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分子生物学 The revised central dogma Gene regulation Gene regulation 基因组的保持基因组的保持 基因组的表达基因组的表达 The structure of DNA.

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Presentation on theme: "分子生物学 The revised central dogma Gene regulation Gene regulation 基因组的保持基因组的保持 基因组的表达基因组的表达 The structure of DNA."— Presentation transcript:

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2 分子生物学

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4 The revised central dogma Gene regulation Gene regulation 基因组的保持基因组的保持 基因组的表达基因组的表达 The structure of DNA

5 Section 1 A brief introduction of gene regulation Section 2 control of gene expression in Prokaryotes Section 3 control of gene expression in Eukaryotes Outline:

6 Key points: Gene expression, activator, repressor,positive gene regulation, negative gene regulation, signal integration, combinatorial Control, cis-acting element, trans-acting factor ( 基因表达、激活蛋白、 阻遏蛋白、正调控与负调控、信号整合、组合控制、 顺式作用元件、反式作用因子等概念; ) Positive and negative control of lac operon; Control mechanism of trp operon; Characteristics and levels of eukaryotic gene regulation, types of DNA binding domains of trans-acting factors ( 真核基因表达调控特点、层次 等,反式作用因子 DNA 结合结构域类型。 )

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8 7 Housekeeping genes ( 管家基因) : expressed constitutively, essential for basic processes involving in cell replication and growth. Inducible genes (可诱导基因) : expressed only when they are activated by inducers or cellular factors. Expression of many genes in cells are regulated

9 constitutive protein( 组成型蛋白质 ) : 1-1 cell proteins are divided into two categories: In cells, many proteins are the necessary enzymes or proteins in the metabolism, the number of these proteins is not almost affected by the environment or metabolic state. regulated or adaptive protein ( 调节型或 适应型蛋白质 ) : the synthesis rate of these proteins is significantly affected by the environment.

10 gene expression (基因表达): 1-2 gene regulation gene regulation or gene control (基 因表达调控) refers to a process in which the gene expression is regulated. refers to the whole process in which the genetic information carried by a gene is converted into a discernible phenotype, generally including two stages, transcription and translation.

11 Gene regulation occurs mainly in two levels 1. transcriptional regulation 转录水平上的调控 2. post- transcriptional regulation 转录后水 平上的调控 processing of RNA transcript mRNA 加工成熟水平上的调控 translation of mRNA 翻译水平上的调控

12 gene regulation is affected by the following factors ( 基因调控的指挥系统 ): In prokaryotes In eukaryotes nutritional status (营养状况) environmental factor (环境因素) hormone level (激素水平) developmental stage (发育阶段)

13 The process of gene expression in prokaryotes and eukaryotes

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15 2-1 theory of operon (操纵子学说): In 1961, Jacob and Monod put forward the theory of operon for the first time after studying on the regulation of lactose metabolism in E.coli. the winners of Nobel Prize in Physiology or Medicine in 1965.

16 Jacob and Monod Regulation of a gene usually occurs through transcriptional control. Operon theory states that genes that function together are often regulated together and are known as an operon.

17 a unit of prokarytoic gene expression and regulation which typically includes promoter, operator gene, one or more structural genes, which is controlled as a unit to produce messenger RNA (mRNA), and regulator gene whose products recognize and control the operator gene. 操纵子:是指细菌基因表达和调节的基本单位,其中含有启动子、操纵基因及 其所控制的一组功能上相关的结构基因,以及调节基因。操纵基因受调节基因 表达产物的控制。

18 structural gene is a gene that codes for any RNA or protein product other than a regulatory element (i.e. regulatory protein).generegulatory protein regulator gene or regulatory gene is a gene involved in controlling the expression of one or more other genes.geneexpression operator gene A gene that interacts with a specific repressor to control the functioning of the adjacent structural genes.

