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1、主讲主讲: 张志军张志军 博士、博导、副教授博士、博导、副教授埃博拉病毒p 截至2014年8月26日,埃博拉已在塞拉利昂、利比亚和几内亚造成1427人死亡。 p 2014年8月9日,中国宣布已掌握埃博拉病毒抗体基因,同时具备对埃博拉病毒进行及时检测的诊断试剂研发能力,这让世界为之惊喜。PC Turner et al. Instant Notes in Molecular Biology (Second edition). BIOS Scientific Publishers Limited, 2000分子生物学分子生物学(第三版第三版)导读版导读版(精要速览系列精要速览系列)朱玉贤,李毅,郑晓
2、峰编著。现代分子生物学(第朱玉贤,李毅,郑晓峰编著。现代分子生物学(第3版)版) 高等教育出版高等教育出版社社 2007-11-1 Robert F. Weaver Molecular biology 2004. (Third edition) 专心听讲 做好笔记 认真思考 学会归纳 注意纪律 预习复习章节名称章节名称理论(学时)理论(学时)实验(学时)实验(学时)1 Cells and macromolecules22 Nucleic acid and Chromosome43 DNA replication64 DNA damage, repair and recombination65
3、Transcription146 Protein synthesis87 Viruses28 Tumor and oncogenes29 Technology of molecular biology416合计合计48161.1 Cellular classification1.2 Subcellular organelles1.3 Macromolecules1.4 Large macromolecular assemblies Prokaryotes: Eubacteria (真细菌真细菌) Acchaea(古细菌古细菌) Eukaryotes: Plants Animals Fungi
4、Protist(原生生物原生生物)Prokaryotes and eukaryotesSchematic of a eukaryotic cell Schematic of a prokaryotic cell Differentiation (分化分化) Nuclei nucleoli(核仁)(核仁), nucleus Mitochondria and Chloroplasts Endoplasmic reticulum Microbodies Organelle isolation Proteins Nucleic acids Polysaccharides Lipids Complex
5、macromolecules Proteins polymers of amino acids, peptide bonds Nucleic acids DNA and RNA, polymers of nucleotides, mRNA, tRNA, rRNA Polysaccharides Polymers of simple sugars, glycoside bonds They functions mainly as nutritional sugar stores and as structural material. Cellulose, starch, chitin, muco
6、polysaccharides(黏多糖黏多糖) Lipids Key components of membranes, protective coats and other structures. Glycerides (甘油酯甘油酯), phospholipids(磷脂磷脂),sphingolipids(鞘脂,如神经氨酰)(鞘脂,如神经氨酰) Complex macromolecules Nucleoproteins(核蛋白核蛋白), ribozyme(核糖体)(核糖体) Glycoproteins Proteoglycans (mucoproteins)蛋白多糖(粘蛋白)蛋白多糖(粘蛋白)
7、 Lipid-linked proteins glycolipids Protein complexes Cytoskeleton: microtubules, microfilaments, intermediate filaments These organize the shape and movement of cells and subcellular organelles. Cilia and flagella (纤毛和鞭毛纤毛和鞭毛 ) are also composed of microtubules complexed with dynein and nexin(动动力蛋白和
8、连接蛋白力蛋白和连接蛋白 ). Nucleoproteins Bacterial 70S ribosomes: 50S larger subunit (23S and 5S RNA + 31 proteins)+30S small subunit (16S RNA + 21 proteins) Eukayotic 80S ribosomes: 60S larger subunit (28S, 5.8S and 5S RNAs + 31 proteins)+40S small subunits (18S RNA) Chromotin contains DNA and the basic hist
