-1 Molecular Biology of the Gene FIFTH EDITION )-l .. �· ��· ' l I f \ l l 1 \ j BAKER BELL LEVINE LOSICK \ The Genetic Code second position Ser UCG uou UNJ ucc UAC UCA I!D l!m ocu :s 8QQ c:: Cl) -� c:: Tyr stop stop CALl CAC His Pro a:G CAA eAG Gin :8 ·;; 0 c. ANJ Asn - I!! .., Thr Lys • Chain-terminating or "nonsense" codons t Also used in bacteria to specify the initiator formyi-Met-tRNAIMet The position lEse and length of the 1"\Ydrogen bonds between the pills. NOT FOR SALE :T a: "C 0 � (j" :I � Cl) :I .S: Molecular Biology of the Gene F I F T H E D IT I O N James D. Watson Cold Spring Harbor Laboratory Tania A. Baker Massachusetts Institute of Technology Stephen P. Bell Massachusetts Institute of Technology Alexander Gann Cold Spring Harbor Laboratory Press Michael Levine University of California, Berkeley Richard Losick Harvard University PEARSO N ---­ Benjamin Cummings NOT FOR SALE Benjamin Cummings Publisher: Jim Smith Associate Project Editors: Alexandra Fellowes, Jeanne Zalesky Senior Production Editor: Corinne Benson Manufacturing Manager: Pam Augspurger Senior Marketing Manager: Josh Frost Production Management and Text Design: Elm Street Publishing Services, Inc. Media Development and Production: Science Technologies and David Marcey, CLU Questions for Website: Peter Follette Art Studio: Dragonfly Media Group Compositor: Progressive Information Technologies Cover Image: Torno Narashima Cold Spring Harbor Laboratory Press Publisher and Sponsoring Editor: John Inglis Editorial Director: Alexander Gann Editorial Development Manager: Jan Argentine Project Manager and Developmental Editor: Kaaren Janssen Project Coordinator: Maryliz Dickerson Editorial Development Assistant: Nora Rice Crystal structure images: Leemor Joshua-Tor Cover concept sketch: Erica Beade, MBC Graphics Cover Designers: Denise Weiss, Ed Atkeson ISBN 0-321-22368-3 Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings, 1301 Sansome Street. San Francisco, CA 94111. All rights reserved. Manufactured in the United States of America. 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PEARSO N -----­ Benjamin Cummings NOT FOR SALE 1 2 3 4 5 6 7 8 9 10 -VHP-07 06 05 04 03 www.aw-bc.com www.cshlpress.com Preface press, completion human genome longer s the fifth editionofofthe is nogoes to Molecular Biologysequence of the Gene news. This was not something that could safely have been A anticipated when the first edition appeared in 1965; even when the fourth edition came out in 1987, few if any foresaw how quickly we would move into a world where whole genomes, not just individual genes, could be visualized and compared. There has been a comparable leap in the elucidation of protein structures as well. Thus, in the last few years, the structures of the huge molecular machines that drive the basic processes discussed in this book-DNA transcription, replication, protein synthesis, and so forth-have largely been solved at the atomic level, and many details of their inner workings revealed. The new edition of Molecular Biology of the Gene reflects these advances, and many others besides. But when we sat down to plan this latest version, we were all of a mind that much of the organization and scope of the original book should be retained. This was not a matter of convenience-inevitably, in light of the dramatic changes that had taken place since the last edition, the vast bulk of the text had to be completely rewritten anyway, and all the art rendered afresh. No, the reasoning was simply that, more than ever in this genomic era, there seemed a need for a book that explained what genes are and how they work, and this was exactly what Molecular Biology of the Gene had originally been designed to do. Thus, we have resisted the temptation to become encyclopedic or to delve into allied disciplines, such as cell biology. Also, we wanted the new edition to retain a focus on principles and concepts, another feature of its predecessors. And so we illustrate our discussion sparingly with experiments, which appear mainly in boxes. These considerations ensured the book did not become unwieldy. As stated by its author in the preface to the first edition: "Often I present a fact, and, because of lack of space, I cannot outline the experiments that demonstrate its validity. Given the choice between deleting an important principle or giving an experimental detail, I am inclined to state the principle." The current incarnation of Molecular Biology of the Gene adheres unapologetically to this philosophy. An outline of this new edition will thus be familiar to anyone who has used the book before. We begin (in Part 1 ) with a series of chapters (modified in the current edition) that place the field of molecular biology in context. These chapters summarize the history of genetics and molecul_ar biology and also present the timeless chemical principles that determine the structure and function of macromolecules. The text thereafter is organized to follow a familiar flow of topics. The nature of the genetic material, its organization and its maintenance, are discussed in Part 2 ; in addition to chapters on DNA structure, replication, recombination, and repair, new to this part of the book is a chapter on chromosomes, chromatin, and the nucleosome. This addition reflects current appreciation of how the context in which a given gene is found influences its function and regulation. NOT FOR SALE The passage of information from gene to protein- so-called gene expression - is covered in Part 3; and in Part 4 we describe the regulation of that process. As well as chapters on the basic mechanisms of gene regulation, Part 4 has chapters on the regulation of gene expression in animal development and in the evolution of animal diversity. These chapters again conform to a tradition established by earlier editions: always there has been a chapter or two linking basic mechanisms of molecular biology to pressing biological questions. In the current edition, these chapters investigate perhaps the most striking revelation to come from comparing the complete genome sequences of various animals: different animalsincluding humans-contain largely the same genes and so differences between those animals must result largely from changes in how those genes are expressed. New to the current edition is the final part-Part 5-comprising chapters on experimental methods-the techniques of molecular biology, genomics, and bioinformatics-and on the model organisms whose study has revealed many of the underlying principles of molecular biology. We alluded to the explosion in the numbers of atomic structures solved in the last few years. These include not only many of the enzymes that mediate the basic processes of molecular biology, and many of the proteins that regulate those processes, but the nucleosome as well. While it remains true that many of the basic concepts in molecular biology can be understood without reliance on structural detail- indeed it is one of the strengths of the field that this is the case-nevertheless, many mechanistic insights come only from seeing these details. Accordingly, where structures shed light on how the molecules in question work, we present them; and we do so in a consistent style throughout the book. Each part opener includes a short text, outlining what will be covered in the coming chapters, and a few photographs. These pictures, from the Cold Spring Harbor Laboratory Archive, were all taken at the Laboratory on Long Island, the great majority at the Symposium hosted there almost every summer since 1933. Captions identify who is in each picture and when it was taken. We thank Clare Bunce and the CSHL Archive for help with these. Parts of the current edition grew out of an introductory course on molecular biology taught by one of us (RL) at Harvard University, and this author is grateful to Steve Harrison and Jim Wang who contributed to this course in past years and whose influence is reflected in Chapter 6 and elsewhere. We have shown sections of the manuscript to various colleagues and their comments have been most valuable, greatly improving the accuracy and accessibility of the text and figures. Specifically we thank: Jamie Cate, Richard Ebright. Mike Eisen, Chris Fromme, Ira Hall, Adrian Krainer, Karolin Luger. Bill McGinnis, Matt Michael, Lily Mirels, Nipam Patel, Craