Molecular Biology of the Gene, 6/E - Watson et al. (2007)Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: Methods1Chapter 9 Techniques of Molecular Biology分子生物学技术分子生物学技术 Yang Yang（杨（杨 洋洋 教授）教授） Huazhong University of Sciences and Technology2Techniques of Molecular Biologyn nTechniques of molecular biology have been one of the major driving forces in the research of molecular biology 3 The methods depend upon understanding of the properties of biological macromolecules themselves.DNA cloning- DNA polymerase, restriction endonucleases and DNA LigasePCR-Thermophilic DNA polymerase4OutlinenTopic 1: Nucleic acids TechniquesNucleic acids TechniquesnTopic 2: Protein TechniquesProtein TechniquesnTopic 3: Techniques for studying Techniques for studying DNA-protein interactionprotein interactionnTopic 4: Techniques for studying Techniques for studying proteinprotein-protein interactionprotein interactionBasic principleBasic procedure application5Topic 1: Nucleic acids Techniques1.Separation by Electrophoresis (电泳分离电泳分离) 2.Cut by Restriction endonuclease (限制性限制性内切酶切割内切酶切割)3.DNA Cloning and gene expression (基因基因克隆和表达技术）克隆和表达技术）4.PCR（聚合酶链式反应）（聚合酶链式反应）5. Hybridization (杂交技术杂交技术): Southern hybridization, hybridization, Northern hybridization, hybridization, Western hybridizationWestern hybridization6Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties1. Gel Electrophoresis （凝胶电泳）凝胶电泳）71.DNA and RNA molecules are negatively charged, thus move in the gel matrix (胶胶支持物支持物) toward the positive pole (正电极正电极)2.Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules.DNA gel mobility (electrophoretic mobility ，DNA在胶上的迁移率在胶上的迁移率)8largemoderate smallRun gel(跑胶跑胶)93.The mobility of circular DNA moleculescircular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋超螺旋) linear (线性线性) nicked or relaxed (缺刻或松散缺刻或松散) 10DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化溴化乙锭乙锭)DNA is separated by gel electrophoresis1 kb0.5 kb2 kb3 kb4 kbDNA marker11Gel matrix (胶支持物胶支持物) is a jello( (果冻状果冻状）-like porous material that supports and allows macromolecules to move through. Gel matrix (胶支持物胶支持物)Polyacrylamide (聚丙烯酰胺聚丙烯酰胺)Agarose (琼脂糖琼脂糖)12Polyacrylamide (聚丙烯酰胺聚丙烯酰胺): (1)has high resolving capability(分辨力高分辨力高), and can resolve DNA/RNA that differ from each other as little as a single base pair.(2)but can separate DNA over a narrow size range (up to a few hundred bp, 几百几百bp).13Agarose (琼脂糖琼脂糖): (1)a much less resolving power than polyacrylamide(2)but can separate DNA molecules of up to tens of kb1 kb0.5 kb2 kb3 kb4 kb14Pulsed-field gel electrophoresis (脉冲电泳脉冲电泳)15pulsed-field gel electrophoresis(PFGE)Switching between two orientations: the larger the DNA is, the longer it takes to reorient1617(1)RNA have a uniform negative charge as DNA does. (2)RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility.(3)RNA can be treated with reagent such as glyoxal (乙二醛乙二醛) to prevent RNA base pairing, so that its mobility only correlates with the molecular weight Electrophoresis is also used to separate RNAs182. Restriction endonucleases (限制性内切酶限制性内切酶) cleave DNA molecules at particular sitesnWhy use endonucleases?nTo make large DNA molecules break into manageable fragmentsnWhy use restriction endonucleases?ncleave DNA molecules at particular sites19n nRestriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific sequences.