9、one proteins. Membranes Membrane phospholipids and sphingolipids (鞘脂鞘脂) form bilayers. Membrane proteins may be peripheral (外周的外周的) or integral (整合的)(整合的)and act as receptors, enzymes, transporters or mediators of cellular interaction. Noncovalent interaction (非共价互作非共价互作) Charge-charge (电荷电荷) intera
10、ctions Charge-dipole(偶极偶极) and dipole-dipole interaction Hydrogen bonds 氢键氢键 Hydrophobic interaction 疏水相互作用疏水相互作用试述原核和真核细胞的异同点。试述原核和真核细胞的异同点。亚细胞器主要有哪些?各自主要结构和功能是亚细胞器主要有哪些?各自主要结构和功能是什么?什么?生物大分子包括哪些?分别叙述其组成结构。生物大分子包括哪些?分别叙述其组成结构。试举出三种大分子的组装形式。试举出三种大分子的组装形式。2.1 Properties of nucleic acid2.2 Prokaryotic
11、 and Eukaryotic chromosome structure2.1.1 Nucleic acid structure2.1.2 Chemical & physical properties2.1.3 Spectroscopic (光谱学光谱学) & thermal (热力学)(热力学) properties2.1.4 DNA supercoiling2.1.1 Nucleic acid structuren Basesn Nucleosides(核苷核苷) base+sugarn Nucleotides (核苷酸核苷酸) base+sugar+phosphaten
12、Phosphodiester bonds n Bases DNA: adenine(A), guanine(G), cytosine(C), thymine(T)RNA: adenine(A), guanine(G), cytosine(C), uracil(U)n DNA/RNA sequence pConventionally, DNA or RNA are represented by their single letter A, T, G, C or U. T, G, C or U.pWrite the sequences with 5-end at the left. DNA: 5-
13、ATAAGCTC-3 or ATAAGCTC RNA: 5-AUAGCUUGA-3 direction: ATAAG is not the same as GAATAn DNA double helixlBase pairinglComplementary base pairslAntiparallellminor groove; major groovelright-handed (clockwise)lB-formlOverwound; underwound The B-form of DNA is a double helix consisting of two polynucleoti
14、de chains that run antiparallel. The nitrogenous bases of each chain are flat purine or pyrimidine rings that face inwards and pair with one another by hydrogen bonding to form A-T or G-C pairs only. The diameter of the double helix is 20 , and there is a complete turn every 34 , with 10 base pairs
15、per turn. The double helix forms a major (wide) groove and a minor (narrow) groove.Key Conceptsn A, B and Z helices Z ZDNADNA:它是左手双螺旋,与右手螺旋的不同是螺距延长:它是左手双螺旋,与右手螺旋的不同是螺距延长(4.5nm(4.5nm左右左右) ),直径变窄,直径变窄(1.8nm)(1.8nm),每个螺旋含,每个螺旋含1212个碱基对,个碱基对,分子长链中磷原子不是平滑延伸而是锯齿形排列,有如分子长链中磷原子不是平滑延伸而是锯齿形排列,有如“之之”字形一样,因此叫它字
16、形一样,因此叫它Z Z构象,这一构象中的重复单位是二核构象,这一构象中的重复单位是二核苷酸而不是单核苷酸;而且苷酸而不是单核苷酸;而且Z ZDNADNA只有一个螺旋沟,它相当只有一个螺旋沟,它相当于于B B构象中的小沟,它狭而深,大沟则不复存在。构象中的小沟,它狭而深,大沟则不复存在。 进一步的分析还证明,进一步的分析还证明,Z ZDNADNA的形成是的形成是DNADNA单链上出现嘌呤单链上出现嘌呤与嘧啶交替排列所成的。比如与嘧啶交替排列所成的。比如CGCGCGCGCGCGCGCG或者或者CACACACACACACACA。双螺旋 碱基倾碱基夹碱基间距螺距 每轮碱 小沟宽nm大沟宽nm 角()角
17、()/nmnm基数小沟宽nm 大沟宽nmB-DNA036.00.3373.4100.570.751.170.85C-DNA638.00.3313.19.3 0.480.791.050.75D-DNA 45.00.303 0.130.67 0.890.58A-DAN20 32.70.2562.8111.100.28 0.271.35总之,DNA的双螺旋结构永远处于动态平衡中,DNA分子构象的变化与糖基和碱基之间空间相对位置有关。2.1.2 Chemical & physical properties of nucleic acidn Stability of nucleic acids
