Lecture 21 Ch 33 Nucleotides and Nucleic Acids 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 1 Central Dogma 2 DNA and RNA differ in the sugar component and one of the bases Base Nucleoside Base sugar Base 3 DNA and RNA differ in the sugar component and one of the bases P Base P Base Nucleotide Base sugar phosphate 4 Backbones of DNA and RNA 5 DNA RNA Phosphodiester bridges 3 5 Nitrogenous Bases Pyrimidines Structure The pyrimidine ring Found in both RNA and DNA Found in RNA only Found in DNA only 6 Nitrogenous Bases Purines Structure The purine ring system Both are found in both RNA and DNA 7 The Deoxyribonucleotides Bases Adenine Guanine Thymine N glycosidic bond links N1 of pyrimidines to C1 of deoxyribose N9 of purines Cytosine 8 The Ribonucleotides Bases Adenine Guanine Uracyl Cytosine N glycosidic bond links N1 of pyrimidines to C1 of ribose N9 of purines 9 Nucleotides High energy phosphoanhydride bonds Low energy phosphomonoester bond 10 11 Cyclic Nucleotides The phosphoric acid moiety is esterified to two of the available OH groups on the ribose ring Cyclic AMP cAMP 12 The polymeric structure of nucleic acids Sense Strand Antisense Strand Antisense Strand Sense Strand 5 3 OH OH RNA DNA axis 13 Chemical Properties of purines and pyramidines 1 UV light absorbance important for measuring DNA concentration 2 Nucleotides are polyprotic acids 14 Purines and Pyrimidines Absorption of UV light Pyrimidines and Purines typically strongly absorb at UV at 260 nm 15 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 16 of the phosphate groups of their component nucleotides Nucleic acids have directionality 5 ACGU 3 5 p ACGU 3 5 pApCpGpU 3 Reading direction 5 P end 3 OH end 17 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 18 Chargaff s Rules 1951 52 ds The GC or AT varies between organisms but always A T and G C Purines Pyrimidines 19 Double Helix Note that the strands are antiparallel 20 Double Helix B form 10 4 nucleotides per turn 21 Axial view looking down the helix axis 22 23 The Major and Minor Grooves are lined sequence specific hydrogen bonding groups Glycosidic bond deoxyribose Grooves arise because glycosidic bonds are not opposite each other Each groove is lined by potential hydrogen bond donor and acceptor atoms 24 Major and Minor groove of the DNA double helix 25 Base Stacking Contribute to the Stability of the Double Helix 26 Right handed A and B DNA A form Pitch 24 6 2 46 nm Base per turn 11 Rise per base 2 3 B form Pitch 34 3 4 nm Base per turn 10 4 Rise per base 3 4 27 Z DNA B DNA Left handed helix Narrower and longer than B DNA Rise per base 3 8 12 bases per turn 45 6 pitch per turn 28 DNA replication is Semi Conservative Antisense Sense nucleotides A S A S What would conservative replication look like 29 A diagram of semiconservative replication Only blue nucleotides nucleotides without blue nucleotides nucleotides without blue nucleotides 30 The resolution of 14N dsDNA and 15N dsDNA by density gradient centrifugation UV absorption Light DNA Heavy DNA ds double strand ss single strand Densitometric tracing 31 Meselson Stahl experiment 1958 N14 N15 32 Denaturation of DNA secondary structure Denaturing agents 1 Heat DNA denaturation DNA melting 2 Extremes of pH 3 Strong H bonding solutes 33 DNA Melting Curves ssDNA dsDNA 34 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 35 Circular DNA from Mitochondria Relaxed dsDNA Most DNA in cells is about 5 underwound relative to B form Supercoiled dsDNA 36 Supercoiling in DNA 260 bp Underwinding Right handed DNA B DNA produces negative lef supercoils These supercoils are right handed 37 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 ve supercoiled or overwound ve 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 38 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 39 Nucleosomes are complexes of DNA and histones Wrapping DNA around histones introduces negative supercoils 40 Eukaryotic DNA is wrapped around histones to form nucleosomes nucleosomes 146 base pair of DNA around each nucleosome core Chromatin DNA histone Histones are rich Lys and Arg 41 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 42 Secondary Structures for Nucleic Acids Stem loop structures 43 Palindromic Sequences Can Form Transient Cruciform Structures Palindrome NOT the same as KAYAK Holiday junctions Play a role in genetic recombination 44 Single strand RNA structure Watson and Crick base pairs Non Watson and Crick base pairs 45 rRNA ribosomal RNA 16S rRNA 2 structure 5 3 rRNA molecules have characteristic 2 structure due to extensive intramolecular base pairing interactions 46
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