What are the differences between

prokaryotes and eukaryotes on the initiation

of TRANSCRIPTION and TRANSLATION?

Why is gene expression tightly regulated?

For prokaryotes, it is the way that organisms adapt to the environment.

For eukaryotesGene expression differs among cell types: cell

differentiation Is involved in development Is associated with physiology and pathology

Genotype+expression level (affected by environment)=phenotype

Gene expression regulation is different between prokaryotes and eukaryotes due to the differences inCellular structureExisting form of genomic DNAComplexity in gene expression

A10 Regulation of gene expression in prokaryotes

Regulation of gene expression Organization of bacterial genes The lac operon Catabolite repression The trp operon Attenuation Regulation by alternative sigma factors

1. Regulation of gene expression Regulation of gene expression allows

bacteria to respond to changes in their environment, typically to the presence or absence of nutrients.

Balance between the rate of synthesis [‘sinθisis] and the rate of degradation

[ degrə'deɪʃən]

Factors that alter the rate of synthesis:

Changes in the rate of gene transcription, the best understood

Changes in mRNA turnover time （更新的时间）

Changes in the rate of translation

2. Organization of bacterial genes

Important feature: operon['ɔpə,rɔn], transcriptional units in which several genes, usually encoding proteins with related functions, are regulated together

操纵子：是原核生物基因表达和调控的单位，由启动子、操纵基因（调控）及其所控制的一组功能上相关的结构基因所组成。操纵基因受调节基因产物的控制。

Operon proposed by Jacob & Monad 1961

In 1961, Jacob and Monod found the difference between two types of sequences in DNA: sequences that code for trans-acting products （反式作用因子） ; and cis-acting sequences （顺式作用序列） that function exclusively within the DNA. Gene activity is regulated by the specific interactions of the trans-acting products (usually proteins) with the cis-acting sequences (usually sites in DNA).

A gene ( 反式作用 products) a sequence of DNA that codes for a product. This product may be protein (as in the case of the majority of genes) or may be RNA (as in the case of genes that code for tRNA and rRNA etc.).

The crucial feature is that the productdiffuses away from its site of synthesis toact elsewhere.

一种基因的蛋白质产物，能影响位于基因组另一条染色体上的（或基因组别处的）另一个基因的活性，这种作用叫做反式作用。参与这种反式作用的特定因子叫做反式作用因子，一般指的是蛋白质，它能够通过与顺式作用元件（ DNA）之间的相互作用而影响基因的表达。

The cis-acting sequence functions exclusively as a DNA sequence, affecting only the DNA to which it is physically linked. (In some cases, a cis-acting sequence functions in an RNA rather than in a DNA molecule, such as RNA termintor)

影响自身基因表达活性的非编码 DNA 序列。

Classification of operons:

Inducible operons （可诱导操纵子） : contain genes that encode enzymes involved in metabolic pathways. e.g. lac operon

Repressible operons （可阻遏操纵子 ): contain genes that encode enzymes involved in biosynthetic pathways and gene expression is controlled by the end product of the pathway e.g. trp operon

Positive regulation （正调控）：有调节蛋白（激活蛋白）存在时基因表达开启，没有调节蛋白存在时则基因的表达关闭 ；Negative regulation （负调控）：在调节蛋白（阻遏蛋白）存在时基因的表达关闭，无调节蛋白时基因的表达得以开启。正、负调控中调节蛋白都是反式作用因子，通常识别基因上有的顺式作用元件

3. The lac operon Three enzymes for the metabolism of

lactose:Lactose permease β 半乳糖苷通透酶 (Lac Y):

transport lactose into the cell β-galatosidase β 半乳糖苷酶 (Lac Z):

hydrolyzes lactose into glucose and galactose

（把乳糖分解为葡萄糖和半乳糖）Transacetylase β 半乳糖苷转乙酰基酶 (Lac A):

hydrolysis [hai’drɔlisis]

Z 编码 β-半乳糖苷酶：将乳糖水解成葡萄糖和半乳糖

Y 编码 β-半乳糖苷透过酶：使外界的 β-半乳糖苷（如乳糖）能透过大肠杆菌细胞壁和原生质膜进入细胞内。

A 编码 β-半乳糖苷乙酰基转移酶：乙酰辅酶 A 上的乙酰基转到 β-半乳糖苷上，形成乙酰半乳糖。

乳糖操纵子的结构

I

OperonRegulatory Gene

P O Z Y A DNA

m-RNA

β-GalactosidasePermease

Transacetylase

Protein

Control elements

-5 +21

repressor

乳糖操纵子的结构

Lac Z, Y and A are transcribed as a single mRNA （多顺反子 mRNA) from a single promoter. Lac I lies upstream of the operon and encodes lac repressor, which regulates the expression of Lac Z, Y and A genes.

