核酶课件 (英文及中文） 最终成稿

Ribozyme Ribozyme Ribozyme Ribozyme 核酶 (Catalytic RNA molecule) (Catalytic RNA molecule) (Catalytic RNA molecule) (Catalytic RNA molecule) 制作：制作：制作：制作：cz12062008cz12062008cz12062008cz12062008 1. 1. 1. 1.What is a Ribozyme? What is a Ribozyme? What is a Ribozyme? What is a Ribozyme? Ribozymes are true enzymes Ribozymes are true enzymes Ribozymes are true enzymes Ribozymes are true enzymes. . . . A ribozyme is an RNA molecule A ribozyme is an RNA molecule A ribozyme is an RNA molecule A ribozyme is an RNA molecule （分子） that is capable that is capable that is capable that is capable（能力） of catalyzing of catalyzing of catalyzing of catalyzing （催化） a chemical a chemical a chemical a chemical reaction. reaction. reaction. reaction. NOT PROTEIN NOT PROTEIN NOT PROTEIN NOT PROTEIN（蛋白） First Ribozyme First Ribozyme First Ribozyme First Ribozyme discovered discovered discovered discovered 上世纪80808080年代，美国科罗拉多大学博尔德分校的 Thomas CechThomas CechThomas CechThomas Cech（The intron inThe intron inThe intron inThe intron in the pre-rRNA ofthe pre-rRNA ofthe pre-rRNA ofthe pre-rRNA of Tetrahemena isTetrahemena isTetrahemena isTetrahemena is self-spliced self-spliced self-spliced self-spliced ）和美国耶鲁大学的Sidnery AltaSidnery AltaSidnery AltaSidnery Altannnn（The M1 The M1 The M1 The M1 RNA RNA RNA RNA in ribonuclease in ribonuclease in ribonuclease in ribonuclease P is catalytic P is catalytic P is catalytic P is catalytic ）各自独立地发现RNARNARNARNA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 Prize1989 Nobel Prize1989 Nobel Prize1989 Nobel Prize In ChemistryIn ChemistryIn ChemistryIn Chemistry Sid Altman Sid Altman Sid Altman Sid Altman Tom Cech Tom Cech Tom Cech Tom Cech I型内含子 剪接型核酶 II型内含子 锤头核酶 剪切型核酶 发夹核酶 自体催化 丁型肝炎病毒（HDV)HDV)HDV)HDV)核酶 RNaseP RNaseP RNaseP RNaseP 异体催化 2.2.2.2.核酶的分类 通过既剪又接 的方式除去内 含子（Intron)Intron)Intron)Intron) 自身催化的 反应是只切 不接。 异体催化能 剪切所tRNAtRNAtRNAtRNA 前体的5555‘‘‘‘端 How many ribozyme ? How many ribozyme ? How many ribozyme ? How many ribozyme ? the hammerhead ribozyme (plant virus) the hammerhead ribozyme (plant virus) the hammerhead ribozyme (plant virus) the hammerhead ribozyme (plant virus) 锤头状核酶（来源：植物病毒） - the hairpin ribozyme (plan virus)- the hairpin ribozyme (plan virus)- the hairpin ribozyme (plan virus)- the hairpin ribozyme (plan virus) 发夹状核酶 - hepatitis delta ribozyme (human virus)- hepatitis delta ribozyme (human virus)- hepatitis delta ribozyme (human virus)- hepatitis delta ribozyme (human virus) 丁型肝炎核酶（来源：人类病毒） - neurospora VS ribozyme (mitochondrial RNA)- neurospora VS ribozyme (mitochondrial RNA)- neurospora VS ribozyme (mitochondrial RNA)- neurospora VS ribozyme (mitochondrial RNA) 孢VSVSVSVS核酶（来源：线粒体RNARNARNARNA） - group I and group II intron ribozyme (rRNA and mt RNA)- group I and group II intron ribozyme (rRNA and mt RNA)- group I and group II intron ribozyme (rRNA and mt RNA)- group I and group II intron ribozyme (rRNA and mt RNA) 内含子IIII和IIIIIIII核酶（来源：rRNArRNArRNArRNA和MT RNAMT RNAMT RNAMT RNA） - RNAse P (tRNA maturation)- RNAse P (tRNA maturation)- RNAse P (tRNA maturation)- RNAse P (tRNA maturation) 核糖核酸酶PPPP（来源：tRNAtRNAtRNAtRNA的成熟） - Ribosome (translation) - Ribosome (translation) - Ribosome (translation) - Ribosome (translation) 核糖体（翻译） - Spliceosome (splicing)- Spliceosome (splicing)- Spliceosome (splicing)- Spliceosome (splicing) 剪接 （拼接） RibozymRibozymRibozymRibozym SequencedSequencedSequencedSequenced Size (nt) Size (nt) Size (nt) Size (nt) Activity Activity Activity Activity SourceSourceSourceSource （核酶） （测序） （大小） （功能） （来源） Group I intronGroup