(剪刀剪刀)nRE used in molecular biology typically recognize (识别识别) short (4-8bp) target sequences that are usually palindromic (回文结构回文结构), and cut (切割切割) at a defined sequence within those sequences. 205.GAATTC.3 .CTTAAG.EcoRI35反向重复序列（回文结构）反向重复序列（回文结构）21na linear DNA molecule with 6 copies of GAATTCnit will be cut into 7 fragments which could be separated by the gel electrophoresis.(The largest fragment)(The smallest fragment)Digestionof a DNA fragment with endonuclease EcoRI22(1)Restriction enzymes differ in the recognition specificityrecognition specificity: target sites are different.(2) Restriction enzymes differ in the length the length they recognizedthey recognized, and thus the frequencies differ: Sau3AI, 5-GATC-3; NotI, 5-GCGGCCGC-3(3) Restriction enzymes differ in the nature the nature of the DNA ends they generateof the DNA ends they generate: blunt ends (平末端平末端), sticky ends (粘性末端粘性末端).23sticky ends(粘性末端粘性末端) blunt ends(平末端平末端) Recognition sequences and cut sites of various endonucleases24Cleavage of an EcoRI site: The 5 protruding ends are said to be “sticky” because they readily anneal (退火退火) through base-pairing to DNA molecules cut with the same enzymeGene BGene A25DNA连接酶连接酶26Discovery of restriction endonucleases Research strategy: from phenomenon to mechanismPhenomenonHypothesisExperimental proofMechanismPractical use of restriction endonucleases27Salvador E. Luria, 1969 Nobel prize in Physiology or Medicine Model: Phage infecting E.coli，噬菌体学派噬菌体学派Phenomenon: restriction and modification in bacteria (限制与修饰现象）限制与修饰现象）28Werner Arber, 1978 Nobel Prize in Physiology or MedicineHe analyzed an apparently obscure phenomenon in bacteria,called host-controlled modification and restriction Arber postulated that bacteria contain restriction enzymes with the capacity to recognize and degrade foreign DNA-phage DNA (protect the host from foreign genes) 29Arbers HypothesisnBacteria have the ability to restrict phage (virus) infection by cutting up the phage DNA once its injected into the cellnThe enzyme that cuts phage DNA is called a restriction enzyme, or more properly, a restriction endonuclease 30Hamilton O. Smith, 1978 Nobel Prize in Physiology or Medicine Hamilton Smith verified Arbers hypothesis- bacteria contain restriction enzymes with the capacity to recognize and degrade foreign DNAHe purified one restriction enzyme and showed that it could cleave foreign DNAHe determined the chemical structure of the regions of DNAwhich were cut by the restriction enzyme(The enzymes dont cut randomly. They bind to specific sequences and only cut at those sites-识别回文结构识别回文结构)31nSmith, H.O. and Wilcox, K.W. A restriction enzyme from Hemophilus influenzae. 1. Purification and general properties. J. Mol. Biol. 51, 379 (1970).nKelly, T.J., Jr. and Smith, H.O. A restriction enzyme from Hemophilus influenzae. 