Preface

xx

Acknowledgments

xxvii

I

FOUNDATIONS

1

Evolution, Science, and Molecular Biology

1

MOMENT OF DISCOVERY Jack Szostak, on his discovery of self-dividing vesicles that mimic growing cells 1

1.1 The Evolution of Life on Earth 2

What Is Life? 2

Evolution Underpins Molecular Biology 4

HIGHLIGHT 1-1 EVOLUTION Observing Evolution in the Laboratory 6

Life on Earth Probably Began with RNA 6

The Last Universal Common Ancestor Is the Root of the Tree of Life 8

Evolution by Natural Selection Requires Variation and Competition 10

1.2 How Scientists Do Science 12

Science Is a Path to Understanding the Natural Universe 12

The Scientific Method Underlies Scientific Progress 13

The Scientific Method Is a Versatile Instrument of Discovery 14

Scientists Work within a Community of Scholars 16

HOW WE KNOW 19

Adenine Could Be Synthesized with Prebiotic Chemistry 19

Clay Had a Role in Prebiotic Evolution 20

Darwin’s World Helped Him Connect the Dots 21

2

DNA: The Repository of Biological Information

23

MOMENT OF DISCOVERY James Berger, on his discovery of the structure and mechanism of topoisomerase II 23

2.1 Mendelian Genetics 25

Mendel’s First Law: Allele Pairs Segregate during Gamete Formation 26

Mendel’s Second Law: Different Genes Assort Independently during Gamete Formation 28

There Are Exceptions to Mendel’s Laws 28

2.2 Cytogenetics: Chromosome Movements during Mitosis and Meiosis 31

Cells Contain Chromosomes and Other Internal Structures 31

Mitosis: Cells Evenly Divide Chromosomes between New Cells 33

Meiosis: Chromosome Number Is Halved during Gamete Formation 35

2.3 The Chromosome Theory of Inheritance 37

Sex-Linked Genes in the Fruit Fly Reveal That Genes Are on Chromosomes 37

Linked Genes Do Not Segregate Independently 38

Recombination Unlinks Alleles 40

Recombination Frequency Can Be Used to Map Genes along Chromosomes 41

2.4 Foundations of Molecular Genetics 43

DNA Is the Chemical of Heredity 43

Genes Encode Polypeptides and Functional RNAs 45

The Central Dogma: Information Flows from DNA to RNA to Protein—Usually 46

Mutations in DNA Give Rise to Phenotypic Change 49

HIGHLIGHT 2-1 MEDICINE The Molecular Biology of Sickle-Cell Anemia, a Recessive Genetic Disease of Hemoglobin 52

HOW WE KNOW 55

Chromosome Pairs Segregate during Gamete Formation in a Way That Mirrors the Mendelian Behavior of Genes 55

Corn Crosses Uncover the Molecular Mechanism of Crossing Over 56

Hershey and Chase Settle the Matter: DNA Is the Genetic Material 57

3

Chemical Basis of Information Molecules

61

MOMENT OF DISCOVERY Roxana Georgescu, on her discovery of how beta processivity clamps bind DNA 61

3.1 Chemical Building Blocks of Nucleic Acids and Proteins 62

Nucleic Acids Are Long Chains of Nucleotides 62

Proteins Are Long Polymers of Amino Acids 64

Chemical Composition Helps Determine Nucleic Acid and Protein Structure 65

Chemical Composition Can Be Altered by Postsynthetic Changes 65

3.2 Chemical Bonds 68

Electrons Are Shared in Covalent Bonds and Transferred in Ionic Bonds 68

Chemical Bonds Are Explainable in Quantum Mechanical Terms 70

Forming and Breaking of Chemical Bonds Involves Energy Transfer 72

Electron Distribution between Bonded Atoms Determines Molecular Behavior 72

3.3 Weak Chemical Interactions 73

Van der Waals Forces Are Nonspecific Contacts between Atoms 74

The Hydrophobic Effect Brings Together Nonpolar Molecules 74

Adjacent Bases in Nucleic Acids Participate in Noncovalent Interactions 75

Hydrogen Bonds Are a Special Kind of Noncovalent Bond 75

Combined Effects of Weak Chemical Interactions Stabilize Macromolecular Structures 76

Weak Chemical Bonds Also Facilitate Macromolecular Interactions 77

3.4 Stereochemistry 78

Three-Dimensional Atomic Arrangements Define Molecules 78

Biological Molecules and Processes Selectively Use One Stereoisomer 79

Proteins and Nucleic Acids Are Chiral 79

HIGHLIGHT 3-1 MEDICINE The Behavior of a Peptide Made of d-Amino Acids 80

3.5 The Role of pH and Ionization 81

The Hydrogen Ion Concentration of a Solution Is Measured by pH 81

Buffers Prevent Dramatic Changes in pH 81

The Henderson-Hasselbalch Equation Estimates the pH of a Buffered Solution 83

3.6 Chemical Reactions in Biology 83

The Mechanism and Speed of Chemical Transformation Define Chemical Reactions 83

Biological Systems Follow the Laws of Thermodynamics 85

Catalysts Increase the Rates of Biological Reactions 86

Energy Is Stored and Released by Making and Breaking Phosphodiester Bonds 86

HIGHLIGHT 3-2 EVOLUTION ATP: The Critical Molecule of Energy Exchange in All Cells 87

HOW WE KNOW 89

Single Hydrogen Atoms Are Speed Bumps in Enzyme-Catalyzed Reactions 89

Peptide Bonds Are (Mostly) Flat 90

4

Protein Structure

93

MOMENT OF DISCOVERY Steve Mayo, on his discovery of the first successful method for computational protein design 93