18、lHydrogen bondinglHydrophobic interactions and dipole-dipole interactionsn Effect of acid lStrong acid -hydrolyzed completely, HClO4, 100lModerate acid-the most easily hydrolyzed bonds are selectively broken, hydrolysis of the purine base glycosylic bonds-apurinic(脱嘌呤脱嘌呤).lMore complex chemistry has
19、 been developed which removes bases specially, and cleaves the DNA or RNA at particular bases.n Effect of alkalilTo change the tautomeric(互变异构的互变异构的) state of the bases.n Chemical denaturation lurea(尿素尿素) and formamide(甲酰胺)(甲酰胺)n Viscosity(粘性)(粘性)l Cellular DNA is very long and thin, solutions have
20、a high viscosity.lLong DNA molecules are easy be damaged by shearing forces or by sonicationn Buoyant density (浮力密度浮力密度)l8M CsCl, 1.7g cm-3lEquilibrium density gradient centrifugation of DNACsCl 密度梯度离心分离密度梯度离心分离DNA l 离心分离密度大于离心分离密度大于1.3g/cm31.3g/cm3的的样品,如样品,如DNADNA、RNARNA,需要使用,需要使用密度比蔗糖和甘油大的介质。密度比蔗糖
21、和甘油大的介质。l 重金属盐氯化铯重金属盐氯化铯(CsCl)(CsCl)是目前是目前使用的最好的离心介质。使用的最好的离心介质。 离心分离细胞组分和生物分子是最常用的分离方法,因为不同的细胞离心分离细胞组分和生物分子是最常用的分离方法,因为不同的细胞器和分子有不同的体积和密度,可在不同离心力的作用下沉降分离。器和分子有不同的体积和密度,可在不同离心力的作用下沉降分离。 不同的细胞器、大分子和病毒的密度及相应的沉降系数不同的细胞器、大分子和病毒的密度及相应的沉降系数 2.1.3 Spectroscopic & thermal propertiesn UV absorption max=2
22、60nm (both DNA and RNA)n Hypochromicity (减色性减色性) lThe extinction coefficient (消光系数消光系数) depends on environment. labsorbance: isolated nucleotides RNA, ss DNA ds DNAlHypochromicity. dsDNA is hypochromicitic with respect to ss DNAn Quantitation(定量)(定量) of nucleic acidslabsorbance of 260 nm. 1mg/ml, 1c
23、m pathlength, dsDNA A260=20; RNA, ss DNA A26025; depended on base composition and secondary structure.n Purity of DNApurity of DNA may be estimated by A260/A280 lA260/A280 =1.8, pure DNA; l A260/A280 1.8, RNA contamination; lA260/A280 AnPromoter efficiencyl-35 sequence- recognition region which enha
24、nces recognition and interaction with the polymerase factor;l-10 region - is important for DNA unwinding;lThe sequence around the start site influences initiation.补充:补充:Promoter 启动子启动子启动子启动子是是RNARNA聚合酶识别、结合和开始转录的一段聚合酶识别、结合和开始转录的一段DNADNA序列,序列,含有含有RNARNA聚合酶特异性结合和转录起始所需保守序列。聚合酶特异性结合和转录起始所需保守序列。转录起点转录起点
25、是指与新生是指与新生RNARNA链第一个核苷酸相对应链第一个核苷酸相对应DNADNA链上的链上的碱基,研究证实通常为一个嘌呤。碱基,研究证实通常为一个嘌呤。启动子一般位于转录起始位点启动子一般位于转录起始位点+1+1的上游。(上下游表示)的上游。(上下游表示)从起点上游约从起点上游约-10-10处找到处找到6bp6bp的保守序列的保守序列TATAATTATAAT,称为,称为PribnowPribnow区(区(boxbox),或称),或称-10 sequence-10 sequence,是转录解旋区。,是转录解旋区。-35-35位置位置- - 保守序列保守序列TTGACATTGACA,称为,称为
26、-35sequence-35sequence或识别区。或识别区。-35-35序列提供了序列提供了RNARNA聚合酶识别的信号,聚合酶识别的信号,-10-10序列则有助于序列则有助于DNADNA局部双链解开。局部双链解开。a.a.-10-10位的位的TATATATA区和区和-35-35位位TTGACATTGACA区是区是RNARNA聚合酶与启动子的聚合酶与启动子的结合位点,能与结合位点,能与因子相互识别而具有很高的亲和力。因子相互识别而具有很高的亲和力。5.1.4 Initiation, Elongation and TerminationpPromoter bindingpDNA unwind