11. Base sequence of the recognition site. J. Mol. Biol. 51, 393 (1970).nRoy, P.H. and Smith, H.O. The DNA methylases of Hemophilus influenzae Rd. 1. Purification and properties. J. Mol. Biol. 81, 427 (1973).nRoy, P.H. and Smith, H.O. The DNA methylases of Hemophilus influenzae Rd. 11. Partial recognition site base sequences. J. Mol. Biol. 81, 445 (1973).nPapers32Daniel Nathans, 1978 Nobel Prize in Physiology or Medicine The last step in this development was taken by Dan NathansHe constructed the first genetic map using restriction enzymes by cleaving the DNA from a monkey virus-SV40He pioneered the application of restriction enzymes in genetics and his work has been a source of inspirationfor scientists who subsequently started to use restriction enzymes3334Discovery of restriction endonucleases PhenomenonHypothesisExperimental proofMechanismpractical use of restriction endonucleases35How does the bacteria prevent its own DNA from being cut by the restriction endonucleases?nModification nOne of the nucleotides in bacteria DNA is modified by an methylase enzyme (甲基化酶，甲基化酶，modification enzyme) that attaches a methyl group to one of the basesnThe restriction enzyme doesnt bind to sites where one of the bases is modified by methylation 36RestrictionModification373. DNA cloning and gene expressionThe ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.38Processes (过程过程) of DNA cloning：1.Constructing the recombinant DNA molecules (重组重组DNA) by inserting your interested DNA fragments into a proper vector (载体载体). (Require restriction enzymes and DNA ligase)2.Transform (转化转化) the recombinant DNA molecules into competent cells (感受态细胞感受态细胞).3.Propagation of the cells containing the recombinant DNA to form a clone (克隆克隆), a set of identical cells containing the same recombinant DNA.4.Select the desired clones using the selective marker. 391.Restriction digestion of your insert and vector using the same enzyme.2.Use ligase (连接酶）连接酶） to join your insert and vector together.3.Transform the ligation products into E. coli competent cells.4.Select the desired clones: Grow the cells on a plate containing tetracycline (四环素四环素).EcoRI40Important components of DNA cloningnVector (载体）载体）nHost organisms/cells ( (宿主细胞）宿主细胞）nRestriction enzymes and DNA ligase(剪刀剪刀+浆糊浆糊)nScreening method for selecting the desired clones （筛选阳性克隆）（筛选阳性克隆） 41General features of a Vector1.They contain an origin of replication and can autonomously replicate DNA independent of hosts genome.2.Easily to be isolated from the host cell. 3.Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. 4.Contains a multiple cloning site (MCS) to be cut by restriction enzymes for DNA manipulation. 42Plasmid(质粒质粒): most used vector in DNA cloning small, extrachromosomal circular molecules, from 2 to 200 kb in size, which exist in multiple copies within the host cells Contain an origin of replication, at least one selective marker and multiple cloning site. Example of selective marker: ampr gene encoding the enzyme b-lactamse which degrades penicillin antibiotics such as ampicillin-氨苄青霉素抗性基因氨苄青霉素抗性基因43multiple cloning site(MCS, 多克隆位点多克隆位点 )MCSselective markerorigin of replication44Important components of DNA cloningnVector (载体）载体）nHost organisms/cells ( (宿主细胞）宿主细胞）nRestriction enzymes and DNA ligase(剪刀剪刀+浆糊浆糊)nScreening method for selecting the desired clones （筛选阳性克隆）（筛选阳性克隆） 45Host organisms/cells: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning.-Prokaryotic host: E. coli ( most cases)-Eukaryotic host: Yeast, Saccharomyces cerevisiae -Vectors can be introduced into competent cells (感受态细胞感受态细胞) by transformation (转化转化)46Important components of DNA cloningnVector (载体）载体）nHost organisms/cells ( (宿主细胞）宿主细胞）nRestriction enzymes and DNA ligase(剪刀剪刀+浆糊浆糊)nScreening method for selecting the desired clones （筛选阳性克隆）（筛选阳性克隆） 47Screening of positive clones481.Antibiotic screening (抗生素选择抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate.2.Blue-white screening (蓝白斑选择蓝白斑选择): DNA insertion in the vector shuts down the LacZ gene expression, and turns the colony to white. Screening methods:49(1).Antibiotic screening (抗生素选择抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate.50multiple cloning site(MCS, 多克隆位点多克隆位点 )MCSselective markerorigin of replication51antibiotic-containing plateHost cell must be sensitive to this antibiotic!52X geneampRampRampRampRAntibiotic screening can not discriminate the clone containing recombinant pasmid and the clonecontaining the religated vector 53(2). Blue-white screening (蓝白蓝白斑选择斑选择)54lacZ encode enzyme bb-galactosidase（bb-半乳糖苷酶）半乳糖苷酶） X-gal(substrate of the enzyme)Blue product55Host E.coli cell : lacZ M15encoding only the C-terminal of aa-peptide of bb-galactosidasePlasmid: lacZ, a shortened derivative of lacZ, encoding N-terminal of aa-peptide of bb-galactosidase aa- complementation (aa-互补互补) 56Host E.coli cellPlasmidlacZ+X-gal(substrate of the enzyme)Blue product bb-半乳糖苷酶半乳糖苷酶57Blue white screeningAmproripUC18(3 kb)MCS (Multiple cloning sites,多克隆位点）多克隆位点）Lac promoterlacZ58AmproripUC18(3 kb)MCS (Multiple cloning sites,多克隆位点）多克隆位点）Lac promoterlacZSelf-ligated vectorAmproripUC18(3 kb)Lac promoterlacZRecombinant plasmidInserted gene59Self-ligated vector: blue transformantsRecombinant plasmid: containing inserted DNA, white transformantsSelf-ligated vector(no insert)Recombinant plasmid (contain insert)6061Herbert W. Boyer Professor in University of California, San Francisco Stanley Cohen Professor in Standford University Invent Recombinant DNA Technology (genetic engineering)Recombined genes that originated from different organisms 62First experiment about Recombinant DNA technology ampRtetR对四环素和氨苄青霉素都有抗性的新细菌菌株对四环素和氨苄青霉素都有抗性的新细菌菌株63Genentech company (Genetic Engineering Technology, Inc.)first biotechnology corporation, which was founded in 1976 by venture capitalist Robert A. Swanson and biochemist Dr.Herbert W. Boyer 64Overexpression of humaninsulin (胰岛素）胰岛素）gene in E.coliBacterial factory producinglarge amounts of Human proteins (insulin)654. 4. PCRPCR（聚合酶链式反应）（聚合酶链式反应）（聚合酶链式反应）（聚合酶链式反应） Molecular Biology Course66The Chemistry of DNADNA synthesis requires deoxynucleoside triphosphates and a primer:template junctionDNA is synthesized by extending the 3 end of the primerHydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis 67Figure 8-3 Substrates required for DNA synthesis68Semiconservative Replication(半保留复制)Three hypotheses for DNA replication69The mechanism of DNA Polymerase (Pol)CHAPTER 8 The replication of DNA70Polymerase chain reaction-An important technique based on DNA Polymerase The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA in vitro, using a pair of primers each complementary to one end of the the DNA target sequence.