4.1 Primary Structure 95

Amino Acids Are Categorized by Chemical Properties 95

Amino Acids Are Connected in a Polypeptide Chain 96

Evolutionary Relationships Can Be Determined from Primary Sequence Comparisons 98

HIGHLIGHT 4-1 A CLOSER LOOK Purification of Proteins by Column Chromatography and SDS-PAGE 100

4.2 Secondary Structure 102

The α Helix Is a Common Form of Secondary Protein Structure 102

The β Conformation Forms Sheetlike Structures 103

Reverse Turns Allow Secondary Structures to Fold 104

4.3 Tertiary and Quaternary Structures 105

Tertiary and Quaternary Structures Can Be Represented in Different Ways 105

Domains Are Independent Folding Units within the Protein 105

Supersecondary Structural Elements Are Building Blocks of Domains 106

Quaternary Structures Range from Simple to Complex 110

Intrinsically Unstructured Proteins Have Versatile Binding Properties 111

Protein Structures Help Explain Protein Evolution 112

HIGHLIGHT 4-2 A CLOSER LOOK Protein Structure Databases 112

4.4 Protein Folding 113

Predicting Protein Folding Is a Goal of Computational Biology 113

Polypeptides Fold through a Molten Globule Intermediate 115

HIGHLIGHT 4-3 MEDICINE Prion-Based Misfolding Diseases 116

Chaperones and Chaperonins Can Facilitate Protein Folding 118

Protein Isomerases Assist in the Folding of Some Proteins 118

4.5 Determining the Atomic Structure of Proteins 120

Most Protein Structures Are Solved by X-Ray Crystallography 120

Smaller Protein Structures Can Be Determined by NMR 122

HOW WE KNOW 126

Sequence Comparisons Yield an Evolutionary Roadmap from Bird Influenza to a Deadly Human Pandemic 126

We Can Tell That a Protein Binds ATP by Looking at Its Sequence 127

Disulfide Bonds Act as Molecular Cross-Braces to Stabilize a Protein 128

5

Protein Function

133

MOMENT OF DISCOVERY Smita Patel, on her early work with the T7 gene 4–encoded DNA helicase 133

5.1 Protein-Ligand Interactions 134

Reversible Binding of Proteins to Other Molecules Follows Defined Principles 134

Protein-Ligand Interactions Can Be Quantified 135

DNA-Binding Proteins Guide Genome Structure and Function 136

5.2 Enzymes: The Reaction Catalysts of Biological Systems 142

Enzymes Catalyze Specific Biological Reactions 142

Enzymes Increase the Rate of a Reaction by Lowering the Activation Energy 145

The Rates of Enzyme-Catalyzed Reactions Can Be Quantified 146

HIGHLIGHT 5-1 A CLOSER LOOK Reversible and Irreversible Inhibition 148

DNA Ligase Activity Illustrates Some Principles of Catalysis 150

5.3 Motor Proteins 151

Helicases Abound in DNA and RNA Metabolism 151

Helicase Mechanisms Have Characteristic Molecular Parameters 155

5.4 The Regulation of Protein Function 157

Modulator Binding Causes Conformational Changes in Allosteric Proteins 158

Allosteric Enzymes Have Distinctive Binding and/or Kinetic Properties 158

Autoinhibition Can Affect Enzyme Activity 159

Some Proteins Are Regulated by Reversible Covalent Modification 160

Phosphoryl Groups Affect the Structure and Catalytic Activity of Proteins 162

Some Proteins Are Regulated by Proteolytic Cleavage 162

HIGHLIGHT 5-2 Medicine HIV Protease: Rational Drug Design Using Protein Structure 164

HOW WE KNOW 166

The Discovery of the Lactose Repressor: One of the Great Sagas of Molecular Biology 166

The lacI Gene Encodes a Repressor 167

Discovery of the Lactose Repressor Helped Give Rise to DNA Sequencing 168

II

NUCLEIC ACID STRUCTURE AND METHODS  

6

DNA and RNA Structure

173

MOMENT OF DISCOVERY Jamie Cate, on determining the molecular structure of the bacterial ribosome 173

6.1 The Structure and Properties of Nucleotides 174

Nucleotides Comprise Phosphates and Characteristic Bases and Sugars 175

Phosphodiester Bonds Link the Nucleotide Units in Nucleic Acids 177

The Properties of Nucleotide Bases Affect the Three-Dimensional Structure of Nucleic Acids 178

Nucleotides Play Additional Roles in Cells 179

6.2 DNA Structure 182

DNA Molecules Have Distinctive Base Compositions 182

DNA Is Usually a Right-Handed Double Helix 183

DNA Adopts Different Helical Forms 185

Certain DNA Sequences Adopt Unusual Structures 187

HIGHLIGHT 6-1 TECHNOLOGY DNA Nanotechnology 190

6.3 RNA Structure 192

RNAs Have Helical Secondary Structures 192

RNAs Form Various Stable Three-Dimensional Structures 193

6.4 Chemical and Thermodynamic Properties of Nucleic Acids 195

HIGHLIGHT 6-2 MEDICINE RNA Structure Governing HIV Gene Expression 196

Double-Helical DNA and RNA Can Be Denatured 196

Nucleic Acids from Different Species Can Form Hybrids 198

Nucleotides and Nucleic Acids Undergo Uncatalyzed Chemical Transformations 199

Base Methylation in DNA Plays an Important Role in Regulating Gene Expression 200

