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Chapter 4 Transcription in Eukaryotes: Mechanism and

Regulation4.1 Eukaryotic RNA

polymerases

4.2 Eukaryotic Promoters

4.3 General Transcription

Factors and Initiation

4.4 Specific Transcription Factors

and Transcriptional Regulation

4.5 Structures of specific

transcription factors

4.6 Experiments

4.1 真核生物 RNA 聚合酶

4.2 真核生物启动子4.3 通用转录因子与 转录起始4.4 特异转录因子与 转录调控4.5 特异转录因子的结构

4.6 实验研究

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Transcription in Eukaryotes: Mechanism and Regulation

真核生物转录：机理与调控

Prokaryote Eukaryote

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Transcription in prokaryotes

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Transcription in eukaryotes

Eukaryotic RNA polymerase II and transcription factors

Specific transcription

factor

General transcription

factorsGeneral

transcription factors

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4.1 Eukaryotic RNA polymerases真核生物 RNA 聚合酶

Table 5.1 Eukaryotic RNA polymerases and their roles

表 5.1 真核生物 RNA 聚合酶 和它们的作用

Polymerase Location Products

Polymerase I Nucleolus A large rRNA precursor for 5.8S, 18S, and 28S rRNAs

Polymerase II Nucleoplasm hnRNAs (precursors for mRNAs) and most of the snRNAs

Polymerase III Nucleoplasm Precursors for 5S rRNA and 4.5S tRNAs, and U6 snRNA, etc.

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Locations of RNA polymerases

Nucleolus ( RNA polymerase I )

Nucleoplasm ( RNA polymerase II and III )

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RNA polymerase I

RNA polymerase IrRNAprecursor

Processing

5.8SrRNA

18SrRNA

28SrRNA

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RNA polymerase III

RNA polymerase IIIRNAprecursor

Processing

5SrRNA4.5S

tRNA

U6snRNA

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RNA polymerase II

RNA polymerase II RNAprecursor

Processing

snRNAs

Translation

Protein

mRNA

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RNA polymerase II

Holoenzyme: Core enzyme (β’, β & α) + Subunit σ

RNA pol II: Core subunits (1, 2 & 3) + Subunits 4-12

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RNA polymerase II

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4.2 Eukaryotic Promoters真核生物启动子

Prokaryoticpromoter

Eukaryotic promoter

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Eukaryotic Promoters真核生物启动子

“I would like to have TATA box and Initiator as

my promoter. What would you like?”

“Well, I prefer BRE and DPE.”

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TATA box

TATAAA-10 boxTATAAT

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Specialized genes have TATA boxes

TATA box

Specialized genes +

Housekeeping genes –

Genes of development –

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DPE: Downstream Promoter Element

G CGAT

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Initiator / 起始子

PyPyAN PyPyTA

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BRE: TFIIB Recognition Element

CGCCGGGCCA

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4.3 General Transcription Factors and Initiation （通用转录因子与转录起始）

Prokaryotic RNApolymerase

pre-initiation complex

IIA

IIFIIBIIE

IIH

RNA pol IITATAbox

IID

Eukaryotic RNApolymerase II:“I need help fromother proteins.”

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4.3.1 TFIID

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TBP: TATA-Binding Protein

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Show 3D structure of TBP

File: TBP-TATA box complex 1.val

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TAFIIs: TBP-Associated Factors II

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TAFIIs: 1) Binding to DPE or Inr

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TAFIIs: 2) Binding to Activator

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4.3.2 Other TFIIs

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Now showing

Initiation of Transcription

File: biophoto7 \ Life \ Initiation of transcription (5’)

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4.3.3 General Transcription factors for RNA Polymerase I and III

RNA 聚合酶 I 和 III 的通用转录因子

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1. General TFs of RNA polymerase I

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2. General TFs of RNA polymerase III

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General TFs of RNA polymerase III

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4.4 Specific Transcription Factors and Transcriptional Regulation

特异转录因子与转录调控

RNA pol II

TATAbox

Specific transcription factor

IIA

IIFIIBIIE

IIHIID

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Specific Transcription Factors and Transcriptional Regulation

特异转录因子与转录调控

4.4.1 Activators / 激活蛋白

4.4.2 Repressors / 阻遏蛋白

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4.4.1 Activators / 激活蛋白

Specific transcription factors that enhance the rate of transcription are called activators.

能提高转录速率的特异转录因子称为激活蛋白。

1. Help recruit the pre-initiation complex.

2. Help organize the subunits of the pre-initiation complex, and maintain cohesion of the complex.

3. Attract enzymes to loosen DNA from proteins, freeing important regions like the promoter.

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1. Help recruit the pre-initiation complex

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2. Help organize the subunits of the pre-initiation complex

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3. Attract enzymes to loosen DNA

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4.4.2 Repressors / 阻遏蛋白Specific transcription factors that prevent the transcription of a gene are called repressors.

能阻碍基因转录的特异转录因子称为阻遏蛋白。

1. Blocking the activity of activators.

2. Blocking access of the pre-initiation complex to the promoter.

3. Recruiting enzymes that tighten DNA around proteins.

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1. Blocking the activity of activators

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2. Blocking access of the pre-initiation complex to the promoter.