19 + Gene Expression is Controlled by Regulatory Proteins ( 调控蛋白 )  Positive regulators or activators (激活蛋白) INCREASE the transcription  Negative regulators or repressors ( 阻遏蛋白 ) DECREASE or ELIMINATE the transcription 2-2 the types and characters of gene regulation in prokaryotes

20 1 、 according to the response of operon to the Regulatory Proteins negative transcription regulation (负转录调控) positive transcription regulation (正转录调控) the products of regulator gene are repressors , decrease the transcription of structural gene. the products of regulator gene are activators , increase the transcription of structural gene. gene regulation include:

21 Regulator geneOperator gene Structural gene repressor acitivator positive transcription regulation If there are no regulatory proteins, the genes are turned off. otherwise, when there are regulatory proteins, the genes are turned on. negative transcription regulation

22 Regulator geneOperator gene Structural gene repressor acitivator positive transcription regulation If there are no regulatory proteins, the genes are turned on. otherwise, when there are regulatory proteins, the genes are turned off. negative transcription regulation

23 + Inducible regulation( 可诱导调节 ): under some specific metabolites or compound, some genes are turned on, namely that these genes are induced by these substances. For example: lac operon of E. coli. The products of the structural genes of these operon are generally involving in catabolism of carbohydrate. 2 、 according to the response of operon to some small molecules with regulatory role gene regulation include:

24 Regulator geneOperator gene Structural gene repressor Regulator gene Operator gene Structural gene repressor inducer mRNA enzyme The model of inducible operon Inducer (诱导 物): be able to induce the bacterium to produce the enzymes to catabolize it, the substance is named as inducer.

25 + Repressible regulation ( 可阻遏调节 ) : the genes are generally turned on. Only when some specific metabolites or compound are accumulated, the genes are turned off, namely that these genes are repressed by these substances. For example: trp operon of E. coli. The products of the structural genes of these operon are generally involving in Synthesis of some substances.

26 The medol of repressible operon Regulator gene Operator gene Structural gene mRNA enzyme Regulator gene Operator gene Structural gene corepressor corepressor (辅阻遏物): be able to repress the bacterium to produce the enzymes to synthesize it, the substance is named as corepressor.

27 3.negative transcription regulation include: 根据其作用特征又分为负控诱导和负控阻遏:

28 负控诱导系统 In this system, when the inducer binds to the repressor, the structural genes are transcribed.

29 负控阻遏系统 In this system, when the corepressor binds to the repressor, the structural genes are not transcribed.

30 4. positive transcription regulation include: 根据激活蛋白的作用分为正控诱导和正控阻 遏

31 正控诱导系统 In this system, when the inducer binds to the actiator, the structural genes are transcribed.

32 正控阻遏系统 In this system, when the corepressor binds to the activator, the structural genes are transcribed.

33 General rules: The inducible operon always encodes the proteins involved in catabolism of some carbohydrates. The levels of these carbohydrates are generally low in bacterium. Therefore, these operons are generally turned off. The repressible operon always encodes the proteins involved in synthesis of some small molecules necessary for the cell metabolism (Such as amino acids, purine and pyrimidine, and so on ). These operons are always turned on because of the importance of these small molecules. The operons are not turned off until these small molecules are enough in the environment.

34 5. The structure of the operator gene : inverted repeat (反向重复) or near repeat (毗邻重复)。

35 Domain of the regulatory protein binding to the operator gene (DNA-binding domain of protein) : 1 ) helix-turn-helix (螺旋 - 转角 - 螺旋, HTH ) In proteins, the helix-turn- helix is a major structural motif capable of binding DNA. It is composed of two α helices joined by a short strand of amino acids and is found in many proteins that regulate gene expression. It should not to be confused with the helix-loop-helix domain.proteinsstructural motifDNAα helicesamino acidsgene expressionhelix-loop-helix

36 In particular, recognition and binding to DNA is done by the two α helices, one occupying the N- terminal end of the motif, the other at the C-terminus. In most cases, the second helix contributes most to DNA recognition, and hence it is often called the "recognition helix". It binds to the major groove of DNA through a series of hydrogen bonds and various Van der Waals interactions with exposed bases. The other α helix stabilizes the interaction between protein and DNA, but does not play a particularly strong role in its recognition.N- terminalC-terminushydrogen bondsVan der Waals interactionsbases

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38 2 ). Zinc finger (锌指) Zinc finger A zinc finger is a large superfamily of protein domains that can bind to DNA. This domain is 30 amino acids long and consists of two antiparallel β strands, and an α helix. The domain also contains four regularly spaced ligands for Zinc (either histidines or cysteines). The Zn ion stabilizes the 3D structure of the domain. Each finger contains one Zn ion and recognizes a specific triplet of DNA basepairs.protein domainsDNAantiparallel β strandsα helix

39 Cartoon representation of the zinc finger motif of proteins, consisting of an α helix and an antiparallel β sheet. The zinc ion (green) is coordinated by two histidine residues and two cysteine residues.α helixβ sheetzinchistidine cysteine