RNA Molecules Are Often Site-Specifically Modified In Vivo 201

The Chemical Synthesis of DNA and RNA Has Been Automated 201

HOW WE KNOW 204

DNA Is a Double Helix 204

DNA Helices Have Unique Geometries That Depend on Their Sequence 205

Ribosomal RNA Sequence Comparisons Provided the First Hints of the Structural Richness of RNA 206

7

Studying Genes

211

MOMENT OF DISCOVERY Norman Arnheim, on the discovery of interspersed CA repeats in genomic DNA 211

7.1 Isolating Genes for Study (Cloning) 212

Genes Are Cloned by Insertion into Cloning Vectors 213

Cloning Vectors Allow Amplification of Inserted DNA Segments 215

DNA Libraries Provide Specialized Catalogs of Genetic Information 220

7.2 Working with Genes and Their Products 221

Gene Sequences Can Be Amplified with the Polymerase Chain Reaction 221

HIGHLIGHT 7-1 TECHNOLOGY A Potent Weapon in Forensic Medicine 224

The Sanger Method Identifies Nucleotide Sequences in Cloned Genes 226

Genomic Sequencing Is Aided by New Generations of DNA Sequencing Methods 228

Cloned Genes Can Be Expressed to Amplify Protein Production 232

Many Different Systems Are Used to Express Recombinant Proteins 232

Alteration of Cloned Genes Produces Altered Proteins 235

Terminal Tags Provide Handles for Affinity Purification 237

7.3 Understanding the Functions of Genes and Their Products 239

Protein Fusions and Immunofluorescence Can Localize Proteins in Cells 239

Proteins Can Be Detected in Cellular Extracts with the Aid of Western Blots 241

Protein-Protein Interactions Can Help Elucidate Protein Function 241

DNA Microarrays Reveal Cellular Protein Expression Patterns and Other Information 244

A Gene’s Function Can Be Elucidated by Examining the Effects of Its Absence 245

HOW WE KNOW 250

New Enzymes Take Molecular Biologists from Cloning to Genetically Modified Organisms 250

A Dreamy Night Ride on a California Byway Gives Rise to the Polymerase Chain Reaction 251

Coelenterates Show Biologists the Light 252

8

Genomes, Transcriptomes, and Proteomes

259

MOMENT OF DISCOVERY Joe DeRisi, on his discovery of the SARS virus 259

8.1 Genomes and Genomics 260

Many Genomes Have Been Sequenced in Their Entirety 260

Annotation Provides a Description of the Genome 262

Genome Databases Provide Information about Every Type of Organism 264

HIGHLIGHT 8-1 TECHNOLOGY Sampling Biodiversity with Metagenomics 266

The Human Genome Contains Many Types of Sequences 267

Genome Sequencing Informs Us about Our Humanity 269

Genome Comparisons Help Locate Genes Involved in Disease 272

8.2 Transcriptomes and Proteomes 275

Special Cellular Functions Are Revealed in a Cell’s Transcriptome 275

High-Throughput DNA Sequencing Is Used in Transcriptome Analysis 276

The Proteins Generated by a Cell Constitute Its Proteome 276

Electrophoresis and Mass Spectrometry Support Proteomics Research 277

Computational Approaches Help Elucidate Protein Function 279

Experimental Approaches Reveal Protein Interaction Networks 280

8.3 Our Genetic History 280

All Living Things Have a Common Ancestor 281

Genome Comparisons Provide Clues to Our Evolutionary Past 281

HIGHLIGHT 8-2 EVOLUTION Phylogenetics Solves a Crime 282

The Human Journey Began in Africa 284

Human Migrations Are Recorded in Haplotypes 287

HIGHLIGHT 8-3 EVOLUTION Getting to Know the Neanderthals 288

HOW WE KNOW 292

Haemophilus influenzae Ushers in the Era of Genome Sequences 292

9

Topology: Functional Deformations of DNA

297

MOMENT OF DISCOVERY Carlos Bustamante, on discovering the elasticity of DNA 297

9.1 Chromosomes: An Overview 298

Chromosome Function Relies on Specialized Genomic Sequences 298

Chromosomes Are Longer Than the Cellular or Viral Packages Containing Them 300

HIGHLIGHT 9-1 MEDICINE The Dark Side of Antibiotics 303

9.2 DNA Supercoiling 304

Most Cellular DNA Is Underwound 305

DNA Underwinding Is Defined by the Topological Linking Number 307

DNA Compaction Requires a Special Form of Supercoiling 309

9.3 The Enzymes That Promote DNA Compaction 311

Topoisomerases Catalyze Changes in the Linking Number of DNA 311

HIGHLIGHT 9-2 MEDICINE Curing Disease by Inhibiting Topoisomerases 312

The Two Bacterial Type II Topoisomerases Have Distinct Functions 313

Eukaryotic Topoisomerases Have Specialized Functions in DNA Metabolism 316

SMC Proteins Facilitate the Condensation of Chromatin 317

HOW WE KNOW 322

The Discovery of Supercoiled DNA Goes through Twists and Turns 322

The First DNA Topoisomerase Unravels Some Mysteries 323

DNA Gyrase Passes the Strand Test 324

10

Nucleosomes, Chromatin, and Chromosome Structure

331

MOMENT OF DISCOVERY C. David Allis, on establishing that p55 from Tetrahymena is a histone acetylase, as is transcription factor Gcn5 331

10.1 Nucleosomes: The Basic Units of DNA Condensation 332

Histone Octamers Organize DNA into Repeating Units 332

DNA Wraps around a Single Histone Octamer 334

Histone Tails Mediate Internucleosome Connections That Regulate the Accessibility of DNA 336

