One of my most exciting educational experiences was my introductory molecular biology course in graduate school. My professor used no textbook, but assigned us readings directly from the scientific literature. It was challenging, but I found it immensely satisfying to meet the challenge and understand, not only the conclusions, but how the evidence supported those conclusions.
When I started teaching my own molecular biology course, I adopted this same approach, but tried to reduce the challenge to a level more appropriate for undergraduate students. I did this by narrowing the focus to the most important experiments in each article, and explaining those carefully in class. I used hand-drawn cartoons and photocopies of the figures as illustrations.
This approach worked well, and the students enjoyed it, but I really wanted a textbook that presented the concepts of molecular biology, along with experiments that led to those concepts. I wanted clear explanations that showed students the relationship between the experiments and the concepts. So, I finally decided that the best way to get such a book would be to write it myself. I had already coauthored a successful introductory genetics text in which I took an experimental approach—as much as possible with a book at that level. That gave me the courage to try writing an entire book by myself and to treat the subject as an adventure in discovery.
Organization
The book begins with a four-chapter sequence that should be a review for most students. Chapter 1 is a brief history of genetics. Chapter 2 discusses the structure and chemical properties of DNA. Chapter 3 is an overview of gene expression, and Chapter 4 deals with the nuts and bolts of gene cloning. All these are topics that the great majority of molecular biology students have already learned in an introductory genetics course. Still, students of molecular-biology need to have a grasp of these concepts and may need to refresh their understanding of them. I do not deal specifically with these chapters in class; instead, I suggest students consult them if they need more work on these topics. These chapters are written at a more basic level than the rest of the book.
Chapter 5 describes a number of common techniques used by molecular biologists. It would not have been possible to include all the techniques described in this book in one chapter, so I tried to include the most common or, in a few cases, valuable techniques that are not mentioned elsewhere in the book. When I teach this course，I do not present Chapter 5 as such. Instead, I refer students to it when we first encounter a technique in a later chapter. I do it that way to avoid boring my students with technique after technique. I also realize that the concepts behind some of these techniques are rather sophisticated, and the students’ appreciation of them is much deeper after they’ve acquired more experience in molecular biology.
Chapters 6-9 describe transcription in bacteria. Chapter 6 introduces the basic transcription apparatus, including promoters, terminators, and RNA polymerase, and shows how transcripts are initiated, elongated, and terminated. Chapter 7 describes the control of transcription in three different operons, then Chapter 8 shows how bacteria and their phages control transcription of many genes at a time, often by providing alternative sigma factors. Chapter 9 discusses the interaction between bacterial DNA-bindiπg proteins, mostly helix-turn-helix proteins, and their DNA targets.
Chapters 10-13 present control of transcription in eukaryotes. Chapter 10 deals with the three eukaryotic RNA polymerases and the promoters they recognize. Chapter 11 introduces the general transcription factors that collaborate with the three RNA polymerases and points out the unifying theme of the TATA-box-binding protein，which participates in transcription by all three polymerases. Chapter 12 explains the functions of gene-specific transcription factors, or activators. This chapter also illustrates the structures of several representative activators and shows how they interact with their DNA targets. Chapter 13 describes the structure of eukaryotic chromatin and shows how activators and silencers can interact with coactivators and corepressors to modify histones, and thereby to activate or repress transcription.
Chapters 14-16 introduce some of the posttranscrip-tioπal events that occur in eukaryotes. Chapter 14 deals with RNA splicing. Chapter 15 describes capping and polyadenylation, and Chapter 16 introduces a collection of fascinating “other posttranscriptional events，” including rRNA and tRNA processing, ^mws-splicing, and RNA editing. This chapter also discusses four kinds of posttranscriptional control of gene expression: (1) RNA interference; (2) modulating mRNA stability (using the transferrin receptor mRNA as the prime example); (3) control by microRNAs, and (4) control of transposons in germ cells by Piwi-interacting RNAs (piRNAs).
Chapters 17-19 describe the translation process in both bacteria and eukaryotes. Chapter 17 deals with initiation of translation, including the control of translation at the initiation step. Chapter 18 shows how polypeptides are elongated, with the emphasis on elongation in bacteria. Chapter 19 provides details on the structure and function of two of the key players in translation： ribosomes and tRNA.
Chapters 20-23 describe the mechanisms of DNA replication, recombination, and translocation. Chapter 20 introduces the basic mechanisms of DNA replication and repair, and some of the proteins (including the DNA polymerases) involved in replication. Chapter 21 provides details of the initiation, elongation, and termination steps in DNA replication in bacteria and eukaryotes. Chapters 22 and 23 describe DNA rearrangements that occur naturally in cells. Chapter 22 discusses homologous recombination and Chapter 23 deals with translocation.