40 Cartoon representation of the protein Zif268 (blue) containing three zinc fingers in complex with DNA (orange). The coordinating amino acid residues of the middle zinc ion (green) are highlighted.Zif268

41 3 ). 亮氨酸拉链( leucine zipper ) A leucine zipper, aka leucine scissors, is a super secondary structural motif found in proteins that creates adhesion forces in parallel alpha helices. It is a common dimerization domain found in some proteins involved in regulating gene expression. The basic leucine zipper (bZIP) domain contains an alpha helix with a leucine at every 7th amino acid. If two such helices find one another, the leucines can interact as the teeth in a zipper, allowing dimerization of two proteins. When binding to the DNA, basic amino acid residues bind to the sugar-phosphate backbone while the helices sit in the major grooves.structural motifproteinsparallelalpha helicesdimerizationgene expression

42 Leucine Zipper (blue) bound to DNA. The leucine residues that represent the 'teeth' of the zipper are colored red

43 Just how do proteins bind to DNA?

44 Gene regulation in prokaryotes mainly happens on the transcription level: 2-3 examples of gene regulation from prokaryotes : Regulation after transcription initiation 转录起始后的调控

45 First example: The Lac Operon ( 乳糖操纵子 ) First example: The Lac Operon ( 乳糖操纵子 ) Regulation of transcription initiation

46 The enzymes encoded by lacZ, lacY, lacA are required for the use of lactose as a carbon source. These genes are only transcribed at a high level when lactose is available as the sole carbon source. Composition of the Lac operon 1. Composition of the Lac operon Lactose operon contains 3 structural genes (结构基 因), operator gene (操纵基因), promoter (启动子), CAP site ( CAP 位点), and regulator gene (调节基 因).

47 lacY encodes a cell membrane protein called lactose permease ( 半乳糖苷透过酶 ) to transport Lactose across the cell wall lacZ codes for β-galactosidase ( 半乳 糖苷酶 ) for lactose hydrolysis lacA encodes a thiogalactoside transacetylase ( 硫代半乳糖苷转 乙酰酶 )to get rid of the toxic thiogalacosides

48 LacZYA transcription unit contains an operator site O lac position between bases -5 and +21 at the 3’-end of P lac Binds with the lac repressor

49 Inducer (诱导物) binds to the repressor tetramer is allostery  Specific binding force declines almost 1000 times The binding repressor  allostery  leaves the O site The free repressor operon on Lack of lactose : repessor tetramer binds to the operator gene operon off allostery  loses the ability binding to the O site 2. Negative regulation of lac operon: The regulaterory protein : lac repressor that is encoded by LacI gene; responses to the lactose. Lactose is present:

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51 Lac repressor binds as a tetramer ( 四聚体 ), 1 55kD Is allosteric protein , consists of DNA, protein, and inducer-binding domain. helix-turn-helix at N- terminal end ( 1-49aa )is DNA-binding domain.

52 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

53 Repressor binding physically prevents RNAP from binding to the promoter Repressor binding physically prevents RNAP from binding to the promoter, because the site bound by lac repressor is called the lac operator (O lac ), and the O lac overlaps promoter (P lac ). Namely repressor only plays a role at the stage of transcription initiation. How does Lac repressor repress the transcription of the lac operon?

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55 + In this system, the direct inducer is not lactose, but allolactose. Lactose is converted into allolactose in E.coli cells.

56 + IPTG ( Isopropyl-β-D-thio-galactoside, 异 丙基 - β –D- 硫代半乳糖苷 ) is a synthetic inducer, can rapidly simulate transcription of the lac operon structural genes. IPTG is used to induce the expression of the cloned gene from LacZ promoter in many vectors, such as pUC19. Isopropyl-β-D-thio-galactoside

57 Amp r ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位点) Lac promoter lacZ’ Gene X No IPTG, little expression of X gene With IPTG, efficient expression of X gene. L1: The LAC operon

58 3. positive regulation of lac operon:  In the lactose operon, even if the inducer inactivates the repressor, the normal expression of operon must have a positive control signal, the cAMP-CAP complex( cAMP-CAP 复合物 ).

