中南大学 生物化学 考研课件 ch12 核酸结构 structure of nucleic acids.ppt

Chapter 12,Structure of nucleic acids 核酸结构,Contents,DNA structure The three levels of DNA structure RNA structure (self-study自学) Difference in structure of mRNA between prokaryotic cells and eucaryotic cells Secondary and tertiary structure of RNA,The three levels of DNA structure （DNA 的三级结构）,The primary structure一级结构 DNA Sequencing 测序 The secondary structure 二级结构 Basic knowledge 基本知识 Comparison among A, B, Z-conformation forms of DNA (DNA的A, B, Z构象形式的比较） Denaturation and renaturation of DNA （DNA）变性与复性 Other forms of DNA conformation (DNA构象的其它形式) The tertiary structure 三级结构 DNA organization in chromosomes 染色体中的DNA构架,The primary structure of DNA （DNA一级结构）,1st structure of DNA is DNA sequence (base sequence) 是DNA序列或碱基序列,DNA sequencing （DNA测序）,Methods Chemical method, also called Maxam-Gilbert method (1977) 化学法 Chain termination method, also called Sanger dideoxy method 链终止法或Sanger双脱氧方法 now the method of choice 常用方法.,Principle of Chain termination method or Sanger dideoxy method 链终止法或Sanger双脱氧方法原理,When a ddNTPs is added to the extending DNA chain, then the chain extension will be terminated due to the nearly added ddNTPs lacks 3-OH. 如果有某一个双脱氧核苷酸连接在延伸的DNA链上，那么，由于新连接上的该双脱氧核苷酸缺乏DNA链延伸所需要的3-OH，DNA链延伸终止。 When the four kinds of ddNTPs (ddATP, ddGTP, ddTTP, ddCTP) bind to the extending DNA at any time during synthesis of DNA according to base pair principle, then a series of DNA pieces can be synthesized, analysis of the size of these DNA pieces can give the sequence of the DNA.当四种双脱氧核苷酸在基于碱基配对原则进行链延伸过程中，随机键合到新延伸的DNA链上，那么，便能形成一系列长短不一的DNA链，分析这些链的分子量大小，会给出整个DNA链碱基序列信息。,Review on the structures of ATP, dATP, ddATP,DNA sequencing （DNA测序） Chain termination method,DNA sequencing （DNA测序） Chain termination method,DNA synthesis,Methodology of Chain termination method or Sanger dideoxy method 链终止法或Sanger双脱氧方法的方法论,Manuel method Autoradiography 放射自显影法 Automatic method Fluorescent dyes 荧光染料（显影） Examples of gels for sequencing of DNA 测序用胶示例,Chain termination method I- autoradiography放射自显影法,Incubation mixture in four different tubes在四个试管中分别进行DNA 链延伸实验 Single-stranded DNA template Primer 引物 DNA polymerase I (DNA聚合酶I) dNTPs, one of which is radioactively labeled, e.g., (a- 32P)dATP ddATP, ddTTP, ddGTP, or ddCTP Electrophoresis 电泳 Membrane transfer and auto-radiophotography 转膜和放射自显影 Analysis of the sequence 序列分析,Sequencing nucleic acids,Ps see figure 12.3, p359,Dideoxy method of Sanger,Note that The DNA fragment to be determined should be in the range 200400bp If the DNA fragment is longer than 400bp, it needs to be cut (excised) into several smaller fragments.,Chain termination method II -autosequencing 自动测序原理,Incubation mixture in four different tubes在四个试管中分别进行DNA 链延伸实验 Single-stranded DNA template Primer 引物 DNA polymerase I (DNA聚合酶I) dNTPs fluorescent dye-labeled ddNTPs Mixing of the cultures of the four tubes混合四个试管中的合成产物 Electrophoresis and real-time tracking bases and give base sequence 电泳，实时示踪碱基的出现，给出碱基序列,Automated DNA sequencing (自动DNA测序),Schematic diagram of automated sequencing of DNA 自动测序的图解,Examples of gels 测序胶示例,automated sequencing gel 自动测序胶,manual sequencing gel 手动测序胶,Basic knowledge about DNA secondary structures,Types A, B Z-conformation forms B-form is standard Something related to DNA structure X-ray diffraction photographs of DNA (Franklin and Wilkins, 1952) DNA X射线衍射图(富兰克林和魏尔金斯) Founder Watson and Crick, 1953 Two antiparallel chains with pairing of A to T, and G to C 通过A与T、G与C配对形成两条反平行链,B-DNA -standard