Molecular biology / Robert Weaver.

B R I E F C O N T E N T S
About the Author iv
Preface xii
Acknowledgments xv
Guide to Experimental Techniques in Molecular
Biology xvi
P A R T I
Introduction
1 A Brief History 1
2 The Molecular Nature of Genes 17
3 Introduction to Gene Function 37
P A R T I I
Methods in Molecular Biology
4 Molecular Cloning Methods 58
5 Molecular Tools for Studying Genes and Gene
Activity 90
P A R T I I I
Transcription in Prokaryotes
6 The Mechanism of Transcription in
Prokaryotes 132
7 Operons: Fine Control of Prokaryotic
Transcription 182
8 Major Shifts in Prokaryotic Transcription 212
9 DNA-Protein Interactions in Prokaryotes 242
P A R T I V
Transcription in Eukaryotes
10 Eukaryotic RNA Polymerases and
Their Promoters 271
11 General Transcription Factors in Eukaryotes 302
12 Transcription Activators in Eukaryotes 342
13 Chromatin Structure and Its Effects on
Transcription 385
P A R T V
Posttranscriptional Events
14 Messenger RNA Processing I: Splicing 424
15 Messenger RNA Processing II: Capping and
Polyadenylation 471
16 Other RNA Processing Events 510
P A R T V I
Translation
17 The Mechanism of Translation I: Initiation 545
18 The Mechanism of Translation II: Elongation and
Termination 585
19 Ribosomes and Transfer RNA 630
20 DNA Replication I: Basic Mechanism and
Enzymology 665
21 DNA Replication II: Detailed Mechanism 711
22 Homologous Recombination 748
23 Transposition 773
P A R T V I I I
Genomes
24 Genomics and Proteomics 804
Glossary 854
Index 879
C O N T E N T S
About the Author iv
Preface xii
Acknowledgments xv
Guide to Experimental Techniques in Molecular
Biology xvi
P A R T I
Introduction
C H A P T E R 1
A Brief History 1
1.1 Transmission Genetics 2
Mendel?s Laws of Inheritance 2
The Chromosome Theory of Inheritance 3
Box 1.1 Cell Structure 4
Genetic Recombination and Mapping 5
Box 1.2 Cell Cycle and Mitosis 6
Box 1.3 Meiosis 8
Physical Evidence for Recombination 10
1.2 Molecular Genetics 10
The Discovery of DNA 10
The Relationship between Genes and Proteins 11
Activities of Genes 11
1.3 The Three Domains of Life 15
C H A P T E R 2
The Molecular Nature of Genes 17
2.1 The Nature of Genetic Material 18
Transformation in Bacteria 18
The Chemical Nature of Polynucleotides 20
2.2 DNA Structure 24
Experimental Background 24
The Double Helix 25
2.3 Genes Made of RNA 28
2.4 Physical Chemistry of Nucleic Acids 28
A Variety of DNA Structures 28
DNAs of Various Sizes and Shapes 32
C H A P T E R 3
An Introduction to Gene
Function 37
3.1 Storing Information 38
Overview of Gene Expression 38
Protein Structure 38
Protein Function 43
Discovery of Messenger RNA 44
Transcription 45
Translation 48
3.2 Replication 53
3.3 Mutations 53
Sickle Cell Disease 54
P A R T I I
Methods of Molecular Biology
C H A P T E R 4
Molecular Cloning Methods 58
4.1 Gene Cloning 59
The Role of Restriction Endonucleases 59
Vectors 62
Identifying a Specific Clone with a Specific Probe 71
4.2 The Polymerase Chain Reaction 72
cDNA Cloning 74
Box 4.1 Jurassic Park: More than a Fantasy? 75
4.3 Methods of Expressing Cloned Genes 78
Expression Vectors 79
Other Eukaryotic Vectors 85
Using the Ti Plasmid to Transfer Genes to Plants 85
C H A P T E R 5
Molecular Tools for Studying Genes
and Gene Activity 90
