分子生物学 Molecular Biology

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Presentation transcript:

分子生物学 Molecular Biology

Chaptor 8 the control of gene expression

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

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

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。)

Section 1 A brief introduction of gene regulation

Expression of many genes in cells are regulated 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. 7

1-1 cell proteins are divided into two categories: constitutive protein(组成型蛋白质): 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.

1-2 gene regulation gene expression(基因表达): 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. gene regulation or gene control(基因表达调控) refers to a process in which the gene expression is regulated.

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

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

The process of gene expression in prokaryotes and eukaryotes

Section 2 gene regulation in prokaryotes

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 .

Regulation of a gene usually occurs through transcriptional control 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. Jacob and Monod

What’s the operon? 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。

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

Gene Expression is Controlled by Regulatory Proteins (调控蛋白) 2-2 the types and characters of gene regulation in prokaryotes 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

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

positive transcription regulation Regulator gene Operator gene Structural gene repressor acitivator positive transcription regulation negative transcription regulation 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.

positive transcription regulation Regulator gene Operator gene Structural gene repressor acitivator positive transcription regulation negative transcription regulation 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.

gene regulation include: 2、according to the response of operon to some small molecules with regulatory role gene regulation include: 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 .

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

Repressible regulation (可阻遏调节): the genes are generally turned on 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.

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

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

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

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

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

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

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

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.

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

1)helix-turn-helix(螺旋-转角- 螺旋,HTH) 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.

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.

2). Zinc finger(锌指) 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.

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.

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.

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.

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

Just how do proteins bind to DNA?

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

First example: The Lac Operon (乳糖操纵子) 2.3.1 Regulation of transcription initiation First example: The Lac Operon (乳糖操纵子)

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). 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.

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

The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (polycistronic mRNA) under the control of a single promoter Plac . LacZYA transcription unit contains an operator site Olac position between bases -5 and +21 at the 3’-end of Plac Binds with the lac repressor

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

Lac repressor binds as a tetramer (四聚体), 155kD Is allosteric protein,consists of DNA, protein, and inducer-binding domain. helix-turn-helix at N-terminal end(1-49aa )is DNA-binding domain.  图5-25 Lac 阻遏蛋白的变构改变。

Molecular model of the lac repressor tetramer Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

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

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

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.

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

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).

Z Y A O P regulation region DNA promoter CAP site cAMP—CAP complex Operator gene structural gene Z Y A O P DNA cAMP—CAP complex

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

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.

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. This cooperative binding stabilizes the binding of polymerase to Plac.

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

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.

Z Y A O P positive gene regulation of lac operon: + + + + trancription CAP Z Y A O P DNA CAP Lack of glucose, the level of cAMP is high→the operon is turned on. CAP When glucose is present, the level of cAMP is low→the operon is turned off.

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

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

4. Some details about lac operon 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。

Very low level of lac mRNA i p o z y a Very low level of lac mRNA Absence of lactose Active 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. Presence of lactose i p o z y a Inactive Permease b-Galactosidase Transacetylase

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 进入细胞 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

乳糖

Detail 2. the pruduct of lac I gene 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。

Detail 3. Lactose is not the preferred carbohydrate source for E. coli Detail 3. Lactose is not the preferred carbohydrate source for E. coli. If lactose and glucose are present, the cell will use all of the glucose before the lac operon is turned on. This type of control is termed catabolite repression(代谢物阻遏效应). If lactose is E. coli’s sole carbon source, three genes are expressed: 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。

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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