10.2 Higher-Order Chromosome Structure 337

Histone H1 Binds the Nucleosome 338

Chromosomes Condense into a Compact Chromatin Filament 338

Higher-Order Chromosome Structure Involves Loops and Coils 341

Bacterial DNA, Like Eukaryotic DNA, Is Highly Organized 341

10.3 Regulation of Chromosome Structure 343

Nucleosomes Are Intrinsically Dynamic 344

ATP-Driven Chromatin Remodeling Complexes Can Reposition Nucleosomes 344

Variant Histone Subunits Alter DNA-Binding Affinity 346

Nucleosome Assembly Requires Chaperones 348

Modifications of Histone Tails Alter DNA Accessibility 348

HIGHLIGHT 10-1 A CLOSER LOOK The Use of a Histone Variant in X Chromosome Inactivation 350

Proteins with Bromodomains and Chromodomains Bind Modified Histones 353

Histone Modifications and Remodeling Complexes May Read a Histone Code 354

Histone Modifying Enzymes Maintain Epigenetic States through Cell Division 355

HIGHLIGHT 10-2 MEDICINE Defects in Epigenetic Maintenance Proteins Associated with Cancer 356

HOW WE KNOW 359

Kornberg Wrapped His Mind around the Histone Octamer 359

A Transcription Factor Can Acetylate Histones 360

III

INFORMATION TRANSFER

11

DNA Replication

363

MOMENT OF DISCOVERY Robert Lehman, on discovering DNA ligase 363

11.1 DNA Transactions during Replication 364

DNA Replication Is Semiconservative 364

Replication Is Initiated at Origins and Proceeds Bidirectionally 366

Replication Is Semidiscontinuous 368

11.2 The Chemistry of DNA Polymerases 369

DNA Polymerases Elongate DNA in the 5′→3′ Direction 369

Most DNA Polymerases Have DNA Exonuclease Activity 371

Five E. coli DNA Polymerases Function in DNA Replication and Repair 373

DNA Polymerase Structure Reveals the Basis for Its Accuracy 373

Processivity Increases the Efficiency of DNA Polymerase Activity 376

11.3 Mechanics of the DNA Replication Fork 377

DNA Polymerase III Is the Replicative Polymerase in E. coli 377

A DNA Sliding Clamp Increases the Speed and Processivity of the Chromosomal Replicase 379

Many Different Proteins Advance a Replication Fork 381

Helicase Activity Is Stimulated by Its Connection to the DNA Polymerase 384

DNA Loops Repeatedly Grow and Collapse on the Lagging Strand 384

Okazaki Fragments Require Removal of RNA and Ligase-Mediated Joining of DNA 386

The Replication Fork Is More Complex in Eukaryotes Than in Bacteria 387

11.4 Initiation of DNA Replication 391

Assembly of the Replication Fork Follows an Ordered Sequence of Events 391

Replication Initiation in E. coli Is Controlled at Multiple Steps 393

Eukaryotic Origins “Fire” Only Once per Cell Cycle 394

HIGHLIGHT 11-1 TECHNOLOGY Two-Dimensional Gel Analysis of Replication Origins 396

11.5 Termination of DNA Replication 398

E. coli Chromosome Replication Terminates Opposite the Origin 398

Telomeres and Telomerase Solve the End Replication Problem in Eukaryotes 399

Telomere Length Is Associated with Immortality and Cancer 401

Telomeres are Protected and Regulated by Proteins 401

HIGHLIGHT 11-2 MEDICINE Short Telomeres Portend Aging Diseases 403

HOW WE KNOW 406

DNA Polymerase Reads the Sequence of the DNA Template to Copy the DNA 406

Polymerase Processivity Depends on a Circular Protein That Slides along DNA 407

Replication Requires an Origin 408

12

DNA Mutation and Repair

413

MOMENT OF DISCOVERY Rose Byrne, on her discovery that E. coli could become a radiation-resistant extremophile 413

12.1 Types of DNA Mutations 414

A Point Mutation Can Alter One Amino Acid 415

Small Insertion and Deletion Mutations Change Protein Length 416

Some Mutations Are Very Large and Form Abnormal Chromosomes 418

12.2 DNA Alterations That Lead to Mutations 420

Spontaneous DNA Damage by Water Can Cause Point Mutations 421

Oxidative Damage and Alkylating Agents Can Create Point Mutations and Strand Breaks 422

The Ames Test Identifies DNA-Damaging Chemicals 423

DNA-Damaging Agents Are Used in Cancer Chemotherapy 425

Solar Radiation Causes Interbase Cross-Links and Strand Breaks 425

Errant Replication and Recombination Lead to DNA Damage 428

12.3 Mechanisms of DNA Repair 428

Mismatch Repair Fixes Misplaced-Nucleotide Replication Errors 428

Direct Repair Corrects a Damaged Nucleotide Base in One Step 430

HIGHLIGHT 12-1 MEDICINE Mismatch Repair and Colon Cancer 433

Base Excision Repairs Subtle Alterations in Nucleotide Bases 435

Nucleotide Excision Repair Removes Bulky Damaged Bases 437

HIGHLIGHT 12-2 MEDICINE Nucleotide Excision Repair and Xeroderma Pigmentosum 439

Recombination Repairs Lesions That Break DNA 440

Specialized Translesion DNA Polymerases Extend DNA Past a Lesion 440

HOW WE KNOW 443

Mismatch Repair in E. coli Requires DNA Methylation 443

UV Lights Up the Pathway to DNA Damage Repair 444

Translesion DNA Polymerases Produce DNA Mutations 445

13

Recombinational DNA Repair and Homologous Recombination

449

MOMENT OF DISCOVERY Lorraine Symington, on discovering how DNA ends are processed to initiate DNA recombination 449