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3. Recruiting enzymes that tighten DNA around proteins

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4.4.3 Enhancers and Silencers增强子和沉默子

Specific transcription factors rely on DNA sequences to tell them how each gene should be regulated. These sequences are called enhancers or silencers, depending whether they bind activators or repressors, respectively.

特异转录因子依靠DNA 序列来告诉它们应该怎样调控每一种基因。这些序列称为增强子或沉默子，分别依赖于它们结合的是激活蛋白还是阻遏蛋白。

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Enhancers and Silencers

Transcriptionis activated

Enhancer

Transcriptionis repressed

Silencer

Repressor

Activator

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DNA looping / DNA 成环

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Sea Urchin Endo 16 gene

A red sea urchin红海胆

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4.5 Structures of Specific Transcription Factors

4.5 特异转录因子的结构

?

?

Prokaryotic

Eukaryotic

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Major Groove vs. Minor Groove

Major grooveA ≠ TG≠ C

Minor grooveA ≈ TG ≈ C

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4.5.1 DNA-Binding Motifs in Prokaryotes原核生物 DNA 结合基序

Recognitionhelix

The helix-turn-helix(HTH) motif

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Negative Regulation – the lac Repressor负调控── lac 阻遏蛋白

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Show 3D structure of the lac repressor

File: lac repressor with DNA.val

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The trp repressor

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The trp repressor

Trp

Trp repressor

DNA

E. coli

Operator

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Show 3D structure of the trp repressor

File: trp repressor.val

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4.5.2 DNA-Binding Motifs in eukaryote真核生物 DNA 结合基序

1. The homeodomain

同源异型域2. Zinc finger motif

锌指基序3. Leucine zipper

亮氨酸拉链4. HLH motif

螺旋 - 环 - 螺旋基序 ?

Eukaryotic

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1. The homeodomain / 同源异型域

Similar to HTHof prokaryotes

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Mutation of homeotic genes

Homeotic genes

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Show 3D structure of homeodomain

File: Homeodomain.val

Baird-Titus JM, et al. The solution structure of the native K50 Bicoid homeodomain bound to the consensus TAATCC DNA-binding site J. Mol. Biol. v356, p.1137-1151

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2. Zinc finger motif / 锌指基序

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Show 3D structure of zinc fingers

Finger 1

Finger 2

Finger 3

File: Zinc finger.val

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3. Leucine zipper / 亮氨酸拉链

Leucines

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Heterodimerization / 异源二聚化作用

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Show 3D structure of leucine zipper

Wu SW, et al. Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates Proteins v60, p.617-628

File: Leucine zipper.val

Yeast cells www.zeiss.de/C12567BE0045ACF1/ContentsWWWInte

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4. Helix-loop-helix motif / 螺旋 - 环 - 螺旋基序

The helix-loop-helix(HLH) motif

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HLH versus HTH

The helix-loop-helix(HLH) motif

The helix-turn-helix(HTH) motif

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Show 3D structure of HLH

Ferre-D'Amare AR, et al. Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain Nature v363, p.38-45

File: HLH.val

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4.6 Experiments / 实验研究

5.5.1 RNA polymerase targets RNA 聚合酶的目标

5.5.2 Modularity of Specific Transcription Factors 特异转录因子的模块化

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4.6.1 RNA polymerase targetsRNA 聚合酶的目标

Table 5.1 Eukaryotic RNA polymerases and their roles

表 5.1 真核生物 RNA 聚合酶 和它们的作用

Polymerase Location Products

Polymerase I Nucleolus A large rRNA precursor for 5.8S, 18S, and 28S rRNAs

Polymerase II Nucleoplasm hnRNAs (precursors for mRNAs) and most of the snRNAs

Polymerase III Nucleoplasm Precursors for 5S rRNA and 4.5S tRNAs, and U6 snRNA, etc.

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α- amanitin / α 鹅膏蕈碱

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Why is α- amanitin poisonous?

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Functions of RNA polymerases

RNA polymerase IRNA polymerase IIRNA polymerase III

rRNA precursor

High concentrationof α-amanitin

RNA polymerase Iproduces rRNAprecursors.

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Functions of RNA polymerases

RNA polymerase IRNA polymerase IIRNA polymerase III

rRNA precursortRNA, 5S rRNA …

Low concentrationof α-amanitin

RNA polymerase IIIproduces tRNA, 5S rRNA and other snRNAs.

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4.6.2 Modularity of Specific Transcription Factors

特异转录因子的模块化

Moduleexchange

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The reporter genes / 报告基因physicalsciences.ucsd.edu/outreach/image_gall...

www.bio-itworld.com/archive/091103/russell.html

Brain cancer models  Human U-87-RFP glioma growing in brain of nude mouse.脑瘤模型：生长在裸鼠脑中的人神经胶质瘤 www.metamouse.com/brain.html

RFP

GFP

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The modularity of Gal4

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The modularity of Gal4

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The modularity of Gal4