Chapters 24 and 25 present concepts of genomics, pro-teomics, and bioinformatics. Chapter 24 begins with an old-fashioned positional cloning story involving the Huntington disease gene and contrasts this lengthy and heroic quest with the relative ease of performing positional cloning with the human genome (and other genomes). Chapter 25 deals with functional genomics (transcriptomics), proteomics, and bioinformatics.
New to the Fifth Edition
The most obvious change in the fifth edition is the splitting of old Chapter 24 (Genomics, Proteomics5 and Bioinformatics) in two. This chapter was already the longest in the book, and the field it represents is growing explosively, so a split was inevitable. The new Chapter 24 deals with classical genomics： the sequencing and comparison of genomes. New material in Chapter 24 includes an analysis of the similarity between the human and chimpanzee genomes, and a look at the even closer similarity between the human and Neanderthal genomes, including recent evidence for interbreeding between humans and Neanderthals. It also includes an update on the new field of synthetic biology, made possible by genomic work on microorganisms, and contains a report of the recent success by Craig Venter and colleagues in creating a living Mycoplasma cell with a synthetic genome.
Chapter 25 deals with fields allied with Genomics: Functional Genomics, Proteomics, and Bioinformatics. New material in Chapter 25 includes new applications of the ChIP-chip and ChIP-seq techniques—the latter using next-generation DNA sequencing; collision-induced dissociation mass spectrometry, which can be used to sequence proteins; and the use of isotope-coded affinity tags (ICATs) and stable isotope labeling by amino acids (SILAC) to make mass spectrometry (MS) quantitative. Quantitative MS in turn enables comparative proteomics, in which the concentrations of large numbers of proteins can be compared between species.
All but the introductory chapters of this fifth edition have been updated. Here are a few highlights:
• Chapter 5: Introduces high-throughput (next generation) DNA sequencing techniques. These have revolutionized the field of genomics. Chromatin immunoprecipitation (ChIP) and the yeast two-hybrid assay have been moved to Chapter 5, in light of their broad applicabilities. A treatment of the energies of the β-electrons from 3H, 14C, 35S, and 32P has been added, and the fluorography technique, which captures information from the lower-energy emissions, is discussed.
• Chapter 6: Adds a discussion of FRET-ALEX (FRET with alternating laser excitation), along with a description of how this technique has been used to support (1) the stochastic release model of the σ-cycle and (2) the scrunching hypothesis to explain abortive transcription. This chapter also updates the structure of the bacterial elongation complex, including a discussion of a two-state model for nucleotide addition.
• Chapter 7: Introduces the riboswitch in the mRNA from the glmS gene of B. subtilis9 in which the end product of the gene turns expression of the gene off by stimulating the mRNA to destroy itself. This chapter also introduces a hammerhead ribozyme as a possible mammalian riboswitch that may operate by a similar mechanism.
• Chapter 8: Introduces the concepts of anti-σ-factors and anti-anti-σ-factors as controllers of transcription during sporulation in B. subtilis∙
• Chapter 9: Emphasizes the dynamic nature of protein structure, and points out that a given crystal structure represents just one of a range of different possible protein conformations.
• Chapter 10: Presents a new study by Roger Korn-berg’s group that identifies the RNA polymerase II trigger loop as a key determinant in transcription specificity, along with a discussion of how the enzyme distinguishes between ribonuncleotides and deoxyribonucleotides. This chapter also introduces the concepts of core promoter and proximal promoter, where the core promoter contains any combination of TFIIB recognition element, TATA box, initiator, downstream promoter element, downstream core element, and motif ten element, and the proximal promoter contains upstream promoter elements.
• Chapter 11: Introduces the concept of core TAFs— those associated with class II preinitiation complexes from a wide variety of eukaryotes, and introduces the new nomenclature (TAF1-TAF13), which replaces the old, confusing nomenclature that was based on molecular masses (e.g∙, TAF∏250). This chapter also describes an experiment that shows the importance of TFΠB in setting the start site of transcription. It also shows that a similar mechanism applies in the archaea, which use a TFIIB homolog known as transcription factor B.
• Chapter 12: Introduces the technique of chromosome conformation capture (3C) and shows how it can be used to detect DNA looping between an enhancer and a promoter. This chapter also introduces the concept of imprinting during gametogenesis, and explains the role of methylation in imprinting, particularly methylation o£ the imprinting control region of the mouse Igf2∕H 19 locus. It also introduces the concept of transcription factories, where transcription of multiple genes occurs. Finally, this chapter refines and updates the concept of the enhaπceosome.
• Chapter 13: Presents a new table showing all the ways histones can be modified in vivo; brings back the solenoid, alongside the two-start helix, as a candidate for the 30-nm fiber structure; and presents evidence that chromatin adopts one or the other structure, depending on its nucleosome repeat length. This chapter also introduces the concept of specific histone methylations as markers for transcription initiation and elongation, and shows how this information can be used to infer that RNA polymerase II is poised between initiation and elongation on many human protein-encoding genes. It also emphasizes the importance of histone modifications in affecting not only histone-DNA interactions, but also πucleosome-nucleosome interactions and recruitment of histone-modifying and chromatinremodeling proteins. Finally, this chapter shows how PARP1 (poly[ADP-ribose] polymerase-1) can facilitate nucleosome loss from chromatin by poly(ADP-ribosyl)atiπg itself.