59 ZYAOP DNA regulation region CAP site promoter Operator gene structural gene cAMP—CAP complex

60 ATP adenylate cyclase cAMP (环腺甘酸) In E. coli : lack of glucose, cAMP has a high level; when glucose is present, cAMP has a low level.  cAMP :

61  CAP ( catabolite gene activator protein , 代谢 降解物基因激活蛋白) / CRP (cAMP Receptor Protein, cAMP 受体蛋白 ); is a dimmer, 45kD , can form the complex with cAMP. The protein has not the effect on the trancription itself, only play a role when it binds together with cAMP.

62  cAMP-CAP complex : binds to a activator site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically interacting with RNAP. binds to a activator site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically interacting with RNAP. This cooperative binding stabilizes the binding of polymerase to P lac.

63 + activator site( 激活位点 ) : locate 60bp upstream the transcription initiation site, consensus sequence TGTGA.

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66 CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif One is the recognition helix that can fits into the major groove of the DNA. Another one sits across the major grove and makes contact with the DNA backbone.

67 trancription Lack of glucose, the level of cAMP is high→the operon is turned on. When glucose is present, the level of cAMP is low→the operon is turned off. ZYAOP DNA CAP positive gene regulation of lac operon:

68 Therefore, the requirements of the transcription of lac operon: The binding of cAMP-CAP complex and leaving of the repressor.

69 Lac operon is controlled by two independent regulation systems, negative and positive regulation system. When lactose and glucose are all present, the operon is turned off. When glucose is present and lactose is absent, the operon is turned off. When lactose and glucose are all absent , the operon is turned off. When glucose is absent and lactose is present, the operon is turned on

70 Detail 1. two conflicts about the theory of lac operon ( 有 两个矛盾是操纵子理论所不能解释的 ) : ① the inducer need to be transferred into the cells by permease ( 透过酶), but the synthesis of permease need to be induced by the inducer. ②the direct inducer of lac operon is allolactose, not lactose. however, allolactose is not converted into lactose until β- galactosidase (β- 半乳糖甘酶 ) is synthesized. 真正的诱导物是异构乳糖而非乳糖,前者是在的催化下由乳糖 形成的,因此,需要有 β- 半乳糖甘酶的预先存在。 4. Some details about lac operon

71 i p o z y a Very low level of lac mRNA Absence of lactose Active i p o z y a  -Galactosidase Permease Transacetylase Presence of lactose Inactiv e Lack of inducer: the lac repressor block all but a very low level of trans- cription of lacZYA.( 本底水 平的表达) Lactose is present, the low basal level of permease allows its uptake, andβ- galactosidase catalyzes the conversion of some lactose to allolactose. Allolactose acts as an inducer, binding to the lac repressor and inactivate it.

72 培养基:甘油,缺少乳糖 按照 lac 操纵子本底水平的表达,每个细胞内有几个 分子的 β- 半乳糖苷酶和 β- 半乳糖苷透过酶; 培养基:加入乳糖 少量乳糖 透过酶 进入细胞 β- 半乳糖苷酶 异构乳糖 诱导物 诱导 lac mRNA 的生物合成大量乳糖进入细胞 多数被降解为葡萄糖和半乳糖(碳源和能源) 异构乳糖

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74 The repressor of lac operon is expressed constitutively under the control of a weak promoter. There are 5-10 molecules per cell. The repressor has 4 subunits, binding 4 IPTG molecules. Lac 操纵子阻遏物 mRNA 是由弱启动子控制下组成型合 成的,每个细胞中有 5-10 个阻遏物分子。该阻遏蛋白 有 4 个相同的亚基,每个亚基均能与 1 分子 IPTG 结合。

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76 如果将葡萄糖和乳糖同时加入培养基中, lac 操纵子处 于阻遏状态,不能被诱导;一旦耗尽外源葡萄糖,乳 糖就会诱导 lac 操纵子表达分解乳糖所需的三种酶。

77 detail 4. the amount of three kinds of the products of lac operon: ß-galactosidase:ß-galactoside permease :ß-galactoside transacetylase =1 : 0.5 : 0.2 β- 半乳糖苷酶:透过酶:乙酰基转移酶 =1 : 0.5 : 0.2 Controlled on the expression level( 翻译水平上受到调节 ) : ( 1 ) the synthesis of the proteins could be terminated early because lac mRNAs disassociates with the ribosomes in the process of translation. lac mRNA 可能与翻译过程中的核糖体相脱离,从而终止蛋白质链 的翻译; ( 2 ) on the lac mRNA molecules, A gene could be degradated more easily than Z gene by the endonuclease. 在 lac mRNA 分子内部, A 基因比 Z 基因更容易受内切酶作用发生 降解。

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