I intronGroup I intronGroup I intron >1>1>1>1’’’’500500500500 210 210 210 210 self-splicing via 3self-splicing via 3self-splicing via 3self-splicing via 3’’’’-OH-OH-OH-OH eukayotic pre-mRNA,eukayotic pre-mRNA,eukayotic pre-mRNA,eukayotic pre-mRNA, of G; transesterificationof G; transesterificationof G; transesterificationof G; transesterification organelles, few bacteria organelles, few bacteria organelles, few bacteria organelles, few bacteria Group II intronGroup II intronGroup II intronGroup II intron >700 >700 >700 >700 500 500 500 500 self-splicing via 3 self-splicing via 3 self-splicing via 3 self-splicing via 3’’’’-OH -OH -OH -OH organelles, few prokaryotes organelles, few prokaryotes organelles, few prokaryotes organelles, few prokaryotes of A; transesterification of A; transesterification of A; transesterification of A; transesterification HammerheadHammerheadHammerheadHammerhead 11 11 11 11 40 40 40 40 self-cleavage via self-cleavage via self-cleavage via self-cleavage via plant viruses plant viruses plant viruses plant viruses Hepatitis delta virus Hepatitis delta virus Hepatitis delta virus Hepatitis delta virus 2 2 2 2 90 transesterification 90 transesterification 90 transesterification 90 transesterification hepatitis B virus hepatitis B virus hepatitis B virus hepatitis B virus (RNAgen.)(RNAgen.)(RNAgen.)(RNAgen.) HairpinHairpinHairpinHairpin 1 1 1 1 70 (2 70 (2 70 (2 70 (2’’’’,3,3,3,3’’’’-cyclic phosphate)-cyclic phosphate)-cyclic phosphate)-cyclic phosphate) satellite RNA of plant virussatellite RNA of plant virussatellite RNA of plant virussatellite RNA of plant virus RNAse PRNAse PRNAse PRNAse P >500 >500 >500 >500 300 300 300 300 pre-tRNA processingpre-tRNA processingpre-tRNA processingpre-tRNA processing prokaryotes and organellesprokaryotes and organellesprokaryotes and organellesprokaryotes and organelles via hydrolysis (3via hydrolysis (3via hydrolysis (3via hydrolysis (3’’’’-OH)-OH)-OH)-OH) of eukaryotesof eukaryotesof eukaryotesof eukaryotes SpliceosomeSpliceosomeSpliceosomeSpliceosome pre-mRNA splicing via pre-mRNA splicing via pre-mRNA splicing via pre-mRNA splicing via eukaryotic pre-mRNAs eukaryotic pre-mRNAs eukaryotic pre-mRNAs eukaryotic pre-mRNAs (U2 + U6 snRNAs) (U2 + U6 snRNAs) (U2 + U6 snRNAs) (U2 + U6 snRNAs) 70; 5070; 5070; 5070; 50 180; 100 ransesterification (3 180; 100 ransesterification (3 180; 100 ransesterification (3 180; 100 ransesterification (3’’’’OH)OH)OH)OH) RibosomeRibosomeRibosomeRibosome >900 >900 >900 >900 2 2 2 2’’’’600600600600 peptidyl transfer (amide) peptidyl transfer (amide) peptidyl transfer (amide) peptidyl transfer (amide) prokaryotes and eukaryotes prokaryotes and eukaryotes prokaryotes and eukaryotes prokaryotes and eukaryotes (23S rRNA)(23S rRNA)(23S rRNA)(23S rRNA) Naturally occurring ribozymes and ribonucleoprotein enzymes (2002)Naturally occurring ribozymes and ribonucleoprotein enzymes (2002)Naturally occurring ribozymes and ribonucleoprotein enzymes (2002)Naturally occurring ribozymes and ribonucleoprotein enzymes (2002) peptidyl transfer (amide)peptidyl transfer (amide)peptidyl transfer (amide)peptidyl transfer (amide)转移肽（酰胺） self-splicingself-splicingself-splicingself-splicing自我剪接 self-cleavageself-cleavageself-cleavageself-cleavage自我裂解 2222’’’’,3,3,3,3’’’’-cyclic phosphate-cyclic phosphate-cyclic phosphate-cyclic phosphate 2'2'2'2'，3' -3' -3' -3' -环磷酸 transesterificationtransesterificationtransesterificationtransesterification酯交换 prokaryotes and eukaryotesprokaryotes and eukaryotesprokaryotes and eukaryotesprokaryotes and eukaryotes 原核生物和真核生物 pre-tRNA processing via hydrolysis (3pre-tRNA processing via hydrolysis (3pre-tRNA processing via hydrolysis (3pre-tRNA processing via hydrolysis (3’’’’-OH)-OH)-OH)-OH) 处理经水解的前氨酰- tRNA- tRNA- tRNA- tRNA（3' - OH3' - OH3' - OH3' - OH） 天然核酶及核糖酶 3.3.3.3.