Molecular Biology

912 Pages·2011·53.01 MB·English

by  Robert Weaver

Molecular Biology
Fifth Edition
R o b e r t F . W e a v e r
University of Kansas
TM
TM
MOLECULAR BIOLOGY, FIFTH EDITION
Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the
Americas, New York, NY 10020. Copyright © 2012 by The McGraw-Hill Companies, Inc. All rights reserved.
Previous editions © 2008, 2005, and 2002. No part of this publication may be reproduced or distributed in
any form or by any means, or stored in a database or retrieval system, without the prior written consent of
The McGraw-Hill Companies, Inc., including, but not limited to, in any network or other electronic storage or
transmission, or broadcast for distance learning.
Some ancillaries, including electronic and print components, may not be available to customers outside the
United States.
This book is printed on acid-free paper.
1 2 3 4 5 6 7 8 9 0 QDB /QDB 1 0 9 8 7 6 5 4 3 2 1
ISBN 978-0-07-352532-7
MHID 0-07-352532-4
Vice President & Editor-in-Chief: Marty Lange
Vice President EDP/Central Publishing Services: Kimberly Meriwether David
Publisher: Janice Roerig-Blong
Executive Marketing Manager: Patrick E. Reidy
Project Manager: Robin A. Reed
Design Coordinator: Brenda A. Rolwes
Cover Designer: Studio Montage, St. Louis, Missouri
Lead Photo Research Coordinator: Carrie K. Burger
Cover Image: © Getty Images RF
Buyer: Sandy Ludovissy
Media Project Manager: Balaji Sundararaman
Compositor: Aptara®, Inc.
Typeface: 10/12 Sabon
Printer: Quad/Graphics
All credits appearing on page or at the end of the book are considered to be an extension of the
copyright page.
Library of Congress Cataloging-in-Publication Data
Weaver, Robert Franklin, 1942-
Molecular biology / Robert F. Weaver.—5th ed.
p. cm.
ISBN 978–0–07–352532–7 (hardcover : alk. paper)
1. Molecular biology. I. Title.
QH506.W43 2011
572.8—dc22
2010051759
www.mhhe.com
To Camilla and Nora
A B O U T T H E A U T H O R
Rob Weaver was born in Topeka, Kansas, and
grew up in Arlington, Virginia. He received his
bachelor’s degree in chemistry from the College
of Wooster in Wooster, Ohio, in 1964. He
earned his Ph.D. in biochemistry at Duke
University in 1969, then spent two years doing
postdoctoral research at the University of
California, San Francisco, where he studied the
structure of eukaryotic RNA polymerases with
William J. Rutter.
He joined the faculty of the University of
Kansas as an assistant professor of biochemistry
in 1971, was promoted to associate professor,
and then to full professor in 1981. In 1984, he
became chair of the Department of Biochemistry,
( Source: Ashvini C. Ganesh) and served in that capacity until he was named
Associate Dean of the College of Liberal Arts
and Sciences in 1995.
Prof. Weaver is the divisional dean for the science and mathematics departments
within the College, which includes supervising 10 different departments and
programs. As a professor of molecular biosciences, he teaches courses in introductory
molecular biology and the molecular biology of cancer. In his research laboratory,
undergraduates and graduate students have participated in research on the molecular
biology of a baculovirus that infects caterpillars.
Prof. Weaver is the author of many scientifi c papers resulting from research
funded by the National Institutes of Health, the National Science Foundation, and
the American Cancer Society. He has also coauthored two genetics textbooks and
has written two articles on molecular biology in the National Geographic
Magazine. He has spent two years performing research in European laboratories
as an American Cancer Society Research Scholar, one year in Zurich, Switzerland,
and one year in Oxford, England.