• Chapter 14： Introduces the exon junction complex (EJC), which is added to mRNAs during splicing in the nucleus, and shows how the EJC can stimulate transcription by facilitating the association of mRNAs with ribosomes. This chapter also introduces exon and intron definition modes of splicing and shows how they can be distinguished experimentally. This test has revealed that higher eukaryotes primarily use exon definition and lower eukaryotes primarily use intron definition.
• Chapter 15: Demonstrates that a subunit of CPSF (CPSF-73) is responsible for cutting a pre-mRNA at a polyadenylation signal. It also shows that serine 7, in addition to serines 2 and 5 in the repeating heptad in the CTD of the largest RNA polymerase subunit, can be phosphorylated, and shows that this serine 7 phosphorylation controls the expression of certain genes (e.g,, the U2 snRNA gene) by controlling the 3'-end processing of their mRNAs.
• Chapter 16: Identifies a single enzyme, tRNA 3' processing endoribonuclease, as the agent that cleaves excess nucleotides from the 3'-end of a eukaryotic tRNA precursor; points out the overwhelming prevalence of trans-splicing in C. elegans; presents a new model for removal of the passenger strand of a double-stranded siRNA—cleavage of the passenger strand by Ago2; introduces Piwi-interacting RNAs (piRNAs) and presents the ping-pong model by which they are assumed to amplify themselves and inactivate transposons in germ cells; introduces plant RNA polymerases IV and V’ and describes their roles in gene silencing. This chapter also greatly expands the coverage of miRNAs, and points out that hundreds of miRNAs control thousands of plant and animal genes, and that mutations in miRNA genes typically have very deleterious effects. Chapter 16 also updates the biogenesis of miRNAs, introducing two pathways to miRNA production： the Drosha and mirtron pathways. Finally, this chapter introduces P-bodies，which are involved in mRNA decay and translational repression.
• Chapter 17: Updates the section on eukaryotic viral internal ribosome entry sequences (IRESs). Some viruses cleave eIF4G, leaving a remnant called pi00. Poliovirus IRESs bind to pi00 and thereby gain access to ribosomes, but hepatitis C virus IRESs bind directly to eIF3, while hepatitis A virus IRESs bind even more directly to ribosomes. This chapter also refines the model describing how the cleavage of eIF4G affects mammalian host mRNA translation. Different cell types respond differently to this cleavage. Finally, this chapter introduces the concept of the pioneer round of translation, and points out that different initiation factors are used in the pioneer round than in all subsequent rounds.
• Chapter 18: Introduces the concept of superwobble, which holds that a single tRNA with a U in its wobble position can recognize codons ending in any of the four bases, and presents evidence that superwobble works. This chapter also introduces the hybrid P/I state as the initial ribosomal binding state for fMet-tRNA ^zlet. In this state, the anticodon is in the P site, but the fMet and acceptor stem are in an “initiator” site between the P site and the E site. This chapter also describes no-go decay, which degrades mRNA containing a stalled ribosome, and introduces the concept of codon bias to explain inefficiency of translation. Finally, this chapter explains how the slowing of translation by rare codons can influence protein folding both negatively and positively.
• Chapter 19： Includes a new section based on recent crystal structures of the ribosome in complex with various elongation factors. One of these structures involves aminoacyl-tRNA and EF-Tu, and has shown that the tRNA is bent by about 30 degrees in forming an A/T complex. This bend is important in fidelity of translation, and also facilitates the GTP hydrolysis that permits EF-Tu to leave the ribosome. Another crystal structure involves EF-G-GDP and shows the ribosome in the post-translocation E∕E,P/P state, as opposed to the spontaneously achieved pre-translocation P∕E, A/P hybrid state. This chapter also provides links to two excellent new movies describing the elongation process and an overview of translation initiation, elongation, and termination. Finally, this chapter describes crystal structures that illustrate the functions of two critical parts of RF1 and RF2 in stop codon recognition and cleavage of polypeptides from their tRNAs∙
• Chapter 20: Introduces the controversial proposal, with evidence, that DNA replication in £. coli is discontinuous on both strands. This chapter also introduces ACL1, a chromatin remodeler recruited via its macrodomain to sites of double-strand breaks by poly(ADP-ribose) formed at these sites by poly(ADP-ribose) polymerase 1 (PARP-1).
• Chapter 21: Presents a co-crystal structure of a β dimer bound to a primed DNA template, showing that the β clamp really does encircle the DNA, but that the DNA runs through the circle at an angle of 20 degrees with respect to the horizontal. This chapter also includes a corrected and updated Figure 21.17 (model of the poll∏* subassembly) to show a single γ-subunit and the two τ-subunits joined to the core polymerases through their flexible C-terminal domains. This section also clarifies that the y- and τ-subunits are products of the same gene, but the former lacks the C-terminal domain of the latter. This chapter also introduces the complex of telomerebinding proteins known as shelterin, and focuses on the six shelterin proteins of mammals and their roles in protecting telomeres, and in preventing inappropriate repair and cell cycle arrest in response to normal chromosome ends.
• Chapter 22: Adds a new figure (Figure 22.3) to show how different nicking patterns to resolve the Holliday junction in the RecBCD pathway lead to different recombination products (crossover or noncrossover recombinants).
• Chapter 23: Reports that piRNAs targeting P element transρosons are likely to be the transposition suppressors in the P-M system. Similarly, piRNAs appear to play the suppressor role in the I-R trans-poson system.
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About the Author iv