结构（Structure Structure Structure Structure ） As with proteins, we considerAs with proteins, we considerAs with proteins, we considerAs with proteins, we consider............ Primary:Primary:Primary:Primary: 一级 GGCCGAACUGGUA GGCCGAACUGGUA GGCCGAACUGGUA GGCCGAACUGGUA SecondarySecondarySecondarySecondary 二级: TertiaryTertiaryTertiaryTertiary 三级: 3.1 3.1 3.1 3.1 Primary Structure Primary Structure Primary Structure Primary Structure 一级结构 Limited to ribonucleotidesLimited to ribonucleotidesLimited to ribonucleotidesLimited to ribonucleotides核苷酸 ( ( ( (basebasebasebase碱基 + ribose + ribose + ribose + ribose核糖 + phosphate + phosphate + phosphate + phosphate磷酸):):):): Common Bases: Common Bases: Common Bases: Common Bases: 常见碱基 Uncommon Bases: Uncommon Bases: Uncommon Bases: Uncommon Bases: 罕见碱基 N N N N O N N N N N N N N O N N N O O A G C UA G C UA G C UA G C U N N O O N N N N O Pseudouridine Pseudouridine Pseudouridine Pseudouridine 假尿苷 Inosine Inosine Inosine Inosine 肌苷 etc... etc... etc... etc... 3.2 3.2 3.2 3.2 Secondary Structure Secondary Structure Secondary Structure Secondary Structure（二级结构） Watson-Crick Base PairingWatson-Crick Base PairingWatson-Crick Base PairingWatson-Crick Base Pairing 沃森----克里克碱基配对 Helix FormationHelix FormationHelix FormationHelix Formation 螺旋形成 B-DNA B-DNA B-DNA B-DNA RNA RNA RNA RNA RNA usually assumesRNA usually assumesRNA usually assumesRNA usually assumes A-form helicesA-form helicesA-form helicesA-form helices………… DNADNADNADNA常常假设A-A-A-A-型螺旋 Small pore along helical axis Small pore along helical axis Small pore along helical axis Small pore along helical axis 沿螺旋轴形成个小孔 “ “ “ “Rungs Rungs Rungs Rungs” ” ” ” stack obliquely to axis stack obliquely to axis stack obliquely to axis stack obliquely to axis 阶梯式的斜上升的中心轴 A-DNA A-DNA A-DNA A-DNA 3′ 5′ -5 B V C U G Y G G A A -5 B V I 10 G A A A C C U U A A H A 40 30 50 20 2-2 发卡型核酶二级结构 Secondary Structure Secondary Structure Secondary Structure Secondary Structure（二级结构） Conserved base-pairing interactions result in...Conserved base-pairing interactions result in...Conserved base-pairing interactions result in...Conserved base-pairing interactions result in... 保守的碱基配对相互作用的导致： • Three Three Three Three ““““stemstemstemstem”””” regions regions regions regions 三个结构域 • Uridine-containing turnUridine-containing turnUridine-containing turnUridine-containing turn 旋转而封闭的尿苷 • An An An An ““““augmenting helixaugmenting helixaugmenting helixaugmenting helix”””” joining stems II and III joining stems II and III joining stems II and III joining stems II and III 结构域II II II II 与 III III III III连接形成的增强螺旋结构 Ribozyme Ribozyme Ribozyme Ribozyme vs. vs. vs. vs. tRNA tRNA tRNA tRNA Phe Phe Phe Phe 核酶 苯丙氨酸tRNA tRNA tRNA tRNA folding folding folding folding折叠 The hammerhead ribozyme (The hammerhead ribozyme (The hammerhead ribozyme (The hammerhead ribozyme (锤头状核酶)))) - discovered in small RNA satellites of small viruses (1986)- discovered in small RNA satellites of small viruses (1986)- discovered in small RNA satellites of small viruses (1986)- discovered in small RNA satellites of small viruses (1986) 是小病毒携带的小的RNARNARNARNA - replication by rolling circle mechanism- replication by rolling circle mechanism- replication by rolling circle