iv
B R I E F C O N T E N T S
About the Author iv
Preface xiii
Acknowledgments xvii
Guide to Experimental Techniques in
Molecular Biology xix
PART I PART V
Introduction Post-Transcriptional Events
1 A Brief History 1 14 RNA Processing I: Splicing 394
2 The Molecular Nature of Genes 12 15 RNA Processing II: Capping and Polyadenylation 436
3 An Introduction to Gene Function 30 16 Other RNA Processing Events and Post-Transcriptional
Control of Gene Expression 471
PART II
PART VI
Methods in Molecular Biology
Translation
4 Molecular Cloning Methods 49
17 The Mechanism of Translation I: Initiation 522
5 Molecular Tools for Studying Genes and Gene
18 The Mechanism of Translation II: Elongation
Activity 75
and Termination 560
19 Ribosomes and Transfer RNA 601
PART III
Transcription in Bacteria
PART VII
6 The Mechanism of Transcription in Bacteria 121 DNA Replication, Recombination,
7 Operons: Fine Control of Bacterial and Transposition
Transcription 167
20 DNA Replication, Damage, and Repair 636
8 Major Shifts in Bacterial Transcription 196
21 DNA Replication II: Detailed Mechanism 677
9 DNA–Protein Interactions in Bacteria 222
22 Homologous Recombination 709
23 Transposition 732
PART IV
Transcription in Eukaryotes
PART VIII
10 Eukaryotic RNA Polymerases and Genomes
Their Promoters 244
24 Introduction to Genomics: DNA Sequencing on
11 General Transcription Factors
a Genomic Scale 759
in Eukaryotes 273
25 Genomics II: Functional Genomics, Proteomics,
12 Transcription Activators in Eukaryotes 314
and Bioinformatics 789
13 Chromatin Structure and Its Effects
on Transcription 355 Glossary 827
Index 856
v
C O N T E N T S
About the Author iv CHAPTER 3
Preface xiii An Introduction to Gene Function 30
Acknowledgments xvii
3.1 Storing Information 31
Guide to Experimental Techniques
Overview of Gene Expression 31
in Molecular Biology xix
Protein Structure 31
Protein Function 35
PART I Discovery of Messenger RNA 37
Transcription 39
Introduction
Translation 40
3.2 Replication 45
CHAPTER 1
3.3 Mutations 45
A Brief History 1
Sickle Cell Disease 45
1.1 Transmission Genetics 2
Mendel’s Laws of Inheritance 2
The Chromosome Theory of Inheritance 3
PART II
Genetic Recombination and Mapping 4
Methods of Molecular Biology
Physical Evidence for Recombination 5
1.2 Molecular Genetics 5
The Discovery of DNA 5 CHAPTER 4
The Relationship Between Genes and Proteins 6 Molecular Cloning Methods 49
Activities of Genes 7
4.1 Gene Cloning 50
1.3 The Three Domains of Life 9
The Role of Restriction Endonucleases 50
Vectors 53
Identifying a Specifi c Clone with a
Specifi c Probe 58
CHAPTER 2
cDNA Cloning 60
The Molecular Nature of Genes 12 Rapid Amplifi cation of cDNA Ends 61
4.2 The Polymerase Chain Reaction 62
2.1 The Nature of Genetic Material 13
Standard PCR 62
Transformation in Bacteria 13
Box 4.1 Jurassic Park: More than a Fantasy? 63
The Chemical Nature of Polynucleotides 15
Using Reverse Transcriptase PCR (RT-PCR)
2.2 DNA Structure 18
in cDNA Cloning 64
Experimental Background 19
Real-Time PCR 64
The Double Helix 19
4.3 Methods of Expressing Cloned Genes 65
2.3 Genes Made of RNA 22
Expression Vectors 65
2.4 Physical Chemistry of Nucleic Acids 23
Other Eukaryotic Vectors 71
A Variety of DNA Structures 23
Using the Ti Plasmid to Transfer Genes
DNAs of Various Sizes and Shapes 27 to Plants 71
vi
Contents vii
CHAPTER 5 5.10 Finding RNA Sequences That Interact
Molecular Tools for Studying Genes with Other Molecules 114
SELEX 114
and Gene Activity 75
Functional SELEX 114
5.1 Molecular Separations 76 5.11 Knockouts and Transgenics 115
Gel Electrophoresis 76 Knockout Mice 115
Two-Dimensional Gel Electrophoresis 79 Transgenic Mice 115
Ion-Exchange Chromatography 80
Gel Filtration Chromatography 80
Affi nity Chromatography 81 PART III
5.2 Labeled Tracers 82 Transcription in Bacteria
Autoradiography 82
Phosphorimaging 83 CHAPTER 6
Liquid Scintillation Counting 84
The Mechanism of Transcription
Nonradioactive Tracers 84
in Bacteria 121
5.3 Using Nucleic Acid Hybridization 85
Southern Blots: Identifying Specifi c DNA Fragments 85 6.1 RNA Polymerase Structure 122
DNA Fingerprinting and DNA Typing 86 Sigma (s) as a Specifi city Factor 122
Forensic Uses of DNA Fingerprinting and 6.2 Promoters 123
DNA Typing 87
Binding of RNA Polymerase to Promoters 123
In Situ Hybridization: Locating Genes in
Promoter Structure 125
Chromosomes 88
6.3 Transcription Initiation 126
Immunoblots (Western Blots) 89
Sigma Stimulates Transcription Initiation 127
5.4 DNA Sequencing and Physical Mapping 89
Reuse of s 128
The Sanger Chain-Termination
Sequencing Method 90 The Stochastic s-Cycle Model 129
Automated DNA Sequencing 91 Local DNA Melting at the Promoter 132
High-Throughput Sequencing 93 Promoter Clearance 134
Structure and Function of s 139
Restriction Mapping 95
5.5 Protein Engineering with Cloned Genes: The Role of the a-Subunit in UP Element
Recognition 142
Site-Directed Mutagenesis 97
6.4 Elongation 144
5.6 Mapping and Quantifying Transcripts 99
Core Polymerase Functions in Elongation 144
Northern Blots 99
Structure of the Elongation Complex 146
S1 Mapping 100
6.5 Termination of Transcription 156
Primer Extension 102
Rho-Independent Termination 156
Run-Off Transcription and G-Less
Cassette Transcription 103 Rho-Dependent Termination 159
5.7 Measuring Transcription Rates in Vivo 104
Nuclear Run-On Transcription 104 CHAPTER 7
Reporter Gene Transcription 105 Operons: Fine Control of Bacterial
Measuring Protein Accumulation in Vivo 106
Transcription 167
5.8 Assaying DNA–Protein Interactions 108
Filter Binding 108 7.1 The lac Operon 168
Gel Mobility Shift 109 Negative Control of the lac Operon 169
DNase Footprinting 109 Discovery of the Operon 169
Repressor–Operator Interactions 173
DMS Footprinting and Other Footprinting
Methods 109 The Mechanism of Repression 174
Chromatin Immunoprecipitation (ChIP) 112 Positive Control of the lac Operon 177
5.9 Assaying Protein–Protein Interactions 112 The Mechanism of CAP Action 178
viii Contents
7.2 The ara Operon 182 9.4 DNA-Binding Proteins: Action at
a Distance 237
The ara Operon Repression Loop 183
Evidence for the ara Operon Repression Loop 183 The gal Operon 237
Autoregulation of araC 185 Duplicated l Operators 237
7.3 The trp Operon 186 Enhancers 238
Tryptophan’s Role in Negative Control of the
trp Operon 186
Control of the trp Operon by Attenuation 187
PART IV
Defeating Attenuation 188
Transcription in Eukaryotes
7.4 Riboswitches 190
CHAPTER 8
CHAPTER 10
Major Shifts in Bacterial
Eukaryotic RNA Polymerases
Transcription 196
and Their Promoters 244
8.1 Sigma Factor Switching 197
10.1 Multiple Forms of Eukaryotic
Phage Infection 197
RNA Polymerase 245
Sporulation 199
Separation of the Three Nuclear Polymerases 245
Genes with Multiple Promoters 201
The Roles of the Three RNA Polymerases 246
