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Molecular Biology PDF

912 Pages·2011·53.01 MB·English
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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

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