DNA,Major conformation of DNA in solution DNA在溶液中的主要构象类型 Right-handed helix 10bp/Pitch 螺距 Pitch length螺距长度 3.4nm,The double chains in the helix are antiparallel,B-DNA,Comparison in structural features among A, B, Z conformation forms of DNA (DNA A,B,Z三种构象形式的结构特征比较),A-DNA,B-DNA,Z-DNA,Ribbon diagrams of A, B, and Z-DNA 彩带图示,Rihgt-handed 11bp/pitch Pitch length 2.46nm Less occurring, but may be in DNA:RNA hybrids,A-DNA,A-DNA,B-DNA,B-DNA,Z-DNA,Left-handed,Right- handed,dG in B-DNA (anti position),dG in Z-DNA (syn position),Change from right-handed to left-handed,Cruciform十字形,Palindrome回形结构,Other forms of DNA conformation (DNA构象的其它形式),DNA can be denatured by heat (DNA 可热变性） Melting curve 溶解曲线 Melting point depends on (G+C)% and ionic strength 熔点与GC含量和离子强度有关 Increase in melting point with rise of GC% Increase in melting point with increase of ionic strength熔点随离子强度增加而增加 Denaturation by heat may be renaturated by slowly cooling again 热变性后的DNA可通过缓慢冷却而复性,Denaturation and renaturation of DNA DNA变性与复性,Melting curve 溶解曲线 Increase in MP with rise of GC%,Increase in melting point with increase of ionic strength熔点随离子强度增加而增加,Denaturation and renaturation of DNA,DNA organization in chromosomes 染色体中的DNA构架,Basic knowledge Comparison in structural features of DNA between prokaryotes and eucaryotes原核生物和真核生物染色体DNA结构特征比较,Location of DNA Prokaryotes原核生物: in nucleoid 类核 Eukaryotes真核生物: chromosome染色体 Chromosome Definition：Complex of nuclear DNA and histone proteins是核DNA和组蛋白的复合物 Histones are very basic proteins 组蛋白是非常碱性的蛋白质 Contains 25% lysine or arginine 含约25%的赖氨酸和精氨酸 Each choromosome contains a single linear double stranded DNA molecule. The length of the packaged DNA molecule varies From species to species, and among the same specie From one mitotic phase to another mitotic phase,Basic knowledge on DNA organization in chromosomes 染色体中的DNA构架的基本知识,Comparison in structural features of DNA between prokaryotes and eucaryotes原核生物和真核生物染色体DNA结构特征比较,Common types of structure in chromosome of a prokaryote cell,50 loops,Left-handed,Negative supercoiling,Positive supercoiling,Left-handed Positive supercoiled 正超螺旋,Right-handed Negative supercoiled 负超螺旋,Toroidal and interwound varieties of DNA supercoiling DNA超螺旋形成的超环面和相互盘绕形式,DNA in eukaryotes,DNA in eukaryotes is linear, but wounding around histones Each core DNA 146 bp Core particle Complex of eight histones-histone octamer 2*(H2A, H2B,H3,H4) Over packing ratio of DNA 7 fold,Levels of chromatin structure in eukaryotic chromosomes,DNA in eukaryotes,A model for the base-pairs compacted to form the chromosome structure,Ps see figure 12.31 p381,tRNA,2nd structure,tRNA,3rd structure: L-conformation,rRNA,Phylogenetic comparison种系比较 of secondary structures of 16S-like rRNAs from representatives of eubacteria, archae, and Lower eucaryotes,Homework:,Problem 1-4, 11,Appendix I,Tertiary structure of DNA：supercoils 超螺旋,Supercoils 超螺旋 Positive supercoils: overwind the helix Negative supercoils: underwind the helix Supercoiling may form varieties of toroidal and interwound structures Linker number (L) characterizes supercoiled DNA,Negative supercoiling,Positive supercoiling,Left-handed Overwound,Right-handed Underwound,Toroidal and interwound varieties of DNA supercoiling DNA超螺旋形成的超环面和相互盘绕形式,Supercoiled DNA in a toroidal form wraps readily around protein “spools:,Two negative supercoils,Toroidal conformation,Toroidal DNA stabilized by the protein spool,Protein spool,Collapse,Assembly,Linker number: L,L=T + W,T (twist): helical turns W(writhe): crossing number