分子生物学 The revised central dogma Gene regulation Gene regulation...

分子生物学

The revised central dogma

Gene regulationGene regulation

基因组的保持

基因组的表达

The structure of DNAThe structure of DNA

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:

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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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

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.

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.

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 ):

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）

The process of gene expression in prokaryotes and eukaryotes

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 .

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.

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 )

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

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:

Regulator gene Operator gene

Structural gene

repressoracitivator

positive 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.

negative transcription regulation

Regulator gene Operator gene

Structural gene

repressoracitivator

positive 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.

negative transcription regulation

+ 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:

Regulator gene Operator 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.

+ 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

Regulator gene

Operator geneStructural gene

mRNA

enzyme

Regulator gene Operator geneStructural 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.

3.negative transcription regulation include:

根据其作用特征又分为负控诱导和负控阻遏：

负控诱导系统

In this system, when the

inducer binds to the

repressor, the structural

genes are transcribed.

负控阻遏系统

In this system, when the

corepressor binds to the

repressor, the structural

genes are not transcribed.

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

正控诱导系统

In this system, when the

inducer binds to the actiator,

the structural genes are

transcribed.

正控阻遏系统

In this system, when the

corepressor binds to

the activator, the

structural genes are

transcribed.

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）。

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. The leucine residues that represent the 'teeth' of the zipper are colored red

Just how do proteins bind to DNA?

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

First example: First example: The The LacLac Operon Operon

(( 乳糖操纵子乳糖操纵子 ))

First example: First example: The The LacLac Operon Operon

(( 乳糖操纵子乳糖操纵子 ))

2.3.1 Regulation of transcription initiation

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.

1. Composition of the Composition of the LacLac operon 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） .

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

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

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 siteThe 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:

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. 　

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

Repressor binding physically prevents Repressor binding physically prevents RNAP from binding to the promoterRNAP 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.

How does Lac repressor repress the transcription of the lac operon?

+ 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.

Ampr

ori

pUC18(3 kb)

MCS (Multiple cloning sites,多克隆位点）

Lac promoter

lacZ’

Gene X

No IPTG, little expression of X geneWith IPTG, efficient expression of X gene.

L1: The LAC operon

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 AOPDNA

regulation region

CAP site

promoter

Operator gene

structural gene

cAMP—CAP complex

ATPadenylate 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 ：

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 binds to a activator sitea activator site upstream of upstream of the promoter, and helps RNA the promoter, and helps RNA polymerase binds to the promoter by polymerase binds to the promoter by physically interacting with RNAP. 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.

+ + + + 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.

Z Y AOPDNA CAP

CAPCAPCAP CAP

CAP

positive gene regulation of lac operon:

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

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

i p o z y a

Very low level of lac mRNA

Absence of lactose

Active

i p o z y a

-Galactosidase

PermeaseTransacetylase

Presence of lactose

Inactive

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.

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

H OH

HO H

OH H

H

CH2OH H O OH

HO O

H O CH2

CH2OH H OH OH H

HO O H 别乳糖 H O OH H H H OH

OH H H ＋ H2O

H H H O OH CH2OH CH2OH

H OH CH2OH H O OH HO O OH

H H

OH H ＋ OH H

HO H H H

H OH H OH

葡萄糖 半乳糖

图 16- 乳糖分解的不同产物

乳糖

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。

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 ： 0.2Controlled 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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