页数：110页文件大小：12.20 MB来源： 生物化学ppt课件
本课件为英文教材第七章，系统讲解核酸的结构层次：先讲一级结构、简写表示与链终止法、化学裂解法等测序方法；再讲 DNA 二级结构的 A 型、B 型与 Z 型，DNA 的变性与复性；随后讲超螺旋与十字形结构等三级结构、染色体的组装方式，最后讲核酸的化学合成。
Chapter 7 Structure of Nucleic Acids"Scherzo in D & E" (detail) by David E. Rodale (1955-1985)
7.1 • The Primary Structure of Nucleic Acids
Structure and Nomenclature of Nucleic Acids5’3’PPTPAPGPCOHC5’3’Shorthand notationof a nucleic acidTwo major classes of nucleic acids:DNA and RNA
Sequencing Nucleic Acids1 chain terminationor dideoxy method2base-specific chemical cleavagemethod
DNAsequencingTheSangerDNAsequencingmethodusesdideoxynucleotidcstoterminateDNAsynthesis,yieldingaseriesofDNAfragmentswhosesizescanbemeasuredbyelectrophoresis.Thelastbaseineachofthesefragmentsisknown,becauseweknowwhichdideoxynucleotidewasusedtoterminateeachreaction.Therefore,orderingthesefragmentsbysize eachfragmentone(known)baselongerthanthenext tellsusthebasesequenceoftheDNA.
DNA sequencing canbe automated
Maxam-GilbertSequencingTheMaxam-Gilbertsequencingmethodfollowsaverydifferentstrategy.InsteadofsynthesizingDNAinvitroandstoppingthesynthesisreactionswithchainterminators,thismethodstartswithfulllength,end-labeledDNAandcleavesitwithbase-specificreagents
TOGWeusedimethylsulfate(DMS)tomethylateguanines.wedothemethylationundermildconditionsthatleadtoanaverageofonlyonemethylatedguanineperDNAstrand.Next,weuseareagent(piperidine，哌啶)thatdoestwothings:Itcauseslossofthemcthylatedbase,thenitbreakstheDNAbackboneatthesiteofthelostbase(theapurinicsite).
TOA+G•wecanweakentheglycosidicbondstobothadenineandguaninewithacid;thenpiperidinewillcausedepurinationandstrandbreakageafterbothA'sandG's.
Maxam-Gilbertsequencingisusedonlyinspecialcasesthesedays,butthemethodsofmodifyingandthenbreakingDNAusedbyMaxamandGilbertarestillusefulforotherpurposes
7.2 •The ABZs of DNA Secondary Structure Structure Overview of Nucleic AcidsUnlike three dimensional structures of proteins, DNA molecules assume simple double helical structures independent on their sequences. There are three kinds of double helices that have been observed in DNA: type A, type B, and type Z, which differ in their geometries. The double helical structure is essential to the coding functional of DNA. Watson (biologist) and Crick (physicist) first discovered double helix structure in 1953 by X-ray crystallography. RNA, on the other, can have as diverse structures as proteins, although they can also hand form double helix of type A. The ability of being both informational and diverse in structure suggests that RNA was the prebiotic molecule that could function in both replication and catalysis (The RNA WorldHypothesis). In fact, some virus encode their genetic materials by RNA (retrovirus)
DNA is a double helixStructure of DNA
Structural Equivalence of Watson–Crick Base Pairs;the A:T pair and G:C pair have virtually identical dimensions.
The DNA Double Helix Is a Stable StructureForces That Stabilize Nucleic Acid Double Helix–Hydrogen bonding in base-pairing–Hydrophobic interactions in base stacking –Electrostatical Interaction with cations in solution such as Mg2+.5’5’3’3’Same strand stackingcross-strand stacking
Types of DNA Double HelixA-DNAA-RNAMajor GrooveMinor Groove
Comparison of the Structural Properties of A-, B-, and Z-DNA•
Intercalating Agents Distort the Double HelixSeveral hydrophobic molecules containing flat aromatic and fused heterocyclic rings can insert between the stacked base pairs of DNA. These molecules are called intercalating agents.Intercalating agentsare potential Cancer-inducing reagents.
Denaturation and Renaturation of DNATmDNA of different sequenceshave different Tm.Tmis higher for DNA that contain more GC pairs; Tmis also directly proportional to the ionic strength of the solution. (salts can shield repulsions of negatively charged phosphate groups)
Structural Changes in DNA Melting•
pH Extremes or Strong H-Bonding Solutes Also Denature DNA DuplexesAt pH values greater than 10, extensive deprotonation of the bases occurs, destroying their hydrogen bonding potential and denaturing the DNA duplex. Similarly, extensive protonation of the bases below pH 2.3 disrupts base pairing. Alkali is the preferred denaturant because, unlike acid, it does not hydrolyze the glycosidic linkages in the sugar–phosphate backbone. Small solutes that readily form H bonds are also DNA denaturants at temperatures below Tmif present in sufficiently high concentrations to compete effectively with the H-bonding between the base pairs. Examples include formamide and urea.
Nucleic Acid Hybridization If DNA from two different species are mixed, denatured, and allowed to cool slowly so that reannealing can occur, artificial hybrid duplexesmay form, provided the DNA from one species is similar in nucleotide sequence to the DNA of the other.