Preface xiii

Acknowledgments xvii

Guide to Experimental Techniques in Molecular Biology xix

PART I Introduction

CHAPTER 1 A Brief History 1

      1.1 Transmission Genetics 2

      Mendel’s Laws of Inheritance 2

      The Chromosome Theory of Inheritance 3

      Genetic Recombination and Mapping 4

      Physical Evidence for Recombination 5

      1.2 Molecular Genetics 5

      The Discovery of DNA 5

      The Relationship Between Genes and Proteins 6

      Activities of Genes 7

      1.3 The Three Domains of Life 9

CHAPTER 2 The Molecular Nature of Genes 12

      2.1 The Nature of Genetic Material 13

      Transformation in Bacteria 13

      The Chemical Nature of Polynucleotides 15

      2.2 DNA Structure 18

      Experimental Background 19

      The Double Helix 19

      2.3 Genes Made of RNA 22

      2.4 Physical Chemistry of Nucleic Acids 23

      A Variety of DNA Structures 23

      DNAs of Various Sizes and Shapes 27

CHAPTER 3 An Introduction to Gene Function 30

      3.1 Storing Information 31

      Overview of Gene Expression 31

      Protein Structure 31

      Protein Function 35

      Discovery of Messenger RNA 37

      Transcription 39

      Translation 40

      3.2 Replication 45

      3.3 Mutations 45

      Sickle Cell Disease 45

PART II Methods of Molecular Biology

CHAPTER 4 Molecular Cloning Methods 49

      4.1 Gene Cloning 50

      The Role of Restriction Endonucleases 50

      Vectors 53

      Identifying a Specific Clone with a Specific Probe 58

      cDNA Cloning 60

      Rapid Amplification of cDNA Ends 61

      4.2 The Polymerase Chain Reaction 62

      Standard PCR 62

      Box 4.1 Jurassic Park: More than a Fantasy? 63

      Using Reverse Transcriptase PCR (RT-PCR) in cDNA Cloning 64

      Real-Time PCR 64

      4.3 Methods of Expressing Cloned Genes 65

      Expression Vectors 65

      Other Eukaryotic Vectors 71

      Using the Ti Plasmid to Transfer Genes to Plants 71

CHAPTER 5 Molecular Tools for Studying Genes and Gene Activity 75

      5.1 Molecular Separations 76

      Gel Electrophoresis 76

      Two-Dimensional Gel Electrophoresis 79

      Ion-Exchange Chromatography 80

      Gel Filtration Chromatography 80

      Affinity Chromatography 81

      5.2 Labeled Tracers 82

      Autoradiography 82

      Phosphorimaging 83

      Liquid Scintillation Counting 84

      Nonradioactive Tracers 84

      5.3 Using Nucleic Acid Hybridization 85

      Southern Blots: Identifying Specific DNA Fragments 85

      DNA Fingerprinting and DNA Typing 86

      Forensic Uses of DNA Fingerprinting and DNA Typing 87

      In Situ Hybridization： Locating Genes in Chromosomes 88

      Immunobiots (Western Blots) 89

      5.4 DNA Sequencing and Physical Mapping 89

      The Sanger Chain-Termination Sequencing Method 90

      Automated DNA Sequencing 91

      High-Throughput Sequencing 93

      Restriction Mapping 95

      5.5 Protein Engineering with Cloned Genes: Site-Directed Mutagenesis 97

      5.6 Mapping and Quantifying Transcripts 99

      Northern Blots 99

      SI Mapping 100

      Primer Extension 102

      Run-Off Transcription and G-Less Cassette Transcription 103

      5.7 Measuring Transcription Rates in Vivo 104

      Nuclear Run-On Transcription 104

      Reporter Gene Transcription 105

      Measuring Protein Accumulation in Vivo 106

      5.8 Assaying DNA-Protein Interactions 108

      Filter Binding 108

      Gel Mobility Shift 109

      DNase Footprinting 109

      DMS Footprinting and Other Footprinting Methods 109

      Chromatin Immunoprecipitation (ChIP) 112

