真核生物转录调控 概念，策略和方法 英文版【2025|PDF下载-Epub版本|mobi电子书|kindle百度云盘下载】

真核生物转录调控 概念，策略和方法 英文版PDF格式电子书版下载

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1 A PRIMER ON TRANSCRIPTIONAL REGULATION IN MAMMALIAN CELLS1

INTRODUCTION2

A general model for regulation of a gene2

Activating a gene3

Inactivating a gene5

Overview5

CONCEPTS AND STRATEGIES：Ⅰ．PROMOTERS AND THE GENERALTRANSCRIPTION MACHINERY5

Core promoter architecture8

The general transcription machinery10

Basal transcription complex assembly11

Conformational changes during transcription complex assembly11

TAFⅡs12

Discovery ofthePol Ⅱ holoenzyme14

The holoenzyme and mediators14

Composition of the yeast holoenzyme15

Mammalian holoenzymes16

ContentsPreface17

CONCEPTS AND STRATEGIES：Ⅱ．ACTIVATORS AND REPRESSORS18

Regulatory promoters and enhancers18

Transcriptional activators20

Modular activators20

Overviewx21

DNA-binding domains21

Activation domains21

Structural aspects of activation domains22

Repressors and corepressors23

General mechanisms23

Sequence-specific repressors24

Abbreviations and Acronyms25

CONCEPTS AND STRATEGIES：Ⅲ．CHROMATIN ANDGENE REGULATION25

Chromatin25

Structure and organization25

Binding of transcription factors to chromatin26

Genetic links between gene actwation and chromatin27

ATP-dependent remodeling complexes27

SWI/SNF complexes27

Mechanisms and targeting29

Acetylation of chromatin31

Mammalian acetylases32

TAFs and chromatin remodeling32

Histone deacetylation,transcriptional repression,and silencing32

Repression and deacetfylases33

Linking deacetylation and ATP-remodeling machines33

Methylation and repression34

Locus control regions,insulators,and matrix attachment regions35

Locus control regions35

Transcriptional silencing35

Boundary elements37

MARs38

CONCEPTS AND STRATEGIES：Ⅳ．THE ENHANCEOSOME38

Combinatorial control,cooperativity,and synergy38

The enhanceosome theory39

The interferon-β enhanceosome40

Biochemical mechanism of activation41

Perspective42

2 INITIAL STRATEGIC ISSUES51

The initial steps in a gene regulation analysis52

CONCEPTS AND STRATEGIES52

INTRODUCTION52

Consider the time commitment and resources needed to reach a defined goal54

Two general strategies that provide preliminary albeit superficial insight into transcriptional regulation mechanisms54

An example ofa rigorous,yet incomplete gene regulation analysis：The immunoglobulin μ heavy-chain gene55

Defining the project goals57

Evaluate the feasibility of the analysis57

Appropriate source of cells for functional studies57

Source of cells for protein extract preparation59

Success in developing an appropriate functional assay59

Initiate an analysis of transcriptional regulation61

Beginning with the promoter or distant control regions61

Summary62

Initiating an analysis of a promoter62

Initiating an analysis of distant control regions62

3 MODES OF REGULATING mRNA ABUNDANCE65

INTRODUCTION66

CONCEPTS AND STRATEGIES66

Transcription initiation versus mRNA stability66

Basic mRNA degradation pathways67

Regulation of mRNA stability and degradation68

Interrelationship between mRNA stability and transcription initiation70

Confirming that the rate of transcription initiation contributes to gene regulation71

Nuclear run-on transcription assay (Box 3.1)72

Measuring mRNA stabilities73

Recommended approach for demonstrating regulation of transcription initiation or mRNA stability77

Transcription elongation78

Basic mechanism of elongation78

Regulation of transcription elongation in prokaryotes79

Regulation of transcription elongation in eukaryotes80

Strategies for distinguishing between regulation of elongation and regulation of initiation82

Recommended approach for demonstrating regulation of transcription initiation or elongation83

