Lecture 21 Chapter 33 Nucleotides 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 left point out features of their model for the structure of DNA Outline Central Dogma review Differences between DNA and RNA Nitrogenous bases Nucleotides Nucleic acids DNA Composition of DNA Chargaff s Rule The DNA double helix Watson Crick Secondary structure of DNA A B Z DNA Denaturation and Renaturation of DNA Higher order packing of DNA in cells chromosomes and histones Nucleic acids RNA secondary and tertiary structures of RNA Central Dogma DNA and RNA differ in the sugar component and one of the bases Alpha or Beta Base Nucleoside Base sugar Base DNA and RNA differ in the sugar component and one of the bases P Base P Nucleotide Base sugar phosphate Base Backbones of DNA and RNA DNA 3 Phosphodiester bridges 5 RNA Nitrogenous Bases Pyrimidines Structure The pyrimidine ring Found in both RNA and DNA Found in RNA only Found in DNA only Nitrogenous Bases Purines Structure The purine ring system Both are found in both RNA and DNA The Deoxyribonucleotides Bases Adenine Guanine Thymine N glycosidic bond links N1 of pyrimidines to C1 of deoxyribose N9 of purines Cytosine The Ribonucleotides Bases Adenine Guanine Uracyl N glycosidic bond links N1 of pyrimidines to C1 of ribose N9 of purines Cytosine Nucleotides High energy phosphoanhydride bonds Low energy phosphomonoester bond Cyclic Nucleotides The phosphoric acid moiety is esterified to two of the available OH groups on the ribose ring cAMP The polymeric structure of nucleic acids Sense Strand Antisense Strand Antisense Strand Sense Strand 5 5 3 3 OH OH RNA DNA axis Chemical Properties of purines and pyramidines 1 UV light absorbance important for measuring DNA concentration 2 Nucleotides are polyprotic acids Purines and Pyrimidines Absorption of UV light Pyrimidines and Purines typically strongly absorb at UV at 260 nm Nucleotides ribo and 2 deoxyribo are polyprotic acids At pH 7 A monomeric nucleotide nucleoside monophosphate has net charge of 2 Nucleic acids polymers of nucleotides derive their name due to the acidic nature of the phosphate groups of their component nucleotides 5 P end O 5 ACGU 3 5 p ACGU 3 5 pApCpGpU 3 Reading direction Nucleic acids have directionality RNA H 3 OH end Conventions in writing DNA RNA sequences Usually if you write a duplex sequence the top strand is 5 3 left to right 5 ATGTCG 3 3 TACAGC 5 Complement to top strand the bottom strand can be written as 5 CGACAT 3 or 3 TACAGC 5 forward backward Chargaff s Rules 1951 52 ds The GC or AT varies between organisms but always A T and G C Purines Pyrimidines The Double Helix Watson Crick Based on Chargaff s rule of DNA base composition X ray diffraction 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 antiparallel phosphate H bonds deoxyribose Watson and Crick basepairs Double Helix B form 10 4 nucleotides per turn Axial view looking down the helix axis Double Helix The Major and Minor Grooves are lined sequence specific hydrogen bonding groups Watson and Crick base pairs S S S deoxyribose S S 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 A form Pitch 3 4 nm B form Z DNA B DNA Left handed helix Thinner and slimmer than B DNA DNA replication is Semi Conservative Antisense Sense nucleotides A S A S What would conservative replication look like A diagram of semiconservative replication Only blue nucleotides nucleotides without blue nucleotides nucleotides without blue nucleotides The resolution of 14N dsDNA and 15N dsDNA by densitygradient centrifugation UV absorption Light DNA Heavy DNA ds double strand ss single strand Densitometric tracing Meselson Stahl experiment 1958 DNA replication is semiconservative hybrids N14 N14 N14 N15 Meselson Stahl experiment 1958 N14 N14 Parental dsDNA Denaturation of DNA secondary structure Denaturing agents 1 Heat DNA denaturation DNA melting 2 Extremes of pH 3 Strong H bonding solutes DNA Melting Curves 1 4 Absorbance ssDNA 1 2 1 0 dsDNA Denatured DNA can renature to re form the double helix Upon cooling returning pH to neutral or when denaturants are diluted out denatured DNA will renature reanneal to re form the duplex Renaturation is dependent on both DNA concentration and time An electron micrograph of part of the E coli genome Circular DNA from Mitochondria Relaxed dsDNA Most DNA in cells is about 5 underwound relative to B form Supercoiled dsDNA Supercoiling in DNA 260 bp Underwinding Right handed DNA B DNA produces negative lef supercoils These supercoils are right handed Supercoils DNA can adopt regular structures of higher complexity 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 Double stranded circular DNA form supercoils when the two strands are underwound vely supercoiled or overwound vely supercoiled Topoisomerases are enzymes that can break one or both strands of the DNA wind them tighter or looser and rejoin the ends DNA Gyrase is a topoisimerase that introduces ve supercoils into DNA DNA in cells occurs in highly folded compact structures Prokaryotic Eukaryotic Single dsDNA molecule length 1 6 mm 23 pairs of ds DNA 46 DNA molecules 3 6 m total E Coli cell 0 002 mm length Human cell 20 m diameter Nucleus 5 m diameter Nucleosomes are complexes of DNA and histones Wrapping DNA around histones introduces negative supercoils Eukaryotic DNA is wrapped around histones to form nucleosomes 2 nm nucleosomes 146 base pair of DNA around each nucleosome core 10 nm Chromatin DNA histone Histones are rich Lys and Arg Still more compaction is required to form condensed mitotic chromosomes Net Result Each DNA molecule has been packaged into a mitotic chromosome that is 50 000X shorter than the simple DNA helix Secondary Structures for Nucleic Acids Stem loop structures Palindromic Sequences Can Form Transient Cruciform Structures palindrome NOT the same as KAYAK Holiday junctions Play a role in genetic recombination Single strand RNA structure Watson and Crick basepairs Non Watson and Crick base pairs rRNA ribosomal RNA 16S rRNA 2 structure 5 3 rRNA molecules have characteristic 2 structure due to
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