      5.9 Assaying Protein-Protein Interactions 112

      5.10 Finding RNA Sequences That Interact with Other Molecules 114

      SELEX 114

      Functional SELEX 114

      5.11 Knockouts and Transgenics 115

      Knockout Mice 115

      Transgenic Mice 115

PART III Transcription in Bacteria

CHAPTER 6 The Mechanism of Transcription in Bacteria 121

      6.1 RNA Polymerase Structure 122

      Sigma (σ) as a Specificity Factor 122

      6.2 Promoters 123

      Binding of RNA Polymerase to Promoters 123

      Promoter Structure 125

      6.3 Transcription Initiation 126

      Sigma Stimulates Transcription Initiation 127

      Reuse of σ 128

      The Stochastic σ-Cycle Model 129

      Local DNA Melting at the Promoter 132

      Promoter Clearance 134

      Structure and Function of σ 139

      The Role of the α-Subunit in UP Element Recognition 142

      6.4 Elongation 144

      Core Polymerase Functions in Elongation 144

      Structure of the Elongation Complex 146

      6.5 Termination of Transcription 156

      Rho-Independent Termination 156

      Rho-Dependent Termination 159

CHAPTER 7 Operons: Fine Control of Bacterial Transcription 167

      7.1 The lac Oρeron 168

      Negative Control of the lac Operon 169

      Discovery of the Operon 169

      Repressor-Operator Interactions 173

      The Mechanism of Repression 174

      Positive Control of the lac Operon 177

      The Mechanism of CAP Action 178

      7.2 The ara Operon 182

      The ara Operon Repression Loop 183

      Evidence for the ara Operon Repression Loop 183

      Autoregulation of araC 185

      7.3 The trp Operon 186

      Tryptophan’s Role in Negative Control of the trp Operon 186

      Control of the trp Operon by Attenuation 187

      Defeating Attenuation 188

      7.4 Riboswitches 190

CHAPTER 8 Major Shifts in Bacterial Transcription 196

      8.1 Sigma Factor Switching 197

      Phage Infection 197

      Sporulation 199

      Genes with Multiple Promoters 201

      Other σ Switches 201

      Anti-σ-Factors 202

      8.2 The RNA Polymerase Encoded in Phage T7 202

      8.3 Infection of E. coli by Phage λ 203

      Lytic Reproduction of Phage λ 204

      Establishing Lysogeny 211

      Autoregulation of the cl Gene During Lysogeny 212

      Determining the Fate of a K Infection: Lysis or Lysogeny 217

      Lysogen Induction 218

CHAPTER 9 DNA-Protein Interactions in Bacteria 222

      9.1 The λ Family of Repressors 223

      Probing Binding Specificity by Site-Directed Mutagenesis 223

      Box 9.1 X-Ray Crystallography 224

      High-Resolution Analysis of λ Repressor-Operator Interactions 229

      High-Resolution Analysis of Phage 434

      Repressor-Operator Interactions 232

      9.2 The trp Repressor 234

      The Role of Tryptophan 234

      9.3 General Considerations on Protein-DNA Interactions 235

      Hydrogen Bonding Capabilities of the Four Different Base Pairs 235

      The Importance of Multimeric DNA-Binding Proteins 236

      9.4 DNA-Binding Proteins: Action at a Distance 237

      The gal Operon 237

      Duplicated λ Operators 237

      Enhancers 238

PART IV Transcription in Eukaryotes

CHAPTER 10 Eukaryotic RNA Polymerases and Their Promoters 244

      10.1 Multiple Forms of Eukaryotic RNA Polymerase 245

      Separation of the Three Nuclear Polymerases 245

      The Roles of the Three RNA Polymerases 246

      RNA Polymerase Subunit Structures 248

      10.2 Promoters 259

      Class II Promoters 259

      Class I Promoters 263

      Class III Promoters 264

      10.3 Enhancers and Silencers 267