Extending an analysis of elongation regulation84

Differential pre-mRNA splicing,mRNA transport,and polyadenylation85

Basic principles85

Identifying regulation of pre-mRNA splicing,transport,and polyadenylation86

Protocol 3.1 Nuclear run-on assay87

TECHNIQUES87

4 TRANSCRIPTION INITIATION SITE MAPPING97

INTRODUCTION98

CONCEPTS AND STRATEGIES99

Initial considerations99

Reagents needed before proceeding99

Information provided by the DNA sequence99

Primer extension102

Advantages and disadvantages102

Design of oligonucleotide primers102

Method(Box 4.1)103

Analysis of example data104

Primer annealing and reverse transcription104

RNase protection105

Advantages and disadvantages105

Probe preparation105

Method(Box 4.2)106

Probe annealing and RNase digestion108

Analysis of example data108

S1 nuclease analysis109

Advantages and disadvantages109

Probe preparation109

Method(Box 4.3)109

Analysis of example data111

Advantages and disadvantages112

Data analysis112

Method(Box 4.4)112

Rapid amplification of cDNA ends112

Effect of introns on the interpretation of start-site mapping results(Box 4.5)114

TECHNIQUES116

Protocol 4.1 Primer extension assay116

Protocol 4.2 RNase protection assay124

Protocol 4.3 S1 nuclease assay130

5 FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS137

I NTRODUCTION138

Choosing an assay：Advantages and disadvantages of each assay141

CONCEPTS AND STRATEGIES141

Transient transfection assay142

Stable transfection assay by integration into host chromosome144

Stable transfection of episomally maintained plasmids145

In vitro transcription assay145

Transgenic assays146

Homologous recombination assay147

Transient transfection assays147

Cells148

Transfection procedures (Box 5.1)148

Reporter genes,vectors,and assays(Boxes 5.2,5.3,5.4)150

Plasmid construction155

Initial transfection experiments157

Assessing appropriate promoter regulation(Boxes 5.5,5.6)159

Stable transfection assays by chromosomal integration160

General strategies160

Cells and transfection procedures162

Reporter genes and assays165

Drug-resistance genes and vectors165

Plasmid construction168

Drug selection169

Controls and interpretation of results171

Common transfection methods for mammalian cells172

TECHNIQUES172

Protocol 5.1 Calcium phosphate transfection of 3T3 fibroblasts174

Protocol 5.2 DEAE-dextran transfection of lymphocyte cell lines176

Protocol 5.3 Transfection by electroporation of RAW264.7 macrophages178

Common reporter enzyme assays180

Protocol 5.4 Luciferase assay181

Protocol 5.5 Chloramphenicol acetyltransferase assay183

Protocol 5.6 β-Galactosidase assay186

6 IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS193

INTRODUCTION194

DNase I hypersensitivity195

Basic principles of DNase I sensitivity and hypersensitivity195

CONCEPTS AND STRATEGIES195

Advantages and disadvantages of using DNase I hypersensitivity to identify control regions197

DNaseI hypersensitivity assay(Box 6.1)198

Data interpretation200

Identification of matrix attachment regions200

Basic principles of the nuclear matrix and of MARs and SARs200

Advantages and disadvantages of usingMARs to identify distant control regions200

Methods for identifying MARs(Box 6.2)201

Functional approaches for the identification of distant control regions201

Basic advantages and disadvantages of functional approaches201

Functionalapproach beginningwith a large genomic DNA fragment203

Functional approach beginning with smaller fragments directing expression of a reportergene204

Functional assays for the characterization of distant control regions205

Transient transfection assays205

Stable transfection assays206

Demonstration of LCR activity208

Demonstration of silencer activity209

Demonstration of insulator activity209

7 IDENTIFYING cis-ACTING DNA ELEMENTS WITHIN A CONTROL REGION213

INTRODUCTION214

CONCEPTS AND STRATEGIES215

Identification of control elements by comprehensive mutant analysis215

Rationale for a comprehensive anialysis215

The Ig μ gene example216

Disadvantages of using mutagenesis to identify control elemen219

Strategies for a comprehensive analysis220

Methodology for mutating a control region235

Identification of control elements using in vivo or in vitro protein-DNA interaction methods235

