical characteristics of different DNA formsBCH261 W 2015434344Describing DNA sequencesAlways write sequence in 5 - > 3 direction:

Left strand:

Right strand:

BCH261 W 2015Unusual DNA sequences45Stretches of six adenosines can bend DNA 18Palindromes: words or phrases that are the same when read backward or forward:

ROTATOR NURSES RUNPalindrome sequencesBCH261 W 2015

46Self-complementary within same strandBCH261 W 201546

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May regulate gene regulation or demarcate specific parts of chromosomes.Palindrome DNA can form hairpins and cruciform conformationsBCH261 W 201547DNA DenaturationCovalent bonds remain intactGenetic code remains intactHydrogen bonds are brokenTwo strands separateBase stacking is lostDenaturation can be induced by high temperature, or change in pH Tm = melting temperature at which half of DNA molecules are denaturedDenaturation may be reversible: __________48BCH261 W 2015

49Body temperatureIncrease temperatureIncrease temperaturefurtherDecrease temperaturefurtherDecrease temperatureBody temperatureBCH261 W 201549

50DNAs with different sequence composition differ in melting temperatureDNA 1DNA 2BCH261 W 201550Factors Affecting DNA DenaturationThe midpoint of melting (Tm) depends on base compositionhigh CG increases Tm

Tm depends on DNA lengthLonger DNA has higher TmImportant for short DNA

Tm depends on pH and ionic strengthHigh salt increases Tm

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52Higher GC content increases Tm of DNA helixBCH261 W 201552Denaturation of VERY Large DNA Molecules is not Uniform AT rich regions melt at a lower temperature than GC-rich regions53

HelixMeltedHelixBCH261 W 2015Molecular Mechanisms of DNA Mutagenesis54

BCH261 W 2015Molecular mechanisms of mutagenesisSpontaneous mutagenesisDeamination (enhanced by certain chemicals)Depurination (enhanced by certain chemicals)

Chemical alkylation Methylation of guanine

Oxidative damage Hydroxylation of guanine Mitochondrial DNA is most susceptible

Radiation damage

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56Deamination Very slow reactions Large number of residues The net effect is significant: 100 C U events /day in a mammalian cellBCH261 W 2015Deamination56

57Depurination N-glycosidic bond is hydrolyzed Significant for purines: 10,000 purines lost/day in a mammalian cellBCH261 W 2015Depurination57Ionizing radiation (X-rays and -rays) causes ring opening and strand breaking.

Cells can repair some of these modifications, but others cause mutations. Accumulation of mutations is linked to aging and carcinogenesis

Radiation-Induced Mutagenesis58UV light induces dimerization of pyrimidines, this may be the main mechanism for skin cancers

BCH261 W 201559RNAIs synthesized using DNA templateContains ribose instead of deoxyriboseContains uracil instead of thymineCan contain modified nucleosidesCan form DNA/RNA hybridsCan form RNA double helices (viruses)Can form complex tertiary structuresSeveral types of RNACan have unusual base pairs (eg. G-U)

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Conformations that RNA can adopt60Messenger RNA: code carrier for the sequence of proteinsIs synthesized using DNA template:TRANSCRIPTIONmRNAs encode for proteinsTRANSLATIONStabilized by base stacking instead of H-bonding but can form base pairsStill helical just not double-helical!

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BCH261 W 2015Transfer RNA: matching amino acids with the mRNA Code tRNA molecules have quite complex structures62

Where the amino acid goesWhere the anti-codon is interacts with mRNA codonBCH261 W 201563The tRNA and mRNA interaction is driven by base pairingCodon - mRNAtRNA anticodon5 A A G 3

3 U U U 5BCH261 W 2015Other structural and enzymatic RNAsRibosomal RNAs (rRNAs) molecules have quite complex structures. Part of the protein synthesis machinery64Small nuclear RNAs (snRNAs) molecules are involved in catalysis, intron splicingMicro RNAs (miRNAs) molecules are involved in gene regulationEnzymatic RNAs are called ribozymesBCH261 W 201565

http://upload.wikimedia.org/wikipedia/commons/e/e4/50S-subunit_of_the_ribosome_3CC2.pngRibosomal RNAs (rRNAs) molecules have quite complex structures. Part of the protein synthesis machineryBCH261 W 201566Nucleotide functionsEnergy carriers

Co-enzymes

Signaling molecules

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8-37Nucleotides are used as energy carriers in cells: ATP is predominant, but GTP, UTP and CTP are also used in some reactionsPhosphoanhydride bonds are high energy: 30 kJ/mol/anhydrideCoupled to synthetic reactions to drive endergonic reactionsATP is the main energy carrier in cells used to drive endergonic reactionsBCH261 W 201568

Nucleotides can function as co-enzymesNAD+ used by many enzymes in redox reactionsBCH261 W 2015e.g. Alcohol dehydrogenase(Vitamin B3/ Niacin)

Nucleotides can function as co-enzymesIt is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle(Vitamin B5)69

FAD is a redox cofactor, more specifically a prosthetic group, involved in several important reactions in metabolism.

Very important in the process known as oxidative phosphorylation (a topic for BCH361).Nucleotides can function as co-enzymes(Vitamin B2)7071

Fig. 8-39cAMP is derived from ATPIt is a second messenger signaling moleculeNucleotides can function as signaling moleculeshormoneATPcAMPAdenylate cyclaseHormone actionsBCH261 W 2015Chapter 8: SummaryFunction of nucleotides and nucleic acidsNames and structures of common nucleotidesStructural basis of DNA function Reversible denaturation of nucleic acidsRNA structures and functionsAspects of mutagenesis

72Questions, 6e1, 2, 3, 5, 8 10, 12 Questions 5e1, 2, 3, 5, 7, 8,10

BCH261 W 2015Questions in blue would be good exam questions.