Ribozyme  核酶 (英文及中文) 最终成稿
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Ribozyme 核酶 (英文及中文) 最终成稿

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Ribozyme

Ribozyme
Catalytic RNA molecule

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  • Very interesting presentation !! Thanks for sharing and congratulations dear Jiang Yh. I wish you and your family Merry Christmas and Happy New Year !! Best greetings from Greece. Nikos
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Ribozyme  核酶 (英文及中文) 最终成稿 Ribozyme 核酶 (英文及中文) 最终成稿 Presentation Transcript

  • Ribozyme 核酶(Catalytic RNA molecule) 制作:cz12062008 制作:cz12062008
  • 1.What is a Ribozyme? 1.WhatRibozymes are true enzymes. enzymes.A ribozyme is an RNA molecule(分子) that is capable capable(能力)of catalyzing (催化)a chemical areaction.NOT PROTEIN PROTEIN(蛋白)
  • First Ribozyme discovered上世纪80 80 80年代,美国科罗拉多大学博尔德分校的Thomas Cech(The intron in the pre-rRNA of Tetrahemena isself-spliced 和美国耶鲁大学的Sidnery Altan(The M1self-spliced) Sidnery Altan TheRNA in ribonuclease P is catalytic 各自独立地发现RNA catalytic) RNA RNA具有生物催化功能.从而改变了生物催比剂的传统概念。 . 1989 Nobel Prize In Chemistry Sid Altman Tom Cech
  • 2. 2.核酶的分类 I型内含子剪接型核酶通过既剪又接 II型内含子的方式除去内含子(Intron) Intron) 锤头核酶剪切型核酶 发夹核酶 自体催化自身催化的 丁型肝炎病毒(HDV)核酶反应是只切不接。 RNaseP 异体催化异体催化能剪切所tRNA tRNA前体的5‘端 5
  • How many ribozyme ? the hammerhead ribozyme (plant virus) 锤头状核酶(来源:植物病毒)- the hairpin ribozyme (plan virus) 发夹状核酶- hepatitis delta ribozyme (human virus) 丁型肝炎核酶(来源:人类病毒)- neurospora VS ribozyme (mitochondrial RNA) 孢VS VS VS核酶(来源:线粒体RNA RNA RNA)- group I and group II intron ribozyme (rRNA and mt RNA) 内含子I和II I II II核酶(来源:rRNA MT RNA rRNA rRNA和MT RNA)- RNAse P (tRNA maturation) 核糖核酸酶P(来源:tRNA P tRNA tRNA的成熟)- Ribosome (translation) 核糖体(翻译)- Spliceosome (splicing) 剪接 (拼接)
  • 天然核酶及核糖酶Ribozym Sequenced Size (nt) Activity Source (核酶) (测序) (大小) (功能) (来源)Group I intron >1’500 >1’ 210 self-splicing via 3’-OH 3’ eukayotic pre-mRNA, of G; transesterification organelles, few bacteriaGroup II intron >700 500 self-splicing via 3’-OH 3’ organelles, few prokaryotes of A; transesterificationHammerhead 11 40 self-cleavage via plant virusesHepatitis delta virus 2 90 transesterification hepatitis B virus (RNAgen.)Hairpin 1 70 (2’,3’-cyclic phosphate) (2’ ,3’ satellite RNA of plant virusRNAse P >500 300 pre-tRNA processing prokaryotes and organelles via hydrolysis (3 ’-OH) (3’ of eukaryotesSpliceosome pre-mRNA splicing via eukaryotic pre-mRNAs(U2 + U6 snRNAs) 70; 50 180; 100 ransesterification (3 ’OH) (3’Ribosome >900 2’600 2’ peptidyl transfer (amide) prokaryotes and eukaryotes(23S rRNA) Naturally occurring ribozymes and ribonucleoprotein enzymes (2002) peptidyl transfer (amide) (amide)转移肽(酰胺) self-splicing self-splicing自我剪接 self-cleavage self-cleavage自我裂解 2’,3’-cyclic phosphate 2 3 - ,3’ 2,3 -环磷酸 transesterification酯交换 prokaryotes and eukaryotes 原核生物和真核生物 transesterificationpre-tRNA processing via hydrolysis (3 ’-OH) 处理经水解的前氨酰- tRNA 3 - OH (3’ - tRNA(3 OH)
  • 3.结构(Structure ) 3. consider...As with proteins, we consider... Primary: GGCCGAACUGGUA 一级 Secondary 二级: Tertiary 三级:
  • 3.1 Primary Structure Structure一级结构 ribonucleotides核苷酸 (baseLimited to ribonucleotides base碱基 + ribose ribose核糖 + phosphate phosphate磷酸): Common Bases: N O N O 常见碱基 N N N N N N N N N N N N O N O A G C U Uncommon Bases: O O 罕见碱基 N N N N O N N Pseudouridine Inosine 假尿苷 肌苷 etc...