27、ingpRNA chain initiationpRNA chain elongationpRNA chain termination pRho-dependent terminationnPromoter bindinglThe factor enhances the specificity of the core RNA polymerase for promoter binding.lThe polymerase finds the promoter -35 and -10 sequences by sliding along the DNA and forming a closed c
28、omplex (封闭复合物封闭复合物) with the promoter.nDNA unwindinglAround 17 bp of the DNA is unwound by the polymerase, forming an open complex.lDNA unwinding at many promoters is enhanced by negative DNA supercoiling. lHowever, the promoters of the genes for DNA gyrase (旋旋转酶转酶) subunits are repressed by negativ
29、e supercoiling. nRNA chain initiationlWithout a primer. lStart with a GTP or ATPlThe first 9 nt is added without enzyme. After each one of these 9 nt is added to the chain, there is a significant probability that the chain will be aborted. lAbortive initiation is important for the overall rate of tr
30、anscription.nRNA chain elongationlTernary complex (三元复合体三元复合体). Polymerase + DNA + nascent (初生的初生的) RNAlThe transcription bubble -the region of unwound DNA. lThe polymerase moves at 40 nt per sec. lRNA-DNA helix nRNA chain terminationlTranscription proceeds until it reaches a terminator sequence (st
31、op signal)-RNA hairpin.lRNA hairpin - self-complementary a stem (G C) and a loop4 or more U residuesnRho-dependent terminationlRho protein ()- mediate transcription启动子选择阶段RNA聚合酶与启动子可逆性结合形成封闭复合物 封闭复合物 DNA解链开放复合物 开放复合物 RNA聚合酶、DNA、新生RNA三元复合物n转录的终止及其终止因子转录的终止及其终止因子 (termination factor)(termination facto
32、r)1 1)定义)定义能提供转录终止信号的能提供转录终止信号的DNADNA序列称为序列称为终止子终止子,协助,协助RNARNA聚合聚合酶识别终止信号的蛋白质因子则称为酶识别终止信号的蛋白质因子则称为终止因子终止因子。有些终止。有些终止子的作用可被特异的因子阻止,使聚合酶能越过终止子继子的作用可被特异的因子阻止,使聚合酶能越过终止子继续转录,称为续转录,称为通读通读(read(readthrough)through),这类引起抗终止作这类引起抗终止作用的蛋白称为用的蛋白称为抗终止因子抗终止因子。大肠杆菌有两类终止子:大肠杆菌有两类终止子:不依赖于不依赖于rho(rho() )因子的终止子因子的终
33、止子;依赖于依赖于因子的终止子因子的终止子。)不依赖于)不依赖于因子的终止因子的终止终止位点上游一般存在一个富含终止位点上游一般存在一个富含GCGC碱基的二重对称区,碱基的二重对称区,由这段由这段DNADNA组成的组成的RNARNA容易形成容易形成发卡式结构发卡式结构(hairpin)(hairpin)。在。在终止位点前面有一段由终止位点前面有一段由4-84-8个个A A组成的序列,所以转录产组成的序列,所以转录产物的物的33端为寡聚端为寡聚U U,这种结构特征的存在决定了转录的终,这种结构特征的存在决定了转录的终止。止。在新生在新生RNARNA中发现发卡式结构会导致中发现发卡式结构会导致RN
34、ARNA聚合酶暂停,破聚合酶暂停,破坏坏RNA-DNARNA-DNA杂合链杂合链5 5端的正常结构。寡聚端的正常结构。寡聚U U的存在使杂合链的存在使杂合链3 3端出现不稳定的区域,使端出现不稳定的区域,使RNARNA从三元复合物中解离出来。从三元复合物中解离出来。终止效率与二重对称区序列和寡聚终止效率与二重对称区序列和寡聚U U的长短有关,随着发的长短有关,随着发卡式结构(至少卡式结构(至少6bp6bp)和寡聚)和寡聚U U 序列(至少序列(至少4 4个个U U)长度的)长度的增加,终止效率逐步提高。增加,终止效率逐步提高。RNA产物具有一个发夹结构和产物具有一个发夹结构和一段富含一段富含U