理论理论技术技术71Denaturation (变性变性): The target DNA (template) is separated into two stands by heating to 95Primer annealing (退火退火): The temperature is reduced to around 55 to allow the primers to anneal.Polymerization (elongation, extension) (延伸延伸): The temperature is increased to 72 for optimal polymerization step which uses dNTPs and requires Mg+.The principle of PCR:Three different steps proceed in each PCR cycle. 7253535353553353535353535353535353535355335535353353535353DenaturationAnnealingExtension (DNA polymerase)Cycle 1Cycle 2Cycle 373InitialDNA8421Number of DNA molecules (2n): 指数扩增指数扩增74Many cycles (25-35 in common) are performed to complete one PCR reaction, which resulted in an exponential amplification of the target DNA in which both forward and reverse primers pair.75DNA template (模板模板)Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR.Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.7677Figure 8-3 Substrates required for DNA synthesis78PCR Primers1.Anneal on opposite strands of the target sequence: forward and reverse primers （正向引物和反向引物）（正向引物和反向引物）2.Have similar G+C contents (Tm) so that they anneal to their complementary sequences at similar temperatures3. (anneal temperature: Tm-5C)795-ATTCCGATCGCTAATCGATGGC- TCCTGTGCA TTTCGCCACTAGAG-33-TAAGGCTAGCGATTAGCTACCG-AGGACACGTAAAGCGGTGATCTC-55-ATTCCGATCGCTAATCGATG-33-CACGTAAAGCGGTGATCTC-5forward primerreverse primer5-CTCTAGTGGCGAAATGCAC-38053535353553353535353535353535353535355335535353353535353DenaturationAnnealingExtension (DNA polymerase)Cycle 1Cycle 2Cycle 381PCR Enzymes82nDenaturation (变性变性): The target DNA (template) is separated into two stands by heating to 95nPrimer annealing (退火退火): The temperature is reduced to around 55 to allow the primers to anneal.nPolymerization (elongation, extension) (延伸延伸): The temperature is increased to 72 for optimal polymerization step which uses dNTPs and requires Mg2+.83nTaq polymerase ：isolated from the thermophilic bacterium Thermus aquaticus (嗜热菌）嗜热菌）, 耐热耐热DNA聚合酶，耐高温聚合酶，耐高温nIt has no 3 to 5 proofreading exonuclease activity nHigh-accuracy DNA polymerase is available commercially(高保真酶）高保真酶）84PCR仪仪85Discovery of PCR techniquen希望大家不仅仅知道希望大家不仅仅知道“是什么是什么”，而且也，而且也了解了解“为什么为什么”和和“结论是怎么得出来的结论是怎么得出来的”86n如果你的研究能力一般，那么，改革本研究领域中大家习以为常的最基本的技术，你就有可能获诺贝尔奖 - Kary Mullis (卡里卡里.穆利斯穆利斯) 87The replication of DNA The replication of DNA 88n哥伦布竖立鸡蛋的故事哥伦布竖立鸡蛋的故事nDNA分子的拷贝术，复印机89nPCR技术的发现充满传奇色彩技术的发现充满传奇色彩nKary Mullis (卡里卡里.穆利斯穆利斯)n1972年获得加州大学伯克利分校生物化学博士学位年获得加州大学伯克利分校生物化学博士学位n1979年进入塞特斯公司：与年进入塞特斯公司：与DNA合成相关的工作合成相关的工作n1983年年4月：月：PCR的最初想法的最初想法n高速公路的灵感高速公路的灵感n230=10亿亿n想法付诸行动想法付诸行动n1983年年12月：第一个月：第一个PCR反应成功反应成功n1984年塞特斯公司申请年塞特斯公司申请PCR技术专利技术专利90nPCR技术引来的官司技术引来的官司n谁发明了谁发明了PCR？DNA聚合酶的发现者？聚合酶的发现者？n纸上谈兵纸上谈兵nKary Mullis (卡里卡里.