Nucleic acid hybridization is a commonly employed procedure in molecular biology. First, it can reveal evolutionary relationships. Second, it gives researchers the power to identify specific genes selectively against a vast background of irrelevant genetic material. An appropriately labeled oligo-or polynucleotide, referred to as a probe,is constructed so that its sequence is complementary to a target gene. The probe specifically base pairs with the target gene, allowing identification and subsequent isolation of the gene. Also, the quantitative expression of genes (in terms of the amount of mRNA synthesized) can be assayed by hybridization experiments.
Buoyant Density of DNANot only the melting temperature of DNA but also its density in solution is dependent on relative G:C content. G:C-rich DNA has a significantly higher density than A:T-rich DNA. Single-stranded DNA is denser than double-helical DNA
Cesium chloride centrifugation is an excellent means of removing RNA and proteins in the purification of DNA. Because of its relatively high density, DNA can be purified from cellular material by a form of density gradientcentrifugationknown asisopycnic centrifugation.
7.4 • Supercoils and Cruciforms: Tertiary Structure in DNA Many DNA molecules are circular (e.g., bacterial chromosomes, all plasmid DNA). Circular DNA can form supercoils. Human chromosome contains 3x109basepairs and are wrapped around proteins to form nucleosomes. Nucleosomes are packed tightly to form helical filament, a structure called chromotin.
DNA SupercoilingSupercoiling meansthe coiling of coiling
Supercoilingof DNARelaxedstate
Supercoiling induced by separating thestrands of a helical structure(duringreplication or transcription)
DNA from a lysed E..coli cell
Relaxed and supercoiled plasmidDNAs. The degree of supercoilingincreases from left to right.
Varieties of Supercoiled DNANote that all black lines represents double stranded DNA
DNAunderwinding isdefined bytopologicallinking number(Lk,or L).
Wr=-2Wr=2Tw=200
DNA Gyrase The bacterial enzyme DNA gyraseis a topoisomerase that introduces negative supercoils into DNA
Mechanism of topoisomerase II(DNA gyrase)
Topoisomerases catalyzechanges inthe linkingnumber ofDNATopoisomerase I treats indifferentlength of time
Superhelix Density The superhelix densityor specific linking differenceis defined as L/L0and is sometimes termed sigma, s . For our example, s = -4/40, or -0.1. As a ratio, s is a measure of supercoiling that is independent of length. Its sign reflects whether the supercoiling tends to unwind (negatives ) or overwind (positives ) the helix.
Toroidal Supercoiled DNA Negatively supercoiled DNA can arrange into a toroidal state. The toroidal state of negatively supercoiled DNA is stabilized by wrapping around proteins which serve as spools for the DNA “ribbon.” This toroidal conformation of DNA is found in protein: DNA interactions that are the basis of phenomena as diverse as chromosome structure and gene expression.
The toroidal state of negatively supercoiled DNA is stabilized by wrapping around proteins which serve as spools for the DNA “ribbon.”
palindromeisaword,phrase,orsentencethatisspelledidenticallyreadingforwardorbackward;twoexamplesareROTATORandNURSESRUN..
Such sequence are self-complementary within eachstrandand therefore havethe potential to form hairpinor cruciform(cross-shaped) structures
Cruciforms
DNA compaction requires a special form of supercoilingThere are two types of supercoiling: plectonemic (from the Greek plektoe, “twisted”, and nema, “thread”) and solenoidal.
PlectonemicSupercoiling(a)An electronmicrograph ofplectonemicallysupercoiledplasmid DNA
(b) An interpretation of theobserved structure. Thepurple lines show the axisof the supercoil.
(c) An indealizedrepresentation of thisstructure
plectonemic and solenoidalsupercoiling.
7.5 • Chromosome StructureHumanchromosomes
Chromatin and nucleoidstructurenucleosomes
Histones are small, basicproteins
Each of histones has variant formsbecause certain amino acid sidechains are enzymatically modified bymethylation, ADP-ribosylation,phosphorylation, or acetylation. Suchmodifications affact the net electriccharge, shape, and other propertiesof histones, as well as the structuraland functional properties ofchromatin, and they play a role in theregulation of transcription.
Nucleosomeare thefundamentalorganizationalunits ofchromatin
Chromatin assembly
The 30 nm fiber, a higher-orderorganization of nucleosomes
A partiallyunraveledhumanchromosome,revealingnumerous loopsof DNA attachedto a scaffoldlikestructure.
Loops of chromosomal DNAattached to a nuclear scaffold
Organization of Chromotin and Chromosomes:
7.6 • Chemical Synthesis of Nucleic Acids
Commercially available automated instruments, called DNA synthesizersor “gene machines,” are capable of carrying out the synthesis of oligonucleotides of 150 bases or more.
Transfer RNA StructuresAnticodonStemD LoopTyC LoopV ariableloopAnticodon LoopSecondary StructureOf large ribosomal RNATertiary StructureOf large ribosome subunit
Ribosomal RNABan et al., Science289 (905-920), 2000Secondary StructureOf large ribosomal RNATertiary StructureOf large ribosome subunit
Catalytic RNASecondary StructureOf large ribosomal RNATertiary StructureOf large ribosome subunit
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