35、的片段的片段)依赖于)依赖于因子的终止因子的终止 因子因子:相对分子量为:相对分子量为46 00046 000的六聚体蛋白,能水解核的六聚体蛋白,能水解核苷酸三磷酸,是一种苷酸三磷酸,是一种NTPNTP酶,通过催化酶,通过催化NTPNTP水解促使新生水解促使新生RNARNA链从三元转录复合物中解离出来,从而终止。链从三元转录复合物中解离出来,从而终止。一般认为一般认为RNARNA合成之后,合成之后,因子即附着在新生的因子即附着在新生的RNARNA链上,链上,靠靠ATPATP水解产生的能量,沿着水解产生的能量,沿着5353方向朝方向朝RNARNA聚合酶移聚合酶移动,到达动,到达RNARNA的的3
36、-OH3-OH端后取代了暂停在终止位点上的端后取代了暂停在终止位点上的RNARNA聚合酶,使之从模板聚合酶,使之从模板DNADNA上释放上释放mRNAmRNA,完成转录过程。,完成转录过程。)抗终止)抗终止抗转录终止主要有两种形式:抗转录终止主要有两种形式:破坏终止位点破坏终止位点RNARNA的茎的茎- -环结构。环结构。介质中某种氨基酸浓度介质中某种氨基酸浓度低时,缺乏相应的氨酰基低时,缺乏相应的氨酰基tRNAtRNA,致使核糖体滞留在串联,致使核糖体滞留在串联密码子上,密码子上,mRNAmRNA不能形成特定的茎不能形成特定的茎- -环结构,出现转录抗环结构,出现转录抗终止现象。终止现象。依
37、赖于蛋白质因子的转录抗终止。依赖于蛋白质因子的转录抗终止。抗转录终止蛋白与抗转录终止蛋白与DNADNA相结合;抗转录终止蛋白与相结合;抗转录终止蛋白与RNARNA聚合酶相结合,并使后者聚合酶相结合,并使后者对转录终止信号不敏感,转录继续进行。对转录终止信号不敏感,转录继续进行。Promoters; transcription complex;terminator; termination factor; read through; Rho-dependent terminationThe E. coli RNA polymerase 的组成及各部分功的组成及各部分功能。能。启动子启动子-10区
38、及区及-35区功能。区功能。叙述原核生物转录过程。叙述原核生物转录过程。RNA hairpin 结构特点。结构特点。5.1 Transcription in prokaryotes5.2 Regulation of transcription in prokaryotes5.3 Transcription in eukaryotes5.4 Regulation of transcription in eukaryotes 5.5 rRNA process and RNPs 5.2.1 The lac operon5.2.2 The trp operon5.2.1 The lac operonn
39、The operon model was proposed by Jacob and Monod in 1961The operon is a unit of gene expression and regulation, includes: lThe structural genes for enzymes involved in a specific biosynthetic pathway whose expression is coordinately (协协调调) controlled.lControl elements such as an operator sequence, w
40、hich is a DNA seq that regulate transcription of the structural genes.lRegulator genes whose products recognize(识别)(识别) the control elements. nThe lactose operonlE. coli can use lactose as a source of carbon. The enzymes required for the use of lactose as a carbon source are only synthesized when la
41、tose is available as the sole carbon source.p3 structure genes: lacZ, lacY, lacAllacZ -galactosidase, hydrolysis of lactose to galactose and glucosellacY galactoside permease, lactose transport across the bacterial cell wallllacA thiogalactoside transacetylase (硫代半乳糖苷转乙酰基酶硫代半乳糖苷转乙酰基酶)pPromoter Plac
42、; pPolycistronic (多顺反子多顺反子) mRNA; pOperator site Olac -5 - +20pLac InThe lac repressorpThe lac I gene encodes the lac repressor, which is active as a tetramer(四聚体四聚体) of identical(相同的相同的) subunits.pThe lac operator site consists of 28bp which is palindromic (回文结构的回文结构的). pIn the absence of lactose,
43、the repressor occupies the operator-binding site. The repressor actually increases the binding of the polymerase to the lac promoter.nInduction pIn the absence of inducer, the lac repressor blocks all but a very low level of transcription of lacZYA. pWhen lactose is added to cells, the low basal lev