5.1 Molecular Separations 91
Gel Electrophoresis 91
Two-Dimensional Gel Electrophoresis 94
wea46119_fm.pdf by Shepherd 1/29/04 10:29 AM Page vi
Contents vii
Ion-Exchange Chromatography 95
Gel Filtration Chromatography 95
5.2 Labeled Tracers 96
Autoradiography 96
Phosphorimaging 97
Liquid Scintillation Counting 98
Nonradioactive Tracers 98
5.3 Using Nucleic Acid Hybridization 99
Southern Blots: Identifying Specific DNA
Fragments 99
DNA Fingerprinting and DNA Typing 101
Forensic Uses of DNA Fingerprinting and DNA
Typing 101
DNA Sequencing 105
Restriction Mapping 107
Protein Engineering with Cloned Genes: Site-Directed
Mutagenesis 112
5.4 Mapping and Quantifying Transcripts 113
S1 Mapping 114
Primer Extension 116
Run-off Transcription and G-Less Cassette
Transcription 117
5.5 Measuring Transcription Rates in Vivo 119
Nuclear Run-on Transcription 119
Reporter Gene Transcription 119
Measuring Protein Accumulation in Vivo 122
5.6 Assaying DNA?Protein Interactions 122
Filter Binding 122
Gel Mobility Shift 123
DNase Footprinting 124
DMS Footprinting and Other Footprinting
Methods 124
5.7 Knockouts 126
P A R T I I I
Transcription in Prokaryotes
C H A P T E R 6
The Mechanism of Transcription
in Prokaryotes 132
6.1 RNA Polymerase Structure 132
Sigma (?) as a Specificity Factor 133
6.2 Promoters 136
Binding of RNA Polymerase to Promoters 136
Promoter Structure 138
6.3 Transcription Initiation 139
The Function of ? 140
Structure and Function of ? 147
The Role of the ?-Subunit in UP Element
Recognition 152
6.4 Elongation 154
Core Polymerase Functions in Elongation 154
Structure of the Elongation Complex 159
6.5 Termination of Transcription 171
Rho-Independent Termination 171
Rho-Dependent Termination 175
C H A P T E R 7
Operons: Fine Control of Prokaryotic
Transcription 182
7.1 The lac Operon 183
Negative Control of the lac Operon 184
Discovery of the Operon 184
Repressor?Operator Interactions 188
The Mechanism of Repression 189
Positive Control of the lac Operon 192
The Mechanism of CAP Action 193
7.2 The ara Operon 198
The ara Operon Repression Loop 198
Evidence for the ara Operon Repression Loop 200
Autoregulation of araC 202
7.3 The trp Operon 202
Tryptophan?s Role in Negative Control of the
trp Operon 202
Control of the trp Operon by Attenuation 203
Defeating Attenuation 204
C H A P T E R 8
Major Shifts in Prokaryotic
Transcription 212
8.1 Modification of the Host RNA Polymerase
during Phage Infection 213
8.2 The RNA Polymerase Encoded in Phage T7 215
8.3 Control of Transcription During Sporulation 216
8.4 Genes with Multiple Promoters 218
The B. subtilis spoVG Gene 218
8.5 The E. coli Heat Shock Genes 220
8.6 Infection of E. coli by Phage k 220
Lytic Reproduction of Phage ? 221
Establishing Lysogeny 229
Autoregulation of the cI Gene during Lysogeny 231
Determining the Fate of a ? Infection: Lysis or
Lysogeny 236
Lysogen Induction 238
C H A P T E R 9
DNA?Protein Interactions
in Prokaryotes 242
9.1 The k Family of Repressors 243
Box 9.1 X-Ray Crystallography 244
High-Resolution Analysis of ? Repressor?Operator
Interactions 249
High-Resolution Analysis of Phage 434
Repressor?Operator Interactions 252
9.2 The trp Repressor 256
The Role of Tryptophan 256
9.3 General Considerations on Protein?DNA