  • 3.2 Secondary Structure Structure(二级结构) Watson-Crick Base Pairing Helix Formation 沃森-克里克碱基配对 - 螺旋形成 B-DNA A-DNA RNA RNA usually assumes A-form helices… helices… DNA DNA常常假设A- A- A-型螺旋 Small pore along helical axis 沿螺旋轴形成个小孔 “Rungs” stack obliquely to axis Rungs” 阶梯式的斜上升的中心轴
  • I -5 -5 CU G3′ B YB 505′ V GV GA A 10 20 C A G U U A A H 40 A A A C 30 2-2 发卡型核酶二级结构
  • Secondary Structure Structure(二级结构) Conserved base-pairing interactions result in... 保守的碱基配对相互作用的导致:• Three “stem” regions stem” 三个结构域• Uridine-containing turn 旋转而封闭的尿苷• An “augmenting helix” helix” joining stems II and III结构域II 与 III II III连接形成的增强螺旋结构
  • Ribozyme vs. tRNAPhe 核酶 苯丙氨酸tRNA tRNA folding folding折叠
  • The hammerhead ribozyme ( ) (锤头状核酶)- discovered in small RNA satellites of small viruses (1986) 是小病毒携带的小的RNA RNA- replication by rolling circle mechanism 通过不断循环机制进行复制折叠 Secondary structure
  • RNAse P(核糖核酸酶P) is a ribozyme P RNAse P cleaves the 5’ end of pre- 5’ tRNAs(核糖核酸酶P切割5 P 5 tRNA 5前体tRNAtRNA的末段) It is composed of 12 kDa P protein and about 400 nt long RNA(它是由12 kDa 12 kDa蛋白质和长约400 400 400元的RNARNA RNA组 成) The catalytic activity lies entirely within RNA part(催化活性中心位于RNA RNA RNA上)
  • RNAse P(核糖核酸酶P) is a ribozyme P Enzyme is efficient without P protein but in high salt conditions(在高的盐条件,酶在没有P蛋白时还是有活性 P 的) P protein or high salt is thought to screen the repulsive electrostatic interactions between RNAse P RNA and substrate pre-tRNA(P 蛋白质或高盐被认为是核糖核酸酶P RNA P 与前体tRNAtRNA tRNA表面之间静电排斥反应的屏障。
  • P(核糖核酸酶P)RNAse P P
  • 3.3 tertiary structure(三级结构)锤头状核酶 发卡状核酶 丁型肝炎病毒 内含子I型核酶 I
  • Hammerhead ribozyme (锤头状核酶)
  • The hammerhead ribozyme (锤头状核酶) )- tertiary structure Scott et al and Klug, Science 1996
  • The hairpin ribozyme ( ) (发夹状核酶) From Lilley TIBS (2003)
  • The hepatitis delta ribozyme (丁型肝炎核酶) ) From Lilley TIBS (2003)
  • Group I &II intron ribozyme (rRNA and mt RNA) 内含子I型和II I II II型核酶 Doudna and Cech Nature, 2002
  • Group I intron ribozyme (rRNA and mt RNA) 内含子I型核酶(rRNA and mt RNA) Golden et al, and cech Science (1998)
  • 4.4.核酶的功能 (The Activity of Ribozym ) The1. 核苷酸转移作用。   2. 水解反应,即磷酸二酯酶作用。   3. 磷酸转移反应,类似磷酸转移酶作 用。   4. 脱磷酸作用,即酸性磷酸酶作用。   5. RNA RNA RNA内切反应,即RNA RNA限制性内切酶 作用。
  • 5.mechanism of catalysis5.mechanism 催化反应机理