LacI 表达阻遏物。操纵基因（ O ）是 DNA上的一小段序列（仅为 26bp），是阻遏物的结合位点。

I

OperonRegulatory Gene

P O Z Y A DNA

m-RNA

β-GalactosidasePermease

Transacetylase

Protein

Control elements

-5 +21

repressor

Lac repressor

Lac repressor is encoded by LacI, which is active as a tetramer of identical subunits. It occupies the operator-binding site Olac (28bp, palindromic) and blocks almostly all transcription of lacZYA when lack of inducer (such as lactose).

Plac

-5 +21

Lactose metabolism in E. coli: An Inducible System

In the absence of lactose Lac repressor binds to operator Blocks the path of the RNA polymerase move

through the lac promoter and operator Prevents the transcription of lac genes

When lactose presentA few molecules of the lac enzymes present

in the cell allow lactose to be taken up and metabolized

Lactose becomes allolactose, the latter binds to the lac repressor and changes its conformation such that it can no longer binds to the operator

The block is removed and the operon is transcribed

When the lactose is used up, lac repressor returns to its original conformation, again block transcription

4. Catabolite repression Catabolite repression: the presence of glucose in the cell

switches off the lac operon 葡糖糖存在时，只利用其作为能量代谢来源物质，对乳糖

的存在视而不见，因而乳糖相关的酶类也无需表达。 葡萄糖可以抑制腺苷酸环化酶的活性。 In the presence of cAMP, CAP (catabolite activator

protein) binds to a DNA sequence upstream of the lac promoter and enhances binding of the RNA polymerase leading to the enhanced transcription of the operon

当细菌处于碳饥饿状态时（葡萄糖缺乏时），腺苷酸环化酶转化 ATP 为 cAMP ， cAMP 的量显著提高，可以与其激活蛋白 CAP （或者叫 CRP ）形成二聚体，并与特定的DNA 序列结合起始操作子进行转录。形成正调控体系

The Lac Operon:When Glucose Is Present But Not Lactose

Repressor Promoter LacY LacALacZOperatorCAPBinding RNA

Pol.

Repressor

Repressor

Repressor mRNA

Hey man, I’m constitutive

Come on, let me through

No wayJose!

CAP

The Lac Operon:When Lactose Is Present But Not Glucose

Repressor Promoter LacY LacALacZOperatorCAPBinding

Repressor

Repressor mRNA

Hey man, I’m constitutive

CAPcAMP

Lac

Repressor

Repressor

X

This lactose has bent me

out of shape

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

RNAPol.

Yipee…!

The Lac Operon:When Neither Lactose Nor Glucose Is Present

Repressor Promoter LacY LacALacZOperatorCAPBinding

CAPcAMP

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

Repressor

Repressor mRNA

Hey man, I’m constitutive

Repressor

STOPRight therePolymerase

Alright, I’m off to the races . . .

Come on, let me through!

乳糖操纵子的正调控(catabolite repression)

When glucose absent

Adenylate cyclase is active and cAMP levels are higher

CAP binds, lac operon transcription level increases

When glucose presentAdenylate cyclase 腺苷酸环化酶） is inhibited and cAMP levels are low 。CAP does not bind, transcription of the lac operon is very low

C A B

Summary

A: RNA polymeraseB: lac repressor C: CRP-cAMP

Glucose/lactoseRepressor/operator bindingcAMP-CAP/promoter

5. The trp operon Structure

five genes encoding enzymes involved in biosynthesis of tryptophan

LeaderTrp operatorTrp promoter

Trp repressor lies upstream of the trp operon

当环境中存在可利用的色氨酸时，细菌会减少和停止自身的合成，通过 Trp 操纵子来实现这种阻遏。

3.4.1.2 Tryptophan Metabolism in E. coli: A Repressible Gene System

trpE 、 D 、 C 、 B 和 A 分别编码： 邻氨基苯甲酸合成酶的 ε 和 δ 亚基、吲哚甘油磷酸合成酶、色氨酸合成酶的 β 和 α 链。它们都是合成色氨酸必需的原料。