Interactions 258
Hydrogen Bonding Capabilities of the Four Different
Base Pairs 258
The Role of DNA Shape in Specific Binding to
Proteins 259
The Importance of Multimeric DNA-Binding
Proteins 260
9.4 DNA-Binding Proteins: Action at a Distance 260
The gal Operon 260
Duplicated ? Operators 261
The lac Operon 264
Enhancers 264
P A R T I V
Transcription in Eukaryotes
C H A P T E R 1 0
Eukaryotic RNA Polymerases
and Their Promoters 271
10.1 Multiple Forms of Eukaryotic RNA
Polymerase 272
Separation of the Three Nuclear Polymerases 272
The Roles of the Three RNA Polymerases 273
RNA Polymerase Subunit Structures 274
10.2 Promoters 287
Class II Promoters 287
Class I Promoters 291
Class III Promoters 292
10.3 Enhancers and Silencers 296
Enhancers 296
Silencers 298
C H A P T E R 1 1
General Transcription Factors
in Eukaryotes 302
11.1 Class II Factors 303
The Class II Preinitiation Complex 303
Structure and Function of TFIID 305
Structure and Function of TFIIB 317
Structure and Function of TFIIF 318
Structure and Function of TFIIH 318
Elongation Factors 323
The Polymerase II Holoenzyme 326
11.2 Class I Factors 327
The Core-Binding Factor 327
The UPE-Binding Factor 328
Structure and Function of SL1 329
11.3 Class III Factors 331
TFIIIA 332
TFIIIB and C 332
The Role of TBP 336
C H A P T E R 1 2
Transcription Activators
in Eukaryotes 342
12.1 Categories of Activators 343
DNA-Binding Domains 343
Transcription-Activating Domains 343
12.2 Structures of the DNA-Binding Motifs
of Activators 344
Zinc Fingers 344
The GAL4 Protein 346
The Nuclear Receptors 347
Homeodomains 349
The bZIP and bHLH Domains 350
12.3 Independence of the Domains of Activators 351
12.4 Functions of Activators 352
Recruitment of TFIID 353
Recruitment of TFIIB 354
Recruitment of Other General Transcription
Factors 357
Recruitment of the Holoenzyme 358
12.5 Interaction among Activators 361
Dimerization 361
Action at a Distance 362
Multiple Enhancers 365
Architectural Transcription Factors 366
Insulators 369
12.6 Regulation of Transcription Factors 372
Coactivators 372
Activator Ubiquitination 375
Activator Sumoylation 377
Activator Acetylation 377
Signal Transduction Pathways 378
wea46119_fm.pdf by Shepherd 1/29/04 10:29 AM Page viii
C H A P T E R 1 3
Chromatin Structure and Its Effects
on Transcription 385
13.1 Histones 386
13.2 Nucleosomes 387
The Nucleosome Filament 390
The 30-nm Fiber 391
The Role of Histone H1 in Chromatin Folding 394
Higher Order Chromatin Folding 394
13.3 Chromatin Structure and Gene Activity 395
The Effects of Histones on 5S rRNA Gene
Transcription 395
The Effects of Histones on Transcription of Class II
Genes 398
Nucleosome Positioning 400
Histone Acetylation 405
Histone Deaceytlation 407
Chromatin Remodeling 410
Heterochromatin and Silencing 414
Nucleosomes and Transcription
Elongation 419
P A R T V
Posttranscriptional Events
C H A P T E R 1 4
Messenger RNA Processing I: Splicing 424
14.1 Genes in Pieces 425
Evidence for Split Genes 425
RNA Splicing 426
Splicing Signals 427
14.2 The Mechanism of Splicing of Nuclear mRNA
Precursors 428
A Branched Intermediate 428
A Signal at the Branch 431
Spliceosomes 432
Snurps 432
Spliceosome Assembly and Function 444
Role of the RNA Polymerase II CTD 454