44、el of the permease allows its uptake, and -galactosidase catalyzes the conversion of some lactose to allolactose (异乳糖异乳糖).pSynthetic inducer: IPTG(异丙基巯基半乳糖苷), TMG(巯甲基半乳糖苷), ONPG(O-硝基半乳糖苷)pAllolactose act as an inducer and binds to the lac repressor. This causes a change in the conformation of the re
45、pressor tetramer, reducing its affinity for the lac operator.pRemoval of the lac repressor from the operator site allows the polymerase to rapidly begin transcription of the lacZYA genes. Thus, the addition of lactose very rapidly stimulates transcription of the lactose operon structural genes.pThe
46、subsequent removal of the inducer leads to an almost immediate inhibition of this induced transcription, since the free lac repressor rapidly re-occupies the operator site and the RNA is extremely unstable.ncAMP receptor protein (CRP)pThe Plac is not a strong promoter.pCRP also be called CAP (catabo
47、lite activator protein)pGluctose is the first carbon source. Gluctose reduces the level of cAMP in the cell. pWhen glucose is absent, the levels of cAMP in E.coli increase and CRP binds to cAMP.cAMPp The CRP-cAMP complex binds to the lactose operon promoter just upstream from the site for RNA polyme
48、rase.p CRP binding is believed to enhance RNA polymerase binding to the promoter, enhancing transcription by 50-fold.CRP-cAMPCRP-cAMP与上游与上游DNADNA位位点结合后造成模板点结合后造成模板DNADNA沿对称序列中央发生沿对称序列中央发生大于大于90900 0的弯曲的弯曲3 3种操纵子上游启动区与种操纵子上游启动区与CRP-cAMPCRP-cAMP结合位点的相对位置分析结合位点的相对位置分析nThe tryptophan operon structureThe
49、 trp operon encodes five structural genes involved in tryptophan biosynthesis.Single promoter (Ptrp) and operator (Otrp)5.2.2 The trp operon 色氨酸操纵子色氨酸操纵子nThe trp repressorA gene product of the separate trpR operon, the trp repressor, specifically interacts with the operator site of the trp operon.Th
50、e core binding site is a palindrome of 18bp (-21 to +3) .The repressor is a dimer of two subunits.Tryptophan acts as a co-repressor and inhibits its own synthesis through end-product inhibition.The repressor reduces the transcription initiation by around 70-fold.nThe attenuator 弱化子弱化子Deletion of a s
51、equence between operator and trpE resulted the increase of transcription. A terminator sequence is present in the 162 bp trp leader before the start of the trpE-coding sequence. This structure is called the attenuator, it is a rho-independent terminator site which has a short GC-rich palindrome foll
52、owed by eight successive U residues.If this sequence is able to form a hairpin in the RNA transcript, then it acts as a highly efficient terminator and causes only a 140bp transcript is synthesized.nLeader RNA structureFour regions1:2, 3:4 attenuator hairpin2:3nThe leader peptideThe leader RNA seque
