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UIUC MCB 450 - MCB450-21 _Spring2015

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Lecture'21'(Chapter'33)'Nucleo4des'and'Nucleic'Acids!“We'have'discovered'the'secret'of'life.”'Francis'Crick,'to'patrons'of'The'Eagle,'a'pub'in'Cambridge,'England'(1953)'Francis'Crick'(right)'and'James'Watson'(leK)'point'out'features'of'their'model'for'the'structure'of'DNA.'Outline'• 'Central'Dogma'review'• 'Differences'between'DNA'and'RNA'• 'Nitrogenous'bases'• 'Nucleo4des'• 'Nucleic'acids'(DNA)''Q Composi4on'of'DNA'(Chargaff’s'Rule)'Q The'DNA'double'helix'(WatsonQCrick)'Q Secondary'structure'of'DNA'(A,'B,'Z'DNA)'Q Denatura4on'and'Renatura4on'of'DNA'Q'Higher'order'packing'of'DNA'in'cells'(chromosomes'and'histones)''• 'Nucleic'acids'(RNA)'secondary'and'ter4ary'structures'of'RNA'Central'Dogma'DNA'and'RNA'differ'in'the'sugar'component'and'one'of'the'bases''Base'Base'Alpha'or'Beta'?'Nucleoside=Base'+'sugar'DNA'and'RNA'differ'in'the'sugar'component'and'one'of'the'bases''Base'Base'P'P'Nucleo4de=Base+sugar+phosphate'DNA'Backbones'of'DNA'and'RNA'Phosphodiester'bridges'RNA'3’!$>'<$!5’!'Nitrogenous'Bases:''PyrimidinesQStructure'Found'in'both'RNA'and'DNA'*'Found'in'RNA'only''The'pyrimidine'ring'Found'in'DNA'only''Nitrogenous'Bases:''PurinesQStructure'Both'are'found'in'both'RNA'and'DNA'The'purine'ring'system'NQglycosidic'bond'links'N1'of'pyrimidines'to'C1'of'deoxyribose''" ' '''''''''''''''''N9'of'purines ' '"'The'Deoxyribonucleo4des'Bases:'Adenine' Guanine' Thymine' Cytosine'NQglycosidic'bond'links'N1'of'pyrimidines'to'C1'of'ribose''" ' '''''''''N9'of'purines '"'The'Ribonucleo4des'Bases:'Adenine' Guanine' Uracyl' Cytosine'Nucleotides “High-energy” phosphoanhydride bonds “Low-energy” phosphomonoester bondCyclic'Nucleo4des'Q'The'phosphoric'acid'moiety'is'esterified'to'two'of'the'available'OH'groups'on'the'ribose'ring'(cAMP)''The'polymeric'structure'of'nucleic'acids'5’'3’'RNA/DNA'axis'5’'QQOH'QQ3’QOH'Sense'Strand'An4sense'Strand'Sense'Strand'An4sense'Strand'Chemical'Proper4es'of'purines'and'pyramidines'1. UV'light'absorbance:'important'for''measuring'DNA'concentra4on'2. Nucleo4des'are'polypro4c'acids'Purines'and'Pyrimidines Q'Abso rp4on' of'UV'l ight'Pyrimidines'and'Purines'typically'strongly'abs orb'at'UV'at'260'nm'At'pH'7:'A'monomeric'nucleo4de'(nucleoside'monophosphate)'has'net'charge'of'Q2'Nucleic'acids'(polymers'of'nucleo4des)'derive'their'name'due'to'the'acidic'nature'of'the'phosphate'groups'of'their'component'nucleo4des'Nucleo4des'(ribo'and'2ʹ′Qdeoxyribo )'are'polypro4c'acids''RNA' 5′-P end  3′-OH end Reading'direc4on'5’QACGUQ'3’'5’QpQACGUQ'3’'5’QpApCpGpUQ'3’'Nucleic'acids''have'direc4onality'QO'H'Conventions in writing DNA/RNA sequences Complement to top strand (the bottom strand) can be written as: 5’ - ATGTCG - 3ʼ' 3’ - TACAGC – 5’'Usually if you write a duplex sequence the top strand is 5ʼ-3ʼ left to right. 