When tryptophan present Tryptophan binds to trp repressor enabling it to binds to

trp operator Obstruct binding of RNA polymerase to the trp promoter Prevent transcription

（变构）

When tryptophan absent

Trp repressor is incapable of binding to trp operator, transcription proceeds

6. Attenuation （衰减作用） Expression level is finely tuned. （与翻

译过程相偶联的转录调控模型）

Structure of attenuator （衰减子的结构）1. Trp mRNA: 14 codons followed by a stop

codon, two consecutive trp codons

2. Trp L: including four complementary

sequences which can form two stem-loop

structures. The small one terminates

transcription （ ρ-factor independent

terminator ） , while the large one not.

终止信号 抗终止信号

If levels of tryptophan are adequateTrp mRNA is

translated, ribosome prevents formation of larger stem-loop, transcription ends

If tryptophan is lackingThe ribosome will be

stalled as it translates the coding region

RNA polymerase will move ahead, form larger stem-loop, transcription can proceed

Low Trp

High Trp

Attenuation determines how efficiency it is transcribed.

Attenuation depends on the fact that transcription and translation are linked in bacteria.

7. Regulation by alternative sigma factors

Sigma factor (σ) is responsible for initiating transcription by recognizing bacterial promoter DNA sequences.

By using different sigma factors, different sets of genes are transcribed to meet different environmental conditions.

factor subunit bound to RNA pol for transcription initiation

Released core enzyme 2’

RNA elongation

factors: is bifunctional protein

Bind to core RNA Pol

识别启动子共有序列并且只为转录起始所需

(-35 and –10) in DNA

factor:

Promoter recognition多种不同的 σ factors

binding to the same RNA Pol

细胞中 RNA 聚合酶可以识别不同的启动子，形成不同的转录物

70 factors is most common one in E. coli under the normal growth condition

Many bacteria produce alternative sets of σ factors to meet the regulation requirements of transcription under normal and extreme growth condition

E. coli: Heat shockσ32 is used to transcribe 17 heat shock related genes

Sporulation in Bacillus subtilisalternative σfactors are used when spore germinates.

bacteriophage σfactorsuse σfactors to instruct host RNA polymerase to transcribe phage genes; produce a series of σfactors that allow temporal control of their own gene

Heat shock

Around 17 proteins are specifically

expressed in E. coli when the temperature is increased above 37ºC.

These proteins are expressed through transcription by RNA polymerase using an alternative factor 32 coded by rhoH gene. 32 has its own specific promoter consensus sequences.

Comparison of the heat-shock 32 and general 70 responsive promoter

Consensus promoter –35 sequence –10 sequence

Standard 70

Heat shock 32

--------------TTGACA-----16~18bp---TATAAT

T--C--C--CTTGAA--13~15bp--CCCCAT--T

Sporulation in Bacillus subtilis

•营养生长的枯草杆菌对不良环境作出反应时形成孢子。孢子形成过程包括细胞非对称地裂解为两部分：一部分为前孢子，它最终形成孢子，另一部分为母细胞，最终被遗弃。

Vegetative B. subtilis cell contains a diverse set of factors 自身含有不同的 因子

Sporulation is regulated by a further set of factors 孢子形成前，在前孢子和母细胞中，细胞分化开始前不同的 σ 具有各自特殊的活性。

Cross-regulation of this compartmentalization permits the forespore and mother cell to tightly coordinate the differentiation process.

Bacteriophage factors

Many bacteriophages synthesize their own factors in order to ‘take over’ the host cell’s own transcription machinery by substituting the normal cellular factor and altering the promoter specificity of the RNA polymerase.

Key points:

1. Definition of an operon

2. Lac operon: P, O, lacZ, lacY and lacA.

3. In E coli, how lactose metabolism is carried out?

Repressor by Lac I and Activation by CRP/CAP

4. Trp operon: structure.

5. In E coli, tryptophan synthesis is regulated both in a repressible form and by attenuation.

6. Sigma factors (normal and alternative).