14.3 Self-Splicing RNAs 459
Group I Introns 459
Group II Introns 462
14.4 tRNA Splicing 464
C H A P T E R 1 5
Messenger RNA Processing II:
Capping and Polyadenylation 470
15.1 Capping 471
Cap Structure 471
Cap Synthesis 472
Functions of Caps 474
15.2 Polyadenylation 477
Poly(A) 477
Functions of Poly(A) 478
Basic Mechanism of Polyadenylation 480
Polyadenylation Signals 482
Cleavage and Polyadenylation of a Pre-mRNA 484
Poly(A) Polymerase 490
Turnover of Poly(A) 491
15.3 Coordination of mRNA Processing Events 493
Effect of Cap on Splicing 493
Effect of Poly(A) on Splicing 494
Binding of the CTD of Rpb1 to mRNA Processing
Proteins 496
Changes in Association of RNA Processing Proteins
with the CTD Correlate with Changes in CTD
Phosphorylation 498
Coupling Transcription Termination with mRNA
3?-End Processing 499
Mechanism of Termination 501
Role of Polyadenylation in mRNA Transport 504
C H A P T E R 1 6
Other RNA Processing Events 510
16.1 Ribosomal RNA Processing 511
Eukaryotic rRNA Processing 511
Prokaryotic rRNA Processing 513
16.2 Transfer RNA Processing 514
Cutting Apart Polycistronic Precursors 514
Forming Mature 5?-Ends 514
Forming Mature 3?-Ends 516
16.3 Trans-Splicing 518
The Mechanism of Trans-Splicing 518
Polycistronic Arrangement of Coding Regions in
Trypanosomes 520
16.4 RNA Editing 521
Mechanism of Editing 521
Editing by Nucleotide Deamination 524
16.5 Posttranscriptional Control of Gene Expression 526
Casein mRNA Stability 526
Transferrin Receptor mRNA Stability 527
16.6 Posttranscriptional Gene Silencing
(RNA Interference) 535
P A R T V I
Translation
C H A P T E R 1 7
The Mechanism of Translation I: Initiation 545
17.1 Initiation of Translation in Prokaryotes 546
tRNA Charging 546
Dissociation of Ribosomes 546
Formation of the 30S Initiation Complex 549
Formation of the 70S Initiation Complex 558
Summary of Initiation in Prokaryotes 559
17.2 Initiation in Eukaryotes 560
The Scanning Model of Initiation 561
Eukaryotic Initiation Factors 565
Overview of Translation Initiation in Eukaryotes 565
17.3 Control of Initiation 572
Prokaryotic Translational Control 572
Eukaryotic Translational Control 573
C H A P T E R 1 8
The Mechanism of Translation II:
Elongation and Termination 585
18.1 The Direction of Polypeptide Synthesis and
of mRNA Translation 586
18.2 The Genetic Code 587
Nonoverlapping Codons 588
No Gaps in the Code 588
The Triplet Code 588
Breaking the Code 590
Unusual Base Pairs between Codon and Anticodon 590
The (Almost) Universal Code 592
18.3 The Elongation Mechanism 594
Overview of Elongation 594
A Three-Site Model of the Ribosome 595
Elongation Step 1: Binding an Aminoacyl-tRNA to the
A Site of the Ribosome 598
Elongation Step 2: Peptide Bond Formation 605
Elongation Step 3: Translocation 608
The Structures of EF-Tu and EF-G 611
GTPases and Translation 612
18.4 Termination 613
Termination Codons 613
Stop Codon Suppression 615
Release Factors 616
Dealing with Aberrant Termination 621
18.5 Posttermination 625
C H A P T E R 1 9
Ribosomes and Transfer RNA 630
19.1 Ribosomes 631
Gross Ribosomal Structure 631
Fine Structure of the 70S Ribosome 632
Ribosome Composition 636
Ribosome Assembly 637
Fine Structure of the 30S Subunit 638
Fine Structure of the 50S Subunit 644
Polysomes 649