穆利斯穆利斯)胜诉胜诉91Kary Mullis won the 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction 心灵的裸舞心灵的裸舞-自传自传9293Topic 2: Protein Techniques1.Protein purification (蛋白质纯化蛋白质纯化) 2.Affinity chromatography can facilitate more rapid protein purification (亲和层析亲和层析纯化纯化)3.Protein separation by PAGE gel electrophoresis (蛋白质分离蛋白质分离) and identification by Western analysis(免疫免疫印迹印迹)94nThe purification of individual proteins is critical to understanding their functionnAlthough there are thousands of proteins in a single cell, each protein has unique properties, such as size, charge (电荷电荷), shape, and in many instance, function, that make its purification somewhat different from others1. Protein purification (蛋白质纯化蛋白质纯化)95nPurification of a protein requires a specific assay (特殊的分析方法特殊的分析方法)to allow you to monitor your purification status, which include a measure of the function of the protein, use of the antibody of the protein.96Column chromatography(柱层析柱层析) is an efficient way to purify proteins In this approach, protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads.97Ion exchange chromatography(离离子交换层析子交换层析)nThe proteins are separated according to their surface charge.nThe beads are modified with either negative-charged or positive-charged chemical groups.nProteins bind more strongly requires more salt to be eluted. 98Fig 20-22-a99Gel filtration chromatography(凝胶过滤层析凝胶过滤层析) nThis technique separate the proteins on the bases of size and shape. size and shape. nThe beads for it have a variety of different sized pores throughout. nSmall proteins can enter all of the pores, and take longer to elute; but large proteins pass quickly.100Fig 20-22-b1012.Affinity chromatography(亲和层析亲和层析)can facilitate more rapid protein purification102Immunoaffinity chromatography(免疫亲和层析免疫亲和层析)nAn antibody that is specific for the target is attached to the bead, and ideally only the target protein can bind to the column.nDisadvantage: sometimes the binding is too tight to elute our target protein.103nSometimes tags (eg. His tag) can be added to the N- or C- terminal of the target protein, using DNA cloning method, to make the fusion protein (融合蛋白）融合蛋白）. 104Affinity chromatographyAntibody-antigen bindingNi2+-His tag-fusion protein binding (镍柱）镍柱）1053. Protein separation by PAGE gel electrophoresis, followed by a western analysisnThe native proteinsnative proteins have neither a uniform charge nor a uniform secondary structure.nIf we treat the protein with a strong detergent SDSSDS, the higher structure is usually eliminated. nSDS confers the polypeptide chain a uniform negative charge.nSometimes, mercaptoethanolmercaptoethanol (巯基乙醇) is need to break the disulphide bond.106nThus, the protein molecules can be resolved by electrophoresiselectrophoresis in the presence of SDS according to the length of individual polypeptide-SDS-PAGEnAfter electrophoresis, the proteins can be visualized with a stainstain, such as Coomassie brilliant blue (考马氏亮蓝考马氏亮蓝).nProteins from the PAGE gel can be transferred to a membrane, followed by a western analysiswestern analysis of the target protein by a corresponding antibodyantibody.107A protein gel stained by Coomassie Blue 108Topic 3: Study the interaction between protein and nucleic acid109 （一）DNA-protein affinity chromatography (DNA-蛋白亲和层析蛋白亲和层析) Affinity chromatographyDNA-protein binding110nDynalbeads M-280链亲和素磁珠链亲和素磁珠 (Invitrogen公公司司)n生物素标记生物素标记DNA (labeled specific DNA sequence)n生物素标记生物素标记DNA结合到链亲和素磁珠上结合到链亲和素磁珠上nDNA-protein binding and elution of binding proteinsnSDS-PAGE纯化及蛋白质测序纯化及蛋白质测序:MS analysis, identify the protein, clone the gene coding for this protein111( (二）二）Gel retardation (凝胶阻凝胶阻 滞滞,EMSA)Confirmation of the specific interactionbetween isolated protein and regulatory DNA elementElectrophoretic Mobility Shift Assay, EMSA（电泳迁移率变动分析）（电泳迁移率变动分析） 112DNA bound totwo proteinsDNA-proteincomplexBare DNAA DNA bound with more than one protein to form a larger complex113nA short labeled nucleic acid (regulatory element) is mixed with the predicted binding proteinnSamples of labeled nucleic acid, with and without being incubated with the protein, are run on a gelnThe DNA-protein complexes are shown by the presence of slowly migrating bands114Bare DNADNA-proteincomplexgel electrophoresis放射自显影放射自显影: 检测检测DNA-protein复合物复合物标记特异标记特异DNA未标记特异未标记特异DNAprotein随机随机DNA序列序列竞争性竞争性EMSA证明证明DNA和某种蛋白质相互作用的特异性和某种蛋白质相互作用的特异性115菠菜叶绿体蛋白质菠菜叶绿体蛋白质与高粱与高粱psbApsbA启动子启动子DNADNA的竞争结合实的竞争结合实验：验：A AEE未标记的未标记的竞争竞争DNADNA量分别是量分别是标记标记DNADNA探针量的探针量的1 1，5 5，1010，3030，5050倍。倍。 竞争性竞争性EMSA116在离体条件下，菠菜叶绿在离体条件下，菠菜叶绿体蛋白与高粱体蛋白与高粱psbApsbA基因启基因启动子动子“-35”“-35”元件元件( (TTGACATTGACA) ) 及其突变体及其突变体( (ATTACTATTACT) )的结的结合效应合效应 A A自由自由DNADNA探针；探针；B BFF逐步增加非标记野逐步增加非标记野生型生型DNADNA探针量而保持菠菜探针量而保持菠菜叶绿体蛋白提取物量不变；叶绿体蛋白提取物量不变；G G以突变子以突变子ATTACAATTACA作为竞作为竞争性争性DNADNA探针，蛋白质的量探针，蛋白质的量同同F FEMSA 检测菠菜叶绿体蛋白与高粱检测菠菜叶绿体蛋白与高粱psbA基因启动子基因启动子“-35”元件元件(TTGACA)之间的相互作用之间的相互作用117Identification of the precise nucleotides withinthe regulatory element which can interact withregulatory protein(精确定位调控序列中与调控蛋白特异性作用的核苷酸精确定位调控序列中与调控蛋白特异性作用的核苷酸): Methods: DNase I footprinting (足迹法足迹法)118DNase I footprinting (DNase I 足迹法足迹法) Identify the actual region of sequence with which the protein interactsAATAAG5*Footprinting(足迹足迹)proteinLabeled DNAThe binding-protein protects DNA from attack by DNase 119+protein-protein+protein-proteinDNase IDNase I1201: 0 pmol protein2: 10 pmol protein3: 18 pmol protein4: 90 pmol protein121015ProteinThe three lanes represent DNA that was bound to 0, 1, and 5 units of protein. The lane with no protein shows a regular ladder of fragments. The lane with one unit shows some protection, The lane with 5 units shows complete protection in the middle.By including sequencing ladders, we can tell exactly where the protein bound.TCGGAGCAACGCAAACAAACGTGCTTGG122Topic 4: Techniques for studying protein-protein interaction123Yeast two-hybrid system(酵母双杂交酵母双杂交)nFinding other proteins which can interact with one proteinnStudying protein-protein interaction124n真核生物的转录需要真核生物的转录需要转录激活因子转录激活因子的参与的参与: 这些转录因子通常由一个这些转录因子通常由一个DNA特异性结合功能特异性结合功能域和一个或多个与其他调控蛋白相互作用的激活域和一个或多个与其他调控蛋白相互作用的激活功能域组成，即功能域组成，即DNA 结合结构域结合结构域(DNA-binding domain, BD)和转录激活结构域和转录激活结构域(activation domain, AD)n转录激活因子的组件式结构特征转录激活因子的组件式结构特征(modular)125Eukaryotic activators-Gal4 in yeastGal4 is the most studied eukaryotic activatorGal4 activates transcription of the galactose genes (eg.GAL1) in the yeast S. cerevisae.Gal4 binds to four sites (UASG) upstream of GAL1, and activates transcription 1,000-fold in the presence of galactose126Gal4 bound to its site on DNA127No transcriptiontranscription报告基因报告基因128targetbait