      Enhancers 267

      Silencers 269

CHAPTER 11 General Transcription Factors in Eukaryotes 273

      11.1 Class II Factors 274

      The Class II Preinitiation Complex 274

      Structure and Function of TFIID 276

      Structure and Function of TFIIB 286

      Structure and Function of TFIIH 288

      The Mediator Complex and the RNA Polymerase II Holoenzyme 295

      Elongation Factors 296

      11.2 Class I Factors 299

      The Core-Binding Factor 299

      The UPE-Binding Factor 300

      Structure and Function of SL1 301

      11.3 Class III Factors 303

      TFIIIA 303

      TFIIIB and C 304

      The Role of TBP 307

CHAPTER 12 Transcription Activators in Eukaryotes 314

      12.1 Categories of Activators 315

      DNA-Binding Domains 315

      Transcription-Activating Domains 315

      12.2 Structures of the DNA-Binding Motifs of Activators 316

      Zinc Fingers 316

      The GAL4 Protein 318

      The Nuclear Receptors 319

      Homeodomains 320

      The bZIP and bHLH Domains 321

      12.3 Independence of the Domains of Activators 323

      12.4 Functions of Activators 324

      Recruitment of TFIID 324

      Recruitment of the Holoenzyme 325

      12.5 Interaction Among Activators 328

      Dimerization 328

      Action at a Distance 329

      Box 12.1 Genomic Imprinting 332

      Transcription Factories 334

      Complex Enhancers 336

      Architectural Transcription Factors 337

      Enhanceosomes 338

      Insulators 339

      12.6 Regulation of Transcription Factors 343

      Coactivators 344

      Activator Ubiquitylation 346

      Activator Sumoylation 347

      Activator Acetylation 348

      Signal Transduction Pathways 348

CHAPTER 13 Chromatin Structure and Its Effects on Transcription 355

      13.1 Chromatin Structure 356

      Histones 356

      Nucleosomes 357

      The 30-nm Fiber 360

      Higher-Order Chromatin Folding 362

      13.2 Chromatin Structure and Gene Activity 364

      The Effects of Histones on Transcription of Class II Genes 365

      Nucleosome Positioning 367

      Histone Acetylation 372

      Histone Deacetylation 373

      Chromatin Remodeling 376

      Heterochromatin and Silencing 383

      Nucleosomes and Transcription Elongation 387

PART V Post-Transcriptional Events

CHAPTER 14 RNA Processing I: Splicing 394

      14.1 Genes in Pieces 395

      Evidence for Split Genes 395

      RNA Splicing 396

      Splicing Signals 397

      Effect of Splicing on Gene Expression 398

      14.2 The Mechanism of Splicing of Nuclear mRNA Precursors 399

      A Branched Intermediate 399

      A Signal at the Branch 401

      Spliceosomes 402

      Spliceosome Assembly and Function 411

      Commitment, Splice Site Selection, and Alternative Splicing 415

      Control of Splicing 425

      14.3 Self-Splicing RNAs 427

      Group I Introns 427

      Group II Introns 430

CHAPTER 15 RNA Processing II: Capping and Polyadenylation 436

      15.1 Capping 437

      Cap Structure 437

      Cap Synthesis 438

      Functions of Caps 440

      15.2 Polyadenylation 442

      Poly(A) 442

      Functions of Poly(A) 443

      Basic Mechanism of Polyadenylation 445

      Polyadenylation Signals 446

      Cleavage and Polyadenylation of a Pre-mRNA 448

      Poly(A) Polymerase 454

      Turnover of Poly(A) 454

      15.3 Coordination of mRNA Processing Events 456

      Binding of the CTD of Rpbl to mRNA-Processing Proteins 457

      Changes in Association of RNA-Processing Proteins with the CTD Correlate with Changes in CTD Phosphorylation 458