Other s Switches 201
RNA Polymerase Subunit Structures 248
Anti-s-Factors 202
10.2 Promoters 259
8.2 The RNA Polymerase Encoded in
Class II Promoters 259
Phage T7 202
Class I Promoters 263
8.3 Infection of E. coli by Phage l 203
Class III Promoters 264
Lytic Reproduction of Phage l 204
10.3 Enhancers and Silencers 267
Establishing Lysogeny 211
Enhancers 267
Autoregulation of the cI Gene During
Lysogeny 212 Silencers 269
Determining the Fate of a l Infection: Lysis or
Lysogeny 217
CHAPTER 11
Lysogen Induction 218
General Transcription Factors
CHAPTER 9 in Eukaryotes 273
DNA–Protein Interactions
11.1 Class II Factors 274
in Bacteria 222
The Class II Preinitiation Complex 274
9.1 The l Family of Repressors 223 Structure and Function of TFIID 276
Probing Binding Specifi city by Site- Structure and Function of TFIIB 286
Directed Mutagenesis 223 Structure and Function of TFIIH 288
Box 9.1 X-Ray Crystallography 224 The Mediator Complex and the
High-Resolution Analysis of l Repressor–Operator RNA Polymerase II Holoenzyme 295
Interactions 229 Elongation Factors 296
High-Resolution Analysis of Phage 434 11.2 Class I Factors 299
Repressor–Operator Interactions 232
The Core-Binding Factor 299
9.2 The trp Repressor 234
The UPE-Binding Factor 300
The Role of Tryptophan 234
Structure and Function of SL1 301
9.3 General Considerations on
11.3 Class III Factors 303
Protein–DNA Interactions 235
TFIIIA 303
Hydrogen Bonding Capabilities of the Four
Different Base Pairs 235 TFIIIB and C 304
The Importance of Multimeric DNA-Binding Proteins 236 The Role of TBP 307
Contents ix
CHAPTER 12 Nucleosome Positioning 367
Transcription Activators Histone Acetylation 372
in Eukaryotes 314 Histone Deacetylation 373
Chromatin Remodeling 376
12.1 Categories of Activators 315
Heterochromatin and Silencing 383
DNA-Binding Domains 315
Nucleosomes and Transcription
Transcription-Activating Domains 315 Elongation 387
12.2 Structures of the DNA-Binding
Motifs of Activators 316
PART V
Zinc Fingers 316
Post-Transcriptional Events
The GAL4 Protein 318
The Nuclear Receptors 319
Homeodomains 320 CHAPTER 14
The bZIP and bHLH Domains 321 RNA Processing I: Splicing 394
12.3 Independence of the Domains
14.1 Genes in Pieces 395
of Activators 323
Evidence for Split Genes 395
12.4 Functions of Activators 324
RNA Splicing 396
Recruitment of TFIID 324
Splicing Signals 397
Recruitment of the Holoenzyme 325
Effect of Splicing on Gene Expression 398
12.5 Interaction Among Activators 328
14.2 The Mechanism of Splicing of Nuclear
Dimerization 328
mRNA Precursors 399
Action at a Distance 329
A Branched Intermediate 399
Box 12.1 Genomic Imprinting 332
A Signal at the Branch 401
Transcription Factories 334
Spliceosomes 402
Complex Enhancers 336
Spliceosome Assembly and Function 411
Architectural Transcription Factors 337
Commitment, Splice Site Selection, and Alternative
Enhanceosomes 338 Splicing 415
Insulators 339 Control of Splicing 425
12.6 Regulation of Transcription Factors 343 14.3 Self-Splicing RNAs 427
Coactivators 344 Group I Introns 427
Activator Ubiquitylation 346 Group II Introns 430
Activator Sumoylation 347
Activator Acetylation 348 CHAPTER 15
Signal Transduction Pathways 348 RNA Processing II: Capping and
Polyadenylation 436
CHAPTER 13 15.1 Capping 437
Chromatin Structure and Its Effects Cap Structure 437
on Transcription 355 Cap Synthesis 438
Functions of Caps 440