Advantages and disadvantages235

Identification of control elements by database analysis237

Advantages and disadvantages237

Mutagenesis techniques(Boxes 7.1-7.6)238

8 IDENTIFICATION OF DNA-BINDING PROTEINS AND ISOLATION OF THEIR GENES249

INTRODUCTION250

Database methods252

CONCEPTS AND STRATEGIES FOR THE IDENTIFICATION OF DNA-BINDING PROTEINS252

Development of a protein-DNA interaction assay for crude cell lysates253

Standard methods for detecting protein-DNA interactions253

Electrophoretic mobility shift assay(Box 8.1)257

DNase I footprinting268

CONCEPTS AND STRATEGIES FOR CLONING GENES ENCODING DNA-BINDING PROTEINS272

Cloning by protein purification and peptide sequence analysis(Box 8.2)276

Amount of starting material276

Conventional chromatography steps277

DNA affinity chromatography277

Identification of the relevant band following SDS-PAGE(Box 8.3)278

Amino acid sequence analysis and gene cloning279

Confirmation that the gene isolated encodes the DNA-binding activity of interest282

Cloning by methods that do not require an initial protein-DNA interaction assay283

One-hybrid screen283

In vitro expression library screening with DNA or antibody probes285

Mammalian expression cloning methods287

Genome database methods and degenerate PCR288

9 CONFIRMING THE FUNCTIONAL IMPORTANCE OF A PROTEIN-DNA INTERACTION291

INTRODUCTION292

CONCEPTS AND STRATEGIES294

Abundance of a protein-DNA complex in vitro294

Relative expression patterns of the DNA-binding protein and target gene295

Correlation between nucleotides required for protein binding and those required for activity of the control element296

trans-Activation of a reporter gene or endogenous gene by overexpression of the DNA-binding protein297

Cooperative binding and synergistic function of proteins bound to adjacent control elements299

Comparison of genomic and in vitro footprinting patterns301

Relative affinity of a protein-DNA interaction302

Gene disruption or antisense experiments304

Dominant-negative mutants305

In vitro transcription strategies308

In vivo protein-DNA crosslinking310

Altered specificity experiments313

10 IN VIVO ANALYSIS OF AN ENDOGENOUS CONTROL REGION319

INTRODUCTION320

DNase I and DMS genomic footprinting(Box 10.1)321

In vivo analysis of sequence-specific protein-DNA interactions321

CONCEPTS AND STRATEGIES321

In vivo protein-DNA crosslinking/immunoprecipitation326

Nucleosome positioning and remodeling326

Model systems326

Low-resolution analysis of nucleosome positioning by the MNase-Southern blot method(Box 10.2)328

High-resolution analysis of nucleosome positioning by an MNase-LM-PCR method and DNase I genomic footprin ting(Box 103)329