      A CTD Code? 460

      Coupling Transcription Termination with mRNA 3'-End Processing 461

      Mechanism of Termination 462

      Role of Polyadenylation in mRNA Transport 466

CHAPTER 16 Other RNA Processing Events and Post-Transcriptional Control of Gene Expression 471

      16.1 Ribosomal RNA Processing 472

      Eukaryotic rRNA Processing 472

      Bacterial rRNA Processing 474

      16.2 Transfer RNA Processing 475

      Cutting Apart Polycistronic Precursors 475

      Forming Mature 5'-Ends 475

      Forming Mature 3’-Ends 476

      16.3 Tnzws-Sρlicing 477

      The Mechanism of Tn7ws-Splicing 477

      16.4 RNA Editing 479

      Mechanism of Editing 479

      Editing by Nucleotide Deamination 482

      16.5 Post-Transcriptional Control of Gene Expression: mRNA Stability 483

      Casein mRNA Stability 484

      Transferrin Receptor mRNA Stability 484

      16.6 Post-Transcriptional Control of Gene Expression: RNA Interference 488

      Mechanism of RNAi 489

      Amplification of siRNA 494

      Role of the RNAi Machinery in Heterochromatin Formation and Gene Silencing 495

      16.7 Piwi-Interacting RNAs and Transposon Control 501

      16.8 Post-Transcriptional Control of Gene Expression: MicroRNAs 502

      Silencing of Translation by miRNAs 502

      Stimulation of Translation by miRNAs 507

      16.9 Translation Repression, mRNA Degradation, and P-Bodies 510

      Processing Bodies 510

      Degradation of mRNAs in P-Bodies 511

      Relief of Repression in P-Bodies 514

      Other Small RNAs 517

PART VI Translation

CHAPTER 17 The Mechanism of Translation I: Initiation 522

      17.1 Initiation of Translation in Bacteria 523

      tRNA Charging 523

      Dissociation of Ribosomes 523

      Formation of the 305 Initiation Complex 525

      Formation of the 70S Initiation Complex 531

      Summary of Initiation in Bacteria 533

      17.2 Initiation in Eukaryotes 533

      The Scanning Model of Initiation 533

      Eukaryotic Initiation Factors 537

      17.3 Control of Initiation 545

      Bacterial Translational Control 545

      Eukaryotic Translational Control 548

CHAPTER 18 The Mechanism of Translation II: Elongation and Termination 560

      18.1 The Direction of Polypeptide Synthesis and of mRNA Translation 561

      18.2 The Genetic Code 562

      Nonoverlapping Codons 562

      No Gaps in the Code 563

      The Triplet Code 563

      Breaking the Code 564

      Unusual Base Pairs Between Codon and Anticodon 566

      The (Almost) Universal Code 567

      18.3 The Elongation Cycle 569

      Overview of Elongation 569

      A Three-Site Model of the Ribosome 570

      Elongation Step 1: Binding an Aminoacyl-tRNA to the A Site of the Ribosome 573

      Elongation Step 2: Peptide Bond Formation 577

      Elongation Step 3: Translocation 580

      G Proteins and Translation 582

      The Structures of EF-Tu and EF-G 583

      18.4 Termination 584

      Termination Codons 584

      Stop Codon Suppression 586

      Release Factors 586

      Dealing with Aberrant Termination 588

      Use of Stop Codons to Insert Unusual Amino Acids 593

      18.5 Posttranslation 593

      Folding Nascent Proteins 594

      Release of Ribosomes from mRNA 595

CHAPTER 19 Ribosomes and Transfer RNA 601

      19.1 Ribosomes 602

      Fine Structure of the 70S Ribosome 602

      Ribosome Composition 605

      Fine Structure of the 30S Subunit 606

      Fine Structure of the 50S Subunit 612

      Ribosome Structure and the Mechanism of Translation 616

      Polysomes 621

      19.2 Transfer RNA 623

      The Discovery of tRNA 623

      tRNA Structure 623

      Recognition of tRNAs by Aminoacyl-tRNA Synthetase: The Second Genetic Code 626

      Proofreading and Editing by Aminoacyl-tRNA Synthetases 630

PART VII DNA Replication, Recombination, and Transposition

CHAPTER 20 DNA Replication, Damage, and Repair 636