19.2 Transfer RNA 650
The Discovery of tRNA 650
tRNA Structure 651
Recognition of tRNAs by Aminoacyl-tRNA Synthetase:
The Second Genetic Code 654
Proofreading and Editing by Aminoacyl-tRNA
Synthetases 659
P A R T V I I
DNA Replication, Recombination, and Transposition
C H A P T E R 2 0
DNA Replication I: Basic Mechanism and Enzymology 665
20.1 General Features of DNA Replication 666
Semiconservative Replication 666
Semidiscontinuous Replication 668
Priming of DNA Synthesis 671
Bidirectional Replication 672
Unidirectional Replication 675
Rolling Circle Replication 675
20.2 Enzymology of DNA Replication 677
Strand Separation 677
Single-Strand DNA-Binding Proteins 678
Topoisomerases 680
Three DNA Polymerases in E. coli 685
Fidelity of Replication 690
Multiple Eukaryotic DNA Polymerases 691
20.3 DNA Damage and Repair 692
Damage Caused by Alkylation of Bases 692
Damage Caused by Ultraviolet Radiation 693
Damage Caused by Gamma and X-Rays 694
Directly Undoing DNA Damage 694
Excision Repair in Prokaryotes 695
Excision Repair in Eukaryotes 696
x Contents
wea46119_fm.pdf by Shepherd 1/29/04 10:29 AM Page x
Double-Strand Break Repair in Eukaryotes 700
Mismatch Repair 701
Failure of Mismatch Repair in Humans 702
Coping with DNA Damage without Repairing It 702
C H A P T E R 2 1
DNA Replication II: Detailed
Mechanism 711
21.1 Speed of Replication 712
21.2 Initiation 713
Priming in E. coli 713
Priming in Eukaryotes 716
21.3 Elongation 721
The Pol II Holoenzyme and Processivity of
Replication 721
21.4 Termination 733
Decatenation: Disentangling Daughter DNAs 733
Termination in Eukaryotes 735
Box 21.1 Telomeres, the Hayflick Limit, and
Cancer 739
C H A P T E R 2 2
Homologous Recombination 748
22.1 The RecBCD Pathway for Homologous
Recombination 749
22.2 Experimental Support for the RecBCD
Pathway 752
RecA 752
RecBCD 756
RuvA and RuvB 757
RuvC 760
22.3 Meiotic Recombination 764
The Mechanism of Meiotic Recombination:
Overview 764
The Double-Stranded DNA Break 764
Creation of Single-Stranded Ends at DSBs 768
22.4 Gene Conversion 768
C H A P T E R 2 3
Transposition 773
23.1 Bacterial Transposons 774
Discovery of Bacterial Transposons 774
Insertion Sequences: The Simplest Bacterial
Transposons 775
Contents xi
More Complex Transposons 777
Mechanisms of Transposition 777
23.2 Eukaryotic Transposons 779
The First Examples of Transposable Elements: Ds and
Ac of Maize 779
P Elements 781
Rearrangement of Immunoglobulin Genes 782
Retrotransposons 787
P A R T V I I I
Genomes
C H A P T E R 2 4
Genomics and Proteomics 804
24.1 Positional Cloning: An Introduction
to Genomics 805
Classical Tools of Positional Cloning 805
Identifying the Gene Mutated in a Human
Disease 807
Box 24.1 Problems in Genetic Screening 811
24.2 Sequencing Genomes 812
The Human Genome Project 815
Vectors for Large-Scale Genome Projects 816
The Clone-by-Clone Strategy 817
Shotgun Sequencing 820
Sequencing Standards 821
Early Progress in Sequencing the Human
Genome 822
Other Vertebrate Genomes 827
The Minimal Genome 829
24.3 Applications of Genomics 830
Functional Genomics Techniques 830
Other Applications 840
24.4 Proteomics 840
Protein Separations 841
Protein Analysis 841
Protein Interactions 842
Bioinformatics 846
Glossary 854
Index 879

B