No transcriptiontargetbait 报告基因报告基因129lacZ encode enzyme bb-galactosidase（bb-半乳糖苷酶）半乳糖苷酶） X-gal(substrate of the enzyme)Blue productReporter gene: lacZ 130131Fishing with the yeast two-hybrid systembait targetdoes X bindwith a unknownprotein?protein XbaitBDADBDBDcDNA library （ cDNA 文库）文库）+AD fusion plasmid transform132酵母双杂交技术的应用：酵母双杂交技术的应用：1）已知蛋白之间相互作用的检测已知蛋白之间相互作用的检测2)获获得得和和已已知知蛋蛋白白相相互互作作用用的的新新蛋蛋白白：将将感感兴兴趣趣的的蛋蛋白白质质基基因因与与BD基基因因构构建建成成“诱诱饵饵”表表达达质质粒粒，将将某某一一器器官官或或组组织织的的cDNA文文库库与与AD基基因因构构建建成成“猎猎物物”基基因因库库，共共转转化化酵酵母母细细胞胞，可可筛筛到到与与感感兴兴趣趣蛋蛋白白质质相相互互作作用用的的蛋蛋白白质的质的cDNA序列，并推测其蛋白质序列序列，并推测其蛋白质序列3）蛋蛋白白质质的的相相互互作作用用功功能能域域研研究究：通通过过对对其其中中某某一一个个蛋蛋白白质质的的氨氨基基酸酸序序列列作作缺缺失失或或定定点点突突变变，再再用用此此系系统统检检测测是是否还存在相互作用，可阐明其功能域或关键氨基酸否还存在相互作用，可阐明其功能域或关键氨基酸133免疫共沉淀技术研究蛋白质蛋白质相互作用免疫共沉淀技术研究蛋白质蛋白质相互作用n当细胞在非变性条件下被裂解时，完整细当细胞在非变性条件下被裂解时，完整细胞内存在的许多蛋白质蛋白质间的相互胞内存在的许多蛋白质蛋白质间的相互作用被保留了下来作用被保留了下来n如果用蛋白质如果用蛋白质X的抗体免疫沉淀的抗体免疫沉淀X，那么与，那么与X在体内结合的蛋白质在体内结合的蛋白质Y也能沉淀下来也能沉淀下来n常用于测定两种目标蛋白质是否在体内结常用于测定两种目标蛋白质是否在体内结合，也可用于确定一种特定蛋白质的新的合，也可用于确定一种特定蛋白质的新的作用搭档作用搭档134Yeast one-hybrid system(酵母单杂交酵母单杂交)n酵母单杂交酵母单杂交(yeast one-hybrid)技术是技术是在酵母细胞内在酵母细胞内分析分析DNA与蛋白质相互作用的一种方法，通过对酵母细与蛋白质相互作用的一种方法，通过对酵母细胞内报告基因表达状况的分析，来鉴别胞内报告基因表达状况的分析，来鉴别DNA结合位点并结合位点并发现潜在的结合蛋白及其编码基因发现潜在的结合蛋白及其编码基因n运用此技术，能筛选到与特定运用此技术，能筛选到与特定DNA 序列结合的蛋白质，序列结合的蛋白质，并可直接从基因文库中得到编码该蛋白质的核苷酸序列，并可直接从基因文库中得到编码该蛋白质的核苷酸序列，而无需复杂的蛋白质分离纯化操作而无需复杂的蛋白质分离纯化操作 n酵母属真核细胞，通过酵母系统得到的结果比其他酵母属真核细胞，通过酵母系统得到的结果比其他体外体外技术技术获得的结果更能体现真核细胞内基因表达调控的真获得的结果更能体现真核细胞内基因表达调控的真实情况实情况135Principle:n真核生物的转录起始需要转录因子的参与真核生物的转录起始需要转录因子的参与.这些转录因子这些转录因子通常由一个通常由一个DNA特异性结合功能域和一个或多个与其他特异性结合功能域和一个或多个与其他调控蛋白相互作用的激活功能域组成，即调控蛋白相互作用的激活功能域组成，即DNA 结合结构结合结构域域(DNA-binding domain, BD)和转录激活结构域和转录激活结构域(activation domain, AD).nGAL4: 酵母酵母转录因子转录因子n酵母单杂交系统由酵母单杂交系统由2 部分组成：部分组成：(1)将待筛选文库蛋白将待筛选文库蛋白 片段与片段与GAL4 转录激活结构域转录激活结构域融合表达的融合表达的cDNA文库质文库质粒；粒；(2)含有已知含有已知DNA序列和下游报告基因的报告质粒序列和下游报告基因的报告质粒.136Libraries of DNA molecules can be created by cloning (Genomic library and cDNA library)A DNA libraryA DNA library (DNA文库文库) is a population of identical vectors that each contains a different DNA insert. Genomic Library Genomic Library (基因组文库基因组文库) : the DNA inserts in a DNA library is derived from restriction digestion of the genomic DNA. cDNAcDNA library library (cDNA文库文库) : the DNA inserts in a DNA library is converted from the mRNAs of a tissue, a cell type or an organism. cDNA stands for the DNA copied from mRNA. 137Digestion of the genomic DNA byrestriction enzymesGenomic Library Genomic Library (基因组文库基因组文库)138cDNAcDNA library library (cDNA文库文库)逆转录酶逆转录酶139ADactivation domain, AD (转录激活结构域转录激活结构域)Pmin: minimal promotor待筛选蛋白待筛选蛋白cDNA+GAL4 AD140Reporter gene: His3, LacZDRE: regulatory element141含有双重报道子的酵母转化子在含有双重报道子的酵母转化子在SD/His - , Ura - ,Leu- 平板平板上的生长情况上的生长情况阳性克隆的鉴定阳性克隆的鉴定142Nucleic acids techniques:Electrophoresis; Restriction digestion; PCR amplification; hybridazation, DNA cloning and gene expressionProtein techniques:Protein purification; affinity chromatography; Protein separation and identification by western blotStudy the interaction between protein and nucleic acid:Gel retardation (EMSA), DNA footprintingStudy the interaction between protein and protein: Yeast two-hybrid system, 免疫共沉淀技术免疫共沉淀技术Summary143