mechanism- replication by rolling circle mechanism 通过不断循环机制进行复制折叠 Secondary structureSecondary structureSecondary structureSecondary structure RNAse PRNAse PRNAse PRNAse P（核糖核酸酶PPPP） is a is a is a is a ribozymeribozymeribozymeribozyme RNAse P cleaves the 5RNAse P cleaves the 5RNAse P cleaves the 5RNAse P cleaves the 5’’’’ end of pre- end of pre- end of pre- end of pre- tRNAstRNAstRNAstRNAs （核糖核酸酶PPPP切割5'5'5'5'前体tRNAtRNAtRNAtRNA的末段） It is composed of 12 kDa P protein and It is composed of 12 kDa P protein and It is composed of 12 kDa P protein and It is composed of 12 kDa P protein and about 400 nt long RNAabout 400 nt long RNAabout 400 nt long RNAabout 400 nt long RNA （它是由12 kDa12 kDa12 kDa12 kDa蛋白质和长约400400400400元的RNARNARNARNA组 成） The catalytic activity lies entirely within The catalytic activity lies entirely within The catalytic activity lies entirely within The catalytic activity lies entirely within RNA partRNA partRNA partRNA part （催化活性中心位于RNARNARNARNA上） RNAse PRNAse PRNAse PRNAse P（核糖核酸酶PPPP） is a is a is a is a ribozymeribozymeribozymeribozyme Enzyme is efficient without P protein but Enzyme is efficient without P protein but Enzyme is efficient without P protein but Enzyme is efficient without P protein but in high salt conditionsin high salt conditionsin high salt conditionsin high salt conditions （在高的盐条件，酶在没有PPPP蛋白时还是有活性 的） P protein or high salt is thought to P protein or high salt is thought to P protein or high salt is thought to P protein or high salt is thought to screen the repulsive electrostatic screen the repulsive electrostatic screen the repulsive electrostatic screen the repulsive electrostatic interactions between RNAse P RNA and interactions between RNAse P RNA and interactions between RNAse P RNA and interactions between RNAse P RNA and substrate pre-tRNAsubstrate pre-tRNAsubstrate pre-tRNAsubstrate pre-tRNA (P (P (P (P 蛋白质或高盐被认为是核糖核酸酶PPPP RNA RNA RNA RNA 与前体tRNAtRNAtRNAtRNA 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 面之间静电排斥反应的屏障。 RNAse PRNAse PRNAse PRNAse P（核糖核酸酶PPPP） 3.3 3.3 3.3 3.3 tertiary structure tertiary structure tertiary structure tertiary structure（三级结构） 锤头状核酶 发卡状核酶 内含子IIII型核酶丁型肝炎病毒 Hammerhead ribozymeHammerhead ribozymeHammerhead ribozymeHammerhead ribozyme （锤头状核酶） The hammerhead ribozyme The hammerhead ribozyme The hammerhead ribozyme The hammerhead ribozyme ((((锤头状核酶)))) - tertiary structure- tertiary structure- tertiary structure- tertiary structure Scott et al and Klug, Science 1996Scott et al and Klug, Science 1996Scott et al and Klug, Science 1996Scott et al and Klug, Science 1996 The hairpin ribozyme (The hairpin ribozyme (The hairpin ribozyme (The hairpin ribozyme (发夹状核酶)))) From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003) The hepatitis delta ribozyme The hepatitis delta ribozyme The hepatitis delta ribozyme The hepatitis delta ribozyme ((((丁型肝炎核酶)))) From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003) Group I &II intron ribozyme (rRNA and mt RNA) Group I &II intron ribozyme (rRNA and mt RNA) Group I &II intron ribozyme (rRNA and mt RNA) Group I &II intron ribozyme (rRNA and mt RNA) 内含子IIII型和IIIIIIII型核酶 Doudna and CechDoudna and CechDoudna and CechDoudna and Cech Nature, 2002Nature, 2002Nature, 2002Nature, 2002 内含子I型核酶(rRNA and mt RNA) Group I intron ribozyme (rRNA and mt RNA) Group I intron ribozyme (rRNA and mt RNA) Group I intron ribozyme (rRNA and mt RNA) Group I intron ribozyme (rRNA and mt RNA) Golden et al, and cechGolden et al, and cechGolden et al, and cechGolden et al, and cech Science (1998)Scienc