53、nce contains an efficient ribosome-binging site and can form a 14-amino-acid leader peptide.The 10th and 11th codons of this peptide encode successive tryptophan residues.Its function is to determine tryptophan availability (可利用可利用性性) and to regulate transcription termination.nAttenuation 弱化作用弱化作用Th
54、e availability of tryptophan is sensed through its being required in translation, and determines whether or not the terminator (3:4) hairpin forms in the mRNA.As transcription, the RNA polymerase pauses at the end of seq 2 until a ribosome begins to translate the leader peptide.High tryptophan, 3:4;
55、 attenuationTryptophan scarce, anti-terminator nImportance of attenuationThe presence of tryptophan gives rise to a 10-fold repression of trp operon transcription through the process of attenuation alone. So tryptophan levels exerts a 700-fold regulatory effect on expression from the trp operon.Atte
56、nuation occurs in at least six operons that encode enzymes concerned with amino acid biosynthesis.His operon -7 successive histidine codons, only mechanism of feedback control.总结补充:总结补充:色氨酸操纵子色氨酸操纵子2 2个水平的阻遏系统个水平的阻遏系统 色氨酸操纵子的阻遏系统色氨酸操纵子的阻遏系统是色氨酸合成途径的第一水平调是色氨酸合成途径的第一水平调控,它主要调控转录的控,它主要调控转录的启动与否启动与否。 色氨
57、酸操纵子的第二水平控制是色氨酸操纵子的色氨酸操纵子的第二水平控制是色氨酸操纵子的弱化系统弱化系统,它决定着它决定着已经启动的转录是否能够继续进行下去已经启动的转录是否能够继续进行下去。nThe The trptrp repressor repressor 色氨酸操纵子的阻遏系统色氨酸操纵子的阻遏系统TrpTrp 操纵子中产生阻遏物的基因是操纵子中产生阻遏物的基因是trpRtrpR,距,距trptrp基因簇很远。基因簇很远。trpRtrpR 基因突变常引起基因突变常引起trptrp mRNA mRNA的组成型合成,该基因产物的组成型合成,该基因产物被成为辅阻遏蛋白(被成为辅阻遏蛋白(aporep
58、ressor)。该辅阻遏蛋白与色)。该辅阻遏蛋白与色氨酸结合形成有活性的阻遏物,与操纵区结合并关闭氨酸结合形成有活性的阻遏物,与操纵区结合并关闭trptrp mRNAmRNA转录。转录。除非培养基中有色氨酸,否则这个辅阻遏蛋白不会与操纵除非培养基中有色氨酸,否则这个辅阻遏蛋白不会与操纵区结合。区结合。Trp操操纵纵区区的的碱碱基基序序列列这个系统这个系统效应物分子效应物分子是是色氨酸色氨酸。培养基中色氨酸过量时,。培养基中色氨酸过量时,它能与阻遏蛋白形成复合物,并结合到操纵基因上阻止结它能与阻遏蛋白形成复合物,并结合到操纵基因上阻止结构基因转录,当培养基中色氨酸供应不足时,阻遏物失去构基因转录
59、,当培养基中色氨酸供应不足时,阻遏物失去色氨酸并从操纵区上解离,色氨酸并从操纵区上解离,trptrp操纵子去阻遏。操纵子去阻遏。阻遏阻遏- -操纵机制对色氨酸来说是一个一级开关,主管转录操纵机制对色氨酸来说是一个一级开关,主管转录是否启动,相当于是否启动,相当于粗调开关粗调开关。trptrp操纵子中对应于色氨酸操纵子中对应于色氨酸生物合成的还有另一个系统进行生物合成的还有另一个系统进行细调控细调控,指示已经启动的,指示已经启动的转录是否继续下去。这个细微调控是通过转录达到第一个转录是否继续下去。这个细微调控是通过转录达到第一个结构基因之前的过早终止来实现的,由色氨酸的浓度来调结构基因之前的过早
60、终止来实现的,由色氨酸的浓度来调节这种过早终止的频率。节这种过早终止的频率。nThe The trptrp attenuator attenuator 色氨酸操纵子的弱化系统色氨酸操纵子的弱化系统l在色氨酸高浓度和低浓度下观察到在色氨酸高浓度和低浓度下观察到trptrp操纵子的表达水平操纵子的表达水平相差约相差约600600倍倍, ,而阻遏作用仅使转录作用降低而阻遏作用仅使转录作用降低7070倍。阻遏物倍。阻遏物失活突变不能完全消除色氨酸对失活突变不能完全消除色氨酸对trptrp操纵子表达的影响。操纵子表达的影响。(1) leader Peptide(前导肽前导肽)及其序列结构及其序列结构在色氨酸在色氨酸mRNA 5mRN
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