In vivo methods for analyzing nucleosome remodeling(Box 10.4)332

DNA methylation335

Subnuclear localization of a gene337

TECHNIQUES338

Protocol 10.1 MNase-Southern blot assay338

Restriction enzyme accessibility to monitor nucleosome remodeling347

DMS genomicfootprinting347

Protocol 10.2 LM-PCR methods347

MNase mapping of nucleosome positioning347

DNase genomic footprinting347

11 APPROACHES FOR THE SYNTHESIS OF RECOMBINANT TRANSCRIPTION FACTORS365

INTRODUCTION366

CONCEPTS AND STRATEGIES367

Prokaryotic expression systems(Boxes 11.1 and 11.2)367

Strategies for overcoming expression problems in E.coli374

Synthesizing large regulatory proteins377

Yeast systems(Box 11.3)377

Baculovirus system(Box 11.4)379

Vaccinia virus(Box 11.5)382

Retroviral expression systems(Box 11.6)384

Synthesizing small quantities of crude protein385

Specialized inducible expression systems(Box 11.7)386

In vitro transcription/translation systems(Box 11.8)388

Mammalian expression vectors(Box 11.9)389

Synthesis and purification of macromolecular complexes390

Choosing an appropriate system391

12 IDENTIFYING AND CHARACTERIZING TRANSCRIPTION FACTOR DOMAINS399

CONCEPTS AND STRATEGIES：DEFINING DOMAINS400

Basic mutagenesis principles400

INTRODUCTION400

Domains of a gene activator402

Separating DNA-binding and activation domains of an activator403

General considerations403

DNA binding404

Activation(Box 12.1)406

Limitations of the domain swap406

Subdividing DNA recognition and oligomerization subdomains(Box 12.2)409

CONCEPTS AND STRATEGIES：PROTEIN-PROTEIN INTERACTIONS410

Interaction of activation domains with coactivators and general factors410

Affinity chromatography413

Principles413

Caveats of the affinityapproach415

Altered specificity genetic systems416

Structure-function analysis of the general transcriptional machinery420

TECHNIQUES422

Protocol 12.1 PCR-mediated site-directed mutagenesis422

13 THEORY,CHARACTE RIZATION,AND MODELING OF DNA BINDING BY REGULATORY TRANSCRIPTION FACTORS433

INTRODUCTION434

CONCEPTS AND STRATEGIES436

General theory and examples of DNA-protein interactions436

Theory of DNA recognition436

Chemical basis of the interactions437

The role of the α-helix in DNA recognition437

Major and minorgroove specificity439

Monomers and dimers；energetic and regulatory considerations441

Dissociation constant analysis(Box 13.1)444

Kd determination447

Analysis and modeling of DNA-protein interactions448

Identification of a high-affinity DNA recognition site448

Basic theory449

General methods(Boxes 13.2 and 133)449

Minor groove/DNA backbone probes (Box 13.4)454

Major groove probes458

Modeling DNA-protein interactions459

Analysis of promoter-specific multicomponent nucleoprotein complexes463

DNA binding cooperativity465

DNA looping and bending466

Mechanisms of DNA bending468

Approaches for studying bending469

TECHNIQUES472

Protocol 13.1 DNase I footprinting472

Protocol 13.2 Hydroxyl-radical footprinting482

Protocol 13.3 Phosphate ethylation interference assay485

Protocol 13.4 Methylation interference assay488

Protocol 13.5 Electrophoretic mobility shift assays493

Protocol 13.6 Preparation of 32P-end-labeled DNA fragments497

14 CRUDE AND FRACTIONATED SYSTEMS FOR IN VITRO TRANSCRIPTION505

INTRODUCTION506

CONCEPTS AND STRATEGIES507

Preparation of extracts507

Cell choice507

Extract preparation method508

Transcription assays510

General considerations(Box 14.1)510

Choice of template514

Chromatin systems516

Optimization of conditions519

Fractionated systems(Box 14.2)519

Holoenzyme520

Partially fractionated systems521

Mediator subcomplexes521

Factor-depleted systems525

Highly fractionated systems526

TECHNIQUES526

Preparation of auclear and whole-cell extracts526

Protocol 14.1 The Dignam and Roeder nuclear extract528

Protocol 14.2 Preparation of nuclear extracts from rat liver532

Protocol 14.3 Preparation of whole-cell extract536

In vitro transcription assays539

Protocol 14.4 In vitro transcription using HeLa cell extracts and primer extension539

Protocol 14.5 G-less cassette in vitro transcription using HeLa cell nuclear extracts545

Transcription factor purification549

Protocol 14.6 Preparation of a crude fractionated system551

Protocol 14.7 Purification of recombinant TFIIB from E.coli556

Protocol 14.8 Purification of recombinant TFIIA560

Protocol 14.9 Affinity purification of RNA Pol Ⅱ562

Protocol 14.10 Purification of epitope-tagged TFIID567

15 APPROACHES FOR STUDYING TRANSCRIPTION COMPLEX ASSEMBLY579

INTRODUCTION580

CONCEPTS AND STRATEGIES582

Formation of the basal preinitiation complex582

Kinetic studies582

Sarkosyl probing582

DNase Ifootprinting and EMSA studies oftranscription complex assembly584

Template commitment experiment584

Photocrosslinking586

Structure-function analyses of the general machinery589

Open complex formation,initiation,and promoter escape589

ATP-analogs and an energy-dependent step589

Permanganate probing590

Premelted templates590

The transition to elongation591

Assembly of activated complexes at a promoter594

The immobilized template approach594

Permanganate probing to study activation596

Gel filtration596

EMSA and DNase I footprinting analyses of the TFIID-TFIIA complex599

Assembly and analysis of TFIID subcomplexes600

Future directions601

TECHNIQUES603

Protocol 15.1 Potassium permanganate probing of Pol Ⅱ open complexes603

Protocol 15.2 Magnesium-agarose EMSA of TFIID binding to DNA607

APPENDICES617

Ⅰ．CAUTIONS617

Ⅱ．SUPPLIERS623

Ⅲ．TRADEMARKS625

INDEX627