5’- CGACAT-3’ or 3ʼTACAGC-5’ (forward)'(backward)'Chargaffʼs Rules (1951-ʼ52) The % GC or AT varies between organisms but always A=T and G=C. (ds)' [Purines] = [Pyrimidines]'The'Double'Helix'(WatsonQCrick)'Based'on'Chargaff’s'rule'of'DNA'base'composi4on,'XQray'diffrac4on'data'obtained'by'Rosalind'Franklin'and'Maurice'Wilkins'James'Watson'and'Francis'Crick'concluded'that:'DNA'is'a'complementary'Double'Helix'Double'Helix'Note'that'the'strands'are'an4parallel'phosphate'deoxyribose'Watson'and'Crick'baseQpairs'HQbonds'Double'Helix'–'BQform'' ~'10.4'nucleo4des'per'turn'Axial'view,'looking'down'the'helix'axis'Double'Helix'The'Major'and'Minor'Grooves'are'lined'sequenceQspecific'hydrogenQbonding'groups'S' S'S'S'Watson'and'Crick'baseQpairs'S'='deoxyribose'Major'and'Minor'groove'of'the'DNA'double'helix'Base'Stacking'Contribute'to'the'Stability'of'the'Double'Helix'Right'handed'–'A'and'B'DNA'Pitch'='2.46'nm'Pitch'''='3.4'nm'A'form'B'form'Z'DNA'BQDNA'Q''LeKQhanded'helix'Q''Thinner'and'slimmer'than'BQDNA'DNA'replica4on'is'SemiQConserva4ve''An4sense'Sense'A'S'S'A'What'would'conserva4ve'replica4on'look'like'+!nucleo/des!A'diagram'of'semiconserva4ve'replica4on.'+!nucleo/des!+!nucleo/des!(without'blue'nucleo4des)'(without'blue'nucleo4des)'(Only'blue'nucleo4des)''The'resolu4on'of'14N'dsDNA'and'15N'dsDNA'by'densityQgradient'centrifuga4on.'UVQabsorp4on'Densitometric'tracing'Heavy'DNA'Light'DNA'ds'='double'strand'ss'='single'strand'MeselsonQStahl'experiment'(1958)'DNA'replica4on'is'semiconserva/ve'N15'N14'N14'N14'hybrids'MeselsonQStahl'experiment'(1958)'N14'N14'Parental'dsDNA'Denatura4on'of'DNA'secondary'structure' Denaturing'agents:'1. Heat'(DNA'denatura4on'='DNA'mel4ng)'2. Extremes'of'pH'3. Strong'HQbonding'solutes'DNA Melting Curves Absorbance 1.0 1.2 1.4 ssDNA'dsDNA'Denatured'DNA'can'renature'to'reQform'the'double'helix'Upon'cooling,'returning'pH'to'neutral'or'when'denaturants'are'diluted'out,'denatured'DNA'will'renature'(reanneal)'to'reQform'the'duplex'Renatura4on'is'dependent'on'both'DNA'concentra4on'and'4me''An'electron'micrograph'of'part'of'the'E.)coli'genome'Circular'DNA'from'Mitochondria'Relaxed'dsDNA'Supercoiled'dsDNA'Most'DNA'in'cells'is'about'5%'underwound'rela4ve'to''B'form.'Supercoiling'in'DNA'le8!Underwinding'RightQhanded'DNA'(BQDNA)'produces'nega4ve'(Q)'supercoils.''These'supercoils'are'rightQhanded'(260'bp)'Supercoils'Q'DNA'can'adopt'regular'structures'of'higher'complexity''• ''Double'stranded'circular'DNA'form'supercoils'when'the'two'strands'are'underwound'(Qvely'supercoiled)'or'overwound'(+vely'supercoiled)'• ''Topoisomerases'are'enzymes'that'can'break'one'or'both'strands'of'the'DNA,'wind'them'4ghter'or'looser'and'rejoin'the'ends'• ''DNA'Gyrase'is'a'topoisimerase'that'introduces'–ve'supercoils'into'DNA'Supercoiling'does'not'mean'the'DNA'is'more'(or'less)'coiled'than'B'form'DNA.'It'means'that'there'is'a'coiling'super$imposed'on'the'coiling'of'B'form'DNA.'DNA'in'cells'occurs'in'highly'folded'compact'structures'''''Prokaryo4c''Single'dsDNA'molecule'length'='1.6'mm'''')E.)Coli'cell'='0.002'mm'length''''Eukaryo4c''23'pairs'of'ds'DNA'(46''DNA'molecules)'='3.6'm'total''''Human'cell'='20'μm'diameter'Nucleus'='5'μm'diameter'Nucleosomes'are'complexes'of'DNA'and'histones'Wrapping'DNA'around'histones'introduces'nega4ve'supercoils''Eukaryo4c'DNA'is'wrapped'around'histones'to'form'nucleosomes'Chroma4n'–'DNA'+'histone'nucleosomes'(146'base'pair'of'DNA'around'each'nucleosome'core)'2'nm'10'nm'Histones'are'rich'Lys'and'Arg'Net Result: Each DNA molecule has been packaged into a mitotic chromosome that is 50,000X shorter than the


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UIUC MCB 450 - MCB450-21 _Spring2015

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