13.1 Recombination as a DNA Repair Process 451

Double-Strand Breaks Are Repaired by Recombination 452

Collapsed Replication Forks Are Reconstructed by Double-Strand Break Repair 453

A Stalled Replication Fork Requires Fork Regression 454

Single-Stranded DNA Regions Are Filled In by Gap Repair 456

13.2 Enzymatic Machines in Bacterial Recombinational DNA Repair 457

RecBCD and RecFOR Initiate Recombinational Repair 457

RecA Protein Is the Bacterial Recombinase 459

RecA Protein Is Subject to Regulation 461

Multiple Enzymes Process DNA Intermediates Created by RecA 463

HIGHLIGHT 13-1 EVOLUTION A Tough Organism in a Tough Environment: Deinococcus radiodurans 464

Repair of the Replication Fork in Bacteria Can Lead to Dimeric Chromosomes 466

13.3 Homologous Recombination in Eukaryotes 467

HIGHLIGHT 13-2 MEDICINE Why Proper Chromosomal Segregation Matters 468

Meiotic Recombination Is Initiated at Double-Strand Breaks 469

Meiotic Recombination Is Completed by a Classic DSBR Pathway 471

Meiotic Recombination Contributes to Genetic Diversity 471

Recombination during Mitosis Is Also Initiated at Double-Strand Breaks 472

Programmed Gene Conversion Events Can Affect Gene Function and Regulation 473

Some Introns Move via Homologous Recombination 475

13.4 Nonhomologous End Joining 475

Nonhomologous End Joining Repairs Double-Strand Breaks 475

Nonhomologous End Joining Is Promoted by a Set of Conserved Enzymes 476

Recombination Systems Are Being Harnessed for Genome Editing 477

HOW WE KNOW 479

A Motivated Graduate Student Inspires the Discovery of Recombination Genes in Bacteria 479

A Biochemical Masterpiece Catches a Recombination Protein in the Act 480

14

Site-Specific Recombination and Transposition

485

MOMENT OF DISCOVERY Wei Yang, on researching the structure and molecular mechanisms of γδ resolvase 485

14.1 Mechanisms of Site-Specific Recombination 487

Precise DNA Rearrangements Are Promoted by Site-Specific Recombinases 487

Site-Specific Recombination Complements Replication 490

Site-Specific Recombination Can Be a Stage in a Viral Infection Cycle 491

Site-Specific Recombination Systems Are Used in Biotechnology 491

Gene Expression Can Be Regulated by Site-Specific Recombination 492

HIGHLIGHT 14-1 TECHNOLOGY Using Site-Specific Recombination to Trace Neurons 494

14.2 Mechanisms of Transposition 496

Transposition Takes Place by Three Major Pathways 496

Bacteria Have Three Common Classes of Transposons 500

Retrotransposons Are Especially Common in Eukaryotes 502

HIGHLIGHT 14-2 EVOLUTION Awakening Sleeping Beauty 503

Retrotransposons and Retroviruses Are Closely Related 504

A Retrovirus Causes AIDS 506

HIGHLIGHT 14-3 MEDICINE Fighting AIDS with HIV Reverse Transcriptase Inhibitors 507

14.3 The Evolutionary Interplay of Transposons and Their Hosts 508

Viruses, Transposons, and Introns Have an Interwoven Evolutionary History 508

A Hybrid Recombination Process Assembles Immunoglobulin Genes 510

HOW WE KNOW 513

Bacteriophage λ Provided the First Example of Site-Specific Recombination 513

If You Leave Out the Polyvinyl Alcohol, Transposition Gets Stuck 514

15

Transcription: DNA-Dependent Synthesis of RNA

519

MOMENT OF DISCOVERY Robert Tjian, on discovering the first specific eukaryotic transcription factor 519

15.1 RNA Polymerases and Transcription Basics 520

RNA Polymerases Differ in Details but Share Many Features 520

HIGHLIGHT 15-1 A CLOSER LOOK The ABCs of RNA: Complexity of the Transcriptome 521

Transcription Initiation, Elongation, and Termination Occur in Discrete Steps 524

DNA-Dependent RNA Polymerases Can Be Specifically Inhibited 524

Transcriptional Regulation Is a Central Mechanism in the Control of Gene Expression 526

15.2 Transcription in Bacteria 527

Promoter Sequences Alter the Strength and Frequency of Transcription 527

Sigma Factors Specify Polymerase Binding to Particular Promoters 529

Structural Changes Lead to Formation of the Transcription-Competent Open Complex 531

Initiation Is Primer-Independent and Produces Short, Abortive Transcripts 531

Transcription Elongation Is Continuous until Termination 533

Specific Sequences in the Template Strand Stop Transcription 535

15.3 Transcription in Eukaryotes 537

Eukaryotic Polymerases Recognize Characteristic Promoters 537

HIGHLIGHT 15-2 MEDICINE Using Transcription Factors to Reprogram Cells 538

Pol II Transcription Parallels Bacterial RNA Transcription 540

Transcription Factors Play Specific Roles in the Transcription Process 540

Transcription Initiation In Vivo Requires the Mediator Complex 543

Termination Mechanisms Vary among RNA Polymerases 544

Transcription Is Coupled to DNA Repair, RNA Processing, and mRNA Transport 545

HOW WE KNOW 547

RNA Polymerase Is Recruited to Promoter Sequences 547

RNA Polymerases Are Both Fast and Slow 548

16

RNA Processing

553

MOMENT OF DISCOVERY Melissa Jurica, on determining the first electron microscopic structures of spliceosomes 553

16.1 Messenger RNA Capping and Polyadenylation 555

Eukaryotic mRNAs Are Capped at the 5′ End 555

Eukaryotic mRNAs Have a Distinctive 3′-End Structure 557

mRNA Capping, Polyadenylation, and Splicing Are Coordinately Regulated during Transcription 557

HIGHLIGHT 16-1 EVOLUTION Eukaryotic mRNAs with Unusual 3′ Tails 558

16.2 Pre-mRNA Splicing and Editing 559

Eukaryotic mRNAs Are Synthesized as Precursors Containing Introns 560

Alternative RNA Splicing Can Generate Multiple Products from a Gene 561

The Spliceosome Catalyzes Most Pre-mRNA Splicing 562

Some Introns Can Self-Splice without Protein or Spliceosome Assistance 564

Exons from Different RNA Molecules Can Be Fused by Trans-Splicing 568

RNA Editing Can Involve the Insertion or Deletion of Bases 569

HIGHLIGHT 16-2 EVOLUTION The Origin of Introns 570

RNA Editing by Substitution Involves Deamination of A or C Residues 571

16.3 RNA Transport and Degradation 573

Different Kinds of RNA Use Different Nuclear Export Pathways 573

mRNA Export from the Nucleus Is Coupled to Pre-mRNA Splicing 574

Some mRNAs Are Localized to Specific Regions of the Cytoplasm 575

Cellular mRNAs Are Degraded at Different Rates 575

Processing Bodies Are the Sites of mRNA Storage and Degradation in Eukaryotic Cells 576

16.4 Processing of Non-Protein-Coding RNAs 577

Maturation of tRNAs Involves Site-Specific Cleavage and Chemical Modification 577

Maturation of rRNA Involves Site-Specific Cleavage and Chemical Modification 578

Small Regulatory RNAs Are Derived from Larger Precursor Transcripts 579

16.5 RNA Catalysis and the RNA World Hypothesis 580

Ribozymes Catalyze Similar Kinds of Reactions But Have Diverse Functions 580

HIGHLIGHT 16-3 EVOLUTION A Viral Ribozyme Derived from the Human Genome? 581

Could RNA Have Formed the Basis for Early Life on Earth? 581

HOW WE KNOW 583

Studying Autoimmunity Led to the Discovery of snRNPs 583

RNA Molecules Are Fine-Tuned for Stability or Function 584

Ribozyme Form Explains Function 585

17

The Genetic Code

589

MOMENT OF DISCOVERY Steve Benner, on discovering that borate minerals stabilize ribose 589

17.1 Deciphering the Genetic Code: tRNA as Adaptor 590

All tRNAs Have a Similar Structure 591

The Genetic Code Is Degenerate 592

Wobble Enables One tRNA to Recognize Two or More Codons 593

Specific Codons Start and Stop Translation 594

The Genetic Code Resists Single-Base Substitution Mutations 595

Some Mutations Are Suppressed by Special tRNAs 596

17.2 The Rules of the Code 597

The Genetic Code Is Nonoverlapping 597

There Are No Gaps in the Genetic Code 598

The Genetic Code Is Read in Triplets 599

Protein Synthesis Is Linear 599

17.3 Cracking the Code 600

Random Synthetic RNA Polymers Direct Protein Synthesis in Cell Extracts 600

RNA Polymers of Defined Sequence Complete the Code 602

The Genetic Code Is Validated in Living Cells 604

17.4 Exceptions Proving the Rules 604

Evolution of the Translation Machinery Is a Mystery 604

Mitochondrial tRNAs Deviate from the Universal Genetic Code 605

HIGHLIGHT 17-1 EVOLUTION The Translation Machinery 606

Initiation and Termination Rules Have Exceptions 608

HOW WE KNOW 610

Transfer RNA Connects mRNA and Protein 610

Proteins Are Synthesized from the N-Terminus to the C-Terminus 611

The Genetic Code In Vivo Matches the Genetic Code In Vitro 612

18

Protein Synthesis

617

MOMENT OF DISCOVERY Harry Noller, on discovering the functional importance of ribosomal RNA 617

18.1 The Ribosome 618

The Ribosome Is an RNA-Protein Complex Composed of Two Subunits 619

Ribosomal Subunits Associate and Dissociate in Each Cycle of Translation 621

The Ribosome Is a Ribozyme 622

The Ribosome Structure Facilitates Peptide Bond Formation 623

HIGHLIGHT 18-1 EVOLUTION Mitochondrial Ribosomes: A Window into Ribosome Evolution? 624

18.2 Activation of Amino Acids for Protein Synthesis 626

Amino Acids Are Activated and Linked to Specific tRNAs 626

Aminoacyl-tRNA Synthetases Attach the Correct Amino Acids to Their tRNAs 626

The Structure of tRNA Allows Accurate Recognition by tRNA Synthetases 628

Proofreading Ensures the Fidelity of Aminoacyl-tRNA Synthetases 628

18.3 Initiation of Protein Synthesis 630

HIGHLIGHT 18-2 TECHNOLOGY Genetic Incorporation of Unnatural Amino Acids into Proteins 631

Base Pairing Recruits the Small Ribosomal Subunit to Bacterial mRNAs 631

Eukaryotic mRNAs Recruit the Small Ribosomal Subunit Indirectly 632

A Specific Amino Acid Initiates Protein Synthesis 632

Initiation in Bacterial Cells Requires Three Initiation Factors 635

Initiation in Eukaryotic Cells Requires Additional Initiation Factors 636

Some mRNAs Use 5’ End–Independent Mechanisms of Initiation 637

18.4 Elongation and Termination of the Polypeptide Chain 639

Peptide Bonds Are Formed in the Translation Elongation Stage 639

Substrate Positioning and the Incoming tRNA Contribute to Peptide Bond Formation 640