  • How to catalyse the reaction ? 催化反应如何进行? From Lilley TIBS (2003)
  • Catalytic efficiency, condition 催化效率和条件- ribozyme follows a Michaelis-Menten kinetics kinetics(米氏动力学) k1 k2 E(酶) + S S(底物) ES ES(过渡态) E + P k-1 k-1+ k2 Km(米氏常数)= = 10-5-10-7 M kcat cat(催化常数)= 0.5-2 min -1 k1 kcat/ Km= 103-106 M-1.min-1 Good catalytic efficiency!! 良好的催化效率 - all ribozyme need cations for activity (Mg 2+ ,Mn2+) 所有活化的核酶的需要阳离子(Mg2+ ,Mn2+)
  • mechanism of catalysis 催化机理 IntactPhosphodiester完整的磷酸二酯 Mg2+Coordinated Transition Mg2 + +协调的过渡态 Cleaved Phosphodiester 结合后的磷酸二酯
  • Catalytic Mechanism(催化机理)New crystal structure shows five Mg 2+ sites:新发现的结晶结构是五个 Mg2+ Mg2+ 2+的结合域
  • 核酶的剪接机制 � 鸟苷的3’-OH攻击 内含子的5’剪接 点,并与Intron的 5’-端磷酸基形成 共价键连接,同时5’ 外显子脱落下来, 但3’外显子此时仍 与Intron相连。 5’-端外显子的 3’-OH进攻内含子 的3’剪接点并使 413nt的内含子脱 落,同时5’外显子 与3’外显子连接成 成熟rRNA
  • 3D structure of ribozyme: mechanism of catalysis hairpin ribozyme Hepatitis delta ribozyme (发夹状核酶) (丁型肝炎核酶)Ruppert et al, Nature 2001, Science 2002 Ferre d’Amare, Nature 1998 d’
  • Structure of the hairpin ribozyme (发夹状核酶的结构) ) hairpin ribozyme Ruppert et al, Nature 2001, Science 2002
  • hairpin ribozyme(发夹状核酶)) Ground state state(基态) Transition state state(过渡态)Ruppert et al, Nature 2001 Ruppert et al, Science 2002
  • hairpin ribozyme (发夹状核酶) Transition state(过渡态) state( )Ruppert et al, Science 2002
  • 5.Ribozyme5.Ribozyme Ribozyme应用 Ribozyme Ribozyme的发现使我们认识到酶不一定都是蛋白质, 这些Ri-bozyme Ri-bozyme RNA Ri-bozyme主要用在RNA RNA分子的切割与降解上。 因为真核生物的突变体很难获得,假如用特制的 Ribozyme Ribozyme破坏切割特定的RNARNA RNA分子,使这种RNA RNA RNA不 能表达出相应的蛋白质,我们再进一步观察其表型变 化,从而了解这个基因的功能。 另外在医学方面,可以用特别的RibozymeRibozyme Ribozyme切割一些对 人体有害的RNA RNA RNA,达到解除有害基因功能的目的,这 也是基因治疗的又一条路子。 在动植物育种方面,可以将具有破坏病毒RNA RNA RNA的 Ribozyme Ribozyme基因导入动植物细胞,提高动植物抗病毒能 力。
  • 举例:Engineered allosteric ribozymes as biosensors Engineered (具有生物传感器功能的变构核酶) a. ATP-sensing aptamer + self-cleaving ribozyme. ribozyme. ATP传感适体+自我切割核酶。 ATP + b. This variant favors ADP binding 100-fold overATP. overATP. 这种变异使对ATP ATP ATP的亲和力是ADP 100 ADP 100倍。 ADP的100 c. Can be used as an in vitro sensor for enzyme activity (or for enzyme inhibition). inhibition). 用作体外传感器的酶激动剂(或酶的抑制剂)。
  • 蓝箭头显示裂解位 点
  • Most common output is Green Fluorescent Protein (GFP) 最常见的输出是绿色荧光蛋白(GFP GFP GFP)