13.1 Chromatin Structure 356
15.2 Polyadenylation 442
Histones 356
Poly(A) 442
Nucleosomes 357
Functions of Poly(A) 443
The 30-nm Fiber 360
Basic Mechanism of Polyadenylation 445
Higher-Order Chromatin Folding 362
Polyadenylation Signals 446
13.2 Chromatin Structure and Gene
Cleavage and Polyadenylation of a Pre-mRNA 448
Activity 364
Poly(A) Polymerase 454
The Effects of Histones on Transcription
of Class II Genes 365 Turnover of Poly(A) 454
x Contents
15.3 Coordination of mRNA Processing Events 456
PART VI
Binding of the CTD of Rpb1 to mRNA-Processing
Translation
Proteins 457
Changes in Association of RNA-Processing Proteins
CHAPTER 17
with the CTD Correlate with Changes in CTD
Phosphorylation 458
The Mechanism of Translation I:
A CTD Code? 460
Initiation 522
Coupling Transcription Termination with mRNA
39-End Processing 461 17.1 Initiation of Translation in Bacteria 523
Mechanism of Termination 462 tRNA Charging 523
Role of Polyadenylation in mRNA Transport 466 Dissociation of Ribosomes 523
Formation of the 30S Initiation Complex 525
CHAPTER 16
Formation of the 70S Initiation Complex 531
Other RNA Processing Events and
Summary of Initiation in Bacteria 533
Post-Transcriptional Control of Gene
17.2 Initiation in Eukaryotes 533
Expression 471
The Scanning Model of Initiation 533
16.1 Ribosomal RNA Processing 472 Eukaryotic Initiation Factors 537
Eukaryotic rRNA Processing 472 17.3 Control of Initiation 545
Bacterial rRNA Processing 474 Bacterial Translational Control 545
16.2 Transfer RNA Processing 475 Eukaryotic Translational Control 548
Cutting Apart Polycistronic Precursors 475
CHAPTER 18
Forming Mature 59-Ends 475
The Mechanism of Translation II:
Forming Mature 39-Ends 476
16.3 Trans-Splicing 477 Elongation and Termination 560
The Mechanism of Trans-Splicing 477
18.1 The Direction of Polypeptide Synthesis and
16.4 RNA Editing 479 of mRNA Translation 561
Mechanism of Editing 479 18.2 The Genetic Code 562
Editing by Nucleotide Deamination 482 Nonoverlapping Codons 562
16.5 Post-Transcriptional Control of Gene No Gaps in the Code 563
Expression: mRNA Stability 483
The Triplet Code 563
Casein mRNA Stability 484
Breaking the Code 564
Transferrin Receptor mRNA Stability 484
Unusual Base Pairs Between Codon and Anticodon 566
16.6 Post-Transcriptional Control of Gene Expression:
The (Almost) Universal Code 567
RNA Interference 488
18.3 The Elongation Cycle 569
Mechanism of RNAi 489
Overview of Elongation 569
Amplifi cation of siRNA 494
A Three-Site Model of the Ribosome 570
Role of the RNAi Machinery in Heterochromatin
Formation and Gene Silencing 495 Elongation Step 1: Binding an Aminoacyl-tRNA to the
A Site of the Ribosome 573
16.7 Piwi-Interacting RNAs and Transposon Control 501
Elongation Step 2: Peptide Bond Formation 577
16.8 Post-Transcriptional Control of Gene
Expression: MicroRNAs 502 Elongation Step 3: Translocation 580
G Proteins and Translation 582
Silencing of Translation by miRNAs 502
Stimulation of Translation by miRNAs 507 The Structures of EF-Tu and EF-G 583
16.9 Translation Repression, mRNA Degradation, 18.4 Termination 584
and P-Bodies 510 Termination Codons 584
Processing Bodies 510 Stop Codon Suppression 586
Degradation of mRNAs in P-Bodies 511 Release Factors 586
Relief of Repression in P-Bodies 514 Dealing with Aberrant Termination 588
Other Small RNAs 517 Use of Stop Codons to Insert Unusual Amino Acids 593