EF-G Drives Translocation by Displacing the A-Site tRNA 641

GTP Binding and Hydrolysis Regulate Successive Elongation Cycles 642

An mRNA Stop Codon Signals Completion of a Polypeptide Chain 643

Ribosome Recycling Factor Prepares Ribosomes for New Rounds of Translation 644

Fast and Accurate Protein Synthesis Requires Energy 644

Antibiotics and Toxins Frequently Target Protein Synthesis 646

HIGHLIGHT 18-3 Medicine Toxins That Target the Ribosome 647

18.5 Translation-Coupled Removal of Defective mRNA 650

Ribosomes Stalled on Truncated mRNAs Are Rescued by tmRNA 650

Eukaryotes Have Other Mechanisms to Detect Defective mRNAs 651

18.6 Protein Folding, Covalent Modification, and Targeting 653

Protein Folding Sometimes Requires the Assistance of Chaperones 653

Covalent Modifications Are Common in Newly Synthesized Proteins 653

Proteins Are Targeted to Correct Locations during or after Synthesis 654

Some Chemical Modifications of Eukaryotic Proteins Take Place in the Endoplasmic Reticulum 654

Glycosylation Plays a Key Role in Eukaryotic Protein Targeting 655

Signal Sequences for Nuclear Transport Are Not Removed 656

Bacteria Also Use Signal Sequences for Protein Targeting 657

HOW WE KNOW 659

The Ribosome Is a Ribozyme 659

Ribosomes Check the Accuracy of Codon-Anticodon Pairing, but Not the Identity of the Amino Acid 660

IV

REGULATION

19

Regulating the Flow of Information

665

MOMENT OF DISCOVERY Lin He, on discovering that microRNA overexpression accelerates tumor development 665

19.1 Regulation of Transcription Initiation 667

Activators and Repressors Control RNA Polymerase Function at a Promoter 667

Transcription Factors Can Function by DNA Looping 668

Regulators Often Work Together for Signal Integration 670

Gene Expression Is Regulated through Feedback Loops 671

Related Sets of Genes Are Often Regulated Together 672

Eukaryotic Promoters Use More Regulators Than Bacterial Promoters 672

Multiple Regulators Provide Combinatorial Control 673

Regulation by Nucleosomes Is Specific to Eukaryotes 674

19.2 The Structural Basis of Transcriptional Regulation 675

Transcription Factors Interact with DNA and Proteins through Structural Motifs 675

Transcription Activators Have Separate DNA-Binding and Regulatory Domains 679

19.3 Posttranscriptional Regulation of Gene Expression 680

Some Regulatory Mechanisms Act on the Nascent RNA Transcript 680

Small RNAs Can Affect mRNA Stability 681

Some Genes Are Regulated at the Level of Translation 681

Some Covalent Modifications Regulate Protein Function 682

Gene Expression Can Be Regulated by Intracellular Localization 682

HIGHLIGHT 19-1 Medicine Insulin Regulation: Control by Phosphorylation 684

Protein Degradation by Ubiquitination Modulates Gene Expression 686

HOW WE KNOW 689

Plasmids Have the Answer to Enhancer Action 689

20

The Regulation of Gene Expression in Bacteria

693

MOMENT OF DISCOVERY Bonnie Bassler, on her discovery of interspecies quorum sensing 693

20.1 Transcriptional Regulation 694

The lac Operon Is Subject to Negative Regulation 694

The lac Operon Also Undergoes Positive Regulation 699

HIGHLIGHT 20-1 TECHNOLOGY Classical Techniques in the Analysis of Gene Regulation 700

CRP Functions with Activators or Repressors to Control Gene Transcription 702

Transcription Attenuation Often Controls Amino Acid Biosynthesis 704

The SOS Response Leads to Coordinated Transcription of Many Genes 705

20.2 Beyond Transcription: Control of Other Steps in the Gene Expression Pathway 707

RNA Sequences or Structures Can Control Gene Expression Levels 707

Translation of Ribosomal Proteins Is Coordinated with rRNA Synthesis 711

HIGHLIGHT 20-2 A CLOSER LOOK T-Box Riboswitches 713

20.3 Control of Gene Expression in Bacteriophages 715

Phage Propagation Can Take One of Two Forms 716

Differential Activation of Promoters Regulates λ Phage Infection 717

The λ Repressor Functions as Both an Activator and a Repressor 718

More Regulation Levels Are Invoked during the λ Phage Life Cycle 719

HOW WE KNOW 722

TRAPped RNA Inhibits Expression of Tryptophan Biosynthetic Genes in Bacillus subtilis 722

Autoinducer Analysis Reveals Possibilities for Treating Cholera 723

21

The Transcriptional Regulation of Gene Expression in Eukaryotes

727

MOMENT OF DISCOVERY Tracy Johnson, on discovering that pre-mRNA splicing requires specific histone acetylation 727

21.1 Basic Mechanisms of Eukaryotic Transcriptional Activation 728

Eukaryotic Transcription Is Regulated by Chromatin Structure 728

Positive Regulation of Eukaryotic Promoters Involves Multiple Protein Activators 730

HIGHLIGHT 21-1 A CLOSER LOOK The Intertwining of Transcription and mRNA Splicing 732

Transcription Activators and Coactivators Help Assemble General Transcription Factors 733

21.2 Combinatorial Control of Gene Expression 736

Combinatorial Control of the Yeast GAL Genes Involves Positive and Negative Regulation 736

HIGHLIGHT 21-2 TECHNOLOGY Discovering and Analyzing DNA-Binding Proteins 738

Combinatorial Control of Transcription Causes Mating-Type Switches in Yeast 738

Combinatorial Mixtures of Heterodimers Regulate Transcription 740

Differentiation Requires Extensive Use of Combinatorial Control 741

21.3 Transcriptional Regulation Mechanisms Unique to Eukaryotes 743

Insulators Separate Adjacent Genes in a Chromosome 743

Some Activators Assemble into Enhanceosomes 744

Gene Silencing Can Inactivate Large Regions of Chromosomes 745

Imprinting Allows Selective Gene Expression from One Allele Only 745

HIGHLIGHT 21-3 A CLOSER LOOK Gene Silencing by Small RNAs 746

Dosage Compensation Balances Gene Expression from Sex Chromosomes 747

Steroid Hormones Bind Nuclear Receptors That Regulate Gene Expression 749

Nonsteroid Hormones Control Gene Expression by Triggering Protein Phosphorylation 750

HOW WE KNOW 753

Transcription Factors Bind Thousands of Sites in the Fruit Fly Genome 753

Muscle Tissue Differentiation Reveals Surprising Plasticity in the Basal Transcription Machinery 754

22

The Posttranscriptional Regulation of Gene Expression in Eukaryotes

759

MOMENT OF DISCOVERY Judith Kimble, on the discovery that noncoding regions of mRNA regulate cell fate 759

22.1 Posttranscriptional Control inside the Nucleus 760

Alternative Splicing Controls Sex Determination in Fruit Flies 761

Multiple mRNA Cleavage Sites Allow the Production of Multiple Proteins 762

Nuclear Transport Regulates Which mRNAs Are Selected for Translation 764

22.2 Translational Control in the Cytoplasm 765

Initiation Can Be Suppressed by Phosphorylation of eIF 2766

The 3′UTR of Some mRNAs Controls Translational Efficiency 766

Upstream Open Reading Frames Control the Translation of GCN4 mRNA 768

mRNA Degradation Rates Can Control Translational Efficiency 769

22.3 The Large-Scale Regulation of Groups of Genes 770

Some Sets of Genes Are Regulated by Pre-mRNA Splicing in the Nucleus 770

5′UTRs and 3′UTRs Coordinate the Translation of Multiple mRNAs 771

HIGHLIGHT 22-1 EVOLUTION Regulation of Splicing in Response to Stress 771

Conserved AU-Rich Elements in 3′UTRs Control Global mRNA Stability for Some Genes 772

22.4 RNA Interference 774

Eukaryotic MicroRNAs Target mRNAs for Gene Silencing 774

Short Interfering RNAs Target mRNAs for Degradation 776

RNAi Pathways Regulate Viral Gene Expression 777

RNAi Provides a Useful Tool for Molecular Biologists 778

HIGHLIGHT 22-2 MEDICINE Viral Takeover Using a Cell Type–Specific miRNA 779

RNAs Regulate a Wide Range of Cellular Processes 780

22.5 Putting It All Together: Gene Regulation in Development 781

Development Depends on Asymmetric Cell Divisions and Cell-Cell Signaling 781

Early Development Is Mediated by Maternal Genes 784

Segmentation Genes Specify the Development of Body Segments and Tissues 785

Homeotic Genes Control the Development of Organs and Appendages 787

Stem Cells Have Developmental Potential That Can Be Controlled 788

22.6 Finale: Molecular Biology, Developmental Biology, and Evolution 791

The Interface of Evolutionary and Developmental Biology Defines a New Field 791

Small Genetic Differences Can Produce Dramatic Phenotypic Changes 792

HOW WE KNOW 794

A Natural Collaboration Reveals a Binding Protein for a 3′UTR 794

Little RNAs Play a Big Role in Controlling Gene Expression 795

Everything Old Is New Again: Beauty at the Turn of a Developmental Switch 796

Model Organisms Appendix

A-1

A Few Organisms Are Models for Understanding Common Life Processes A-1

Three Approaches Are Used to Study Human Disease A-2

Bacterium, Escherichia coli

A-6

Early Studies of E. coli as a Model Organism A-6

Life Cycle A-6

Genetic Techniques A-7

E. coli as a Model Organism Today A-7

Budding Yeast, Saccharomyces cerevisiae

A-8

Early Studies of Yeast as a Model Organism A-8

Life Cycle A-8

Genetic Techniques A-8

Yeast as a Model Organism Today A-9

Bread Mold, Neurospora crassa

A-10

Early Studies of Neurospora as a Model Organism A-10

Life Cycle A-10

Genetic Techniques A-11

Neurospora as a Model Organism Today A-11

Nematode, Caenorhabditis elegans

A-12

Early Studies of C. elegans as a Model Organism A-12

Life Cycle A-12

Genetic Techniques A-13

C. elegans as a Model Organism Today A-13

Mustard Weed, Arabidopsis thaliana

A-14

Early Studies of Arabidopsis as a Model Organism A-14

Life Cycle A-14

Genetic Techniques A-15

Arabidopsis as a Model Organism Today A-15

Fruit Fly, Drosophila melanogaster

A-16

Early Studies of Drosophila as a Model Organism A-16

Life Cycle A-16

Genetic Techniques A-17

Drosophila as a Model Organism Today A-17

House Mouse, Mus musculus

A-18

Early Studies of the Mouse as a Model Organism A-18

Life Cycle A-18

Genetic Techniques A-18

The Mouse as a Model Organism Today A-19

Glossary

G-1

Solutions to Problems

S-1

Index

I-1