      20.1 General Features of DNA Replication 637

      Semiconservative Replication 637

      At Least Semidiscontinuous Replication 639

      Priming of DNA Synthesis 641

      Bidirectional Replication 642

      Rolling Circle Replication 645

      20.2 Enzymology of DNA Replication 646

      Three DNA Polymerases in E. coli 646

      Fidelity of Replication 649

      Multiple Eukaryotic DNA Polymerases 650

      Strand Separation 651

      Single-Strand DNA-Binding Proteins 651

      Topoisomerases 653

      20.3 DNA Damage and Repair 656

      Damage Caused by Alkylation of Bases 657

      Damage Caused by Ultraviolet Radiation 658

      Damage Caused by Gamma and X-Rays 658

      Directly Undoing DNA Damage 659

      Excision Repair 660

      Double-Strand Break Repair in Eukaryotes 665

      Mismatch Repair 667

      Failure of Mismatch Repair in Humans 668

      Coping with DNA Damage Without Repairing It 668

CHAPTER 21 DNA Replication II:Detailed Mechanism 677

      21.1 Initiation 678

      Priming in E. coli 678

      Priming in Eukaryotes 679

      21.2 Elongation 683

      Speed of Replication 683

      The Pol III Holoenzyme and Processivity of Replication 683

      21.3 Termination 694

      Decatenation： Disentangling Daughter DNAs 694

      Termination in Eukaryotes 695

      Box 21.1 Telomeres, the Hayflick Limit, and Cancer 699

      Telomere Structure and Telomere-Binding Proteins in Lower Eukaryotes 702

CHAPTER 22 Homologous Recombination 709

      22.1 The RecBCD Pathway for Homologous Recombination 710

      22.2 Experimental Support for the RecBCD Pathway 712

      RecA 712

      RecBCD 715

      RuvA and RuvB 717

      RuvC 719

      22.3 Meiotic Recombination 721

      The Mechanism of Meiotic Recombination: Overview 721

      The Double-Stranded DNA Break 722

      Creation of Single-Stranded Ends at DSBs 728

      22.4 Gene Conversion 728

CHAPTER 23 Transposition 732

      23.1 Bacterial Transposons 733

      Discovery of Bacterial Transposons 733

      Insertion Sequences: The Simplest Bacterial Transposons 733

      More Complex Transposons 734

      Mechanisms of Transposition 734

      23.2 Eukaryotic Transposons 737

      The First Examples of Transposable Elements:Ds and Ac of Maize 737

      P Elements 739

      23.3 Rearrangement of Immunoglobulin Genes 740

      Recombination Signals 742

      The Recombinase 743

      Mechanism of V(D)J Recombination 743

      23.4 Retrotransposons 745

      Retroviruses 745

      Retrotransposons 749

PART VIII Genomes

CHAPTER 24 Introduction to Genomics: DNA Sequencing on a Genomic Scale 759

      24.1 Positional Cloning: An Introduction to Genomics 760

      Classical Tools of Positional Cloning 760

      Identifying the Gene Mutated in a Human Disease 762

      24.2 Techniques in Genomic Sequencing 765

      The Human Genome Project 767

      Vectors for Large-Scale Genome Projects 769

      The Clone-by-Clone Strategy 770

      Shotgun Sequencing 773

      Sequencing Standards 774

      24.3 Studying and Comparing Genomic Sequences 774

      The Human Genome 774

      Personal Genomics 779

      Other Vertebrate Genomes 779

      The Minimal Genome 782

      The Barcode of Life 784

CHAPTER 25 Genomics II: Functional Genomics, Proteomics, and Bioinformatics 789

      25.1 Functional Genomics: Gene Expression on a Genomic Scale 790

      Transcriptomics 790

      Genomic Functional Profiling 799

      Single-Nucleotide Polymorphisms:Pharmacogenomics 810

      25.2 Proteomics 812

      Protein Separations 812

      Protein Analysis 813

      Quantitative Proteomics 814

      Protein Interactions 816

      25.3 Bioinformatics 820

      Finding Regulatory Motifs in Mammalian Genomes 820

      Using the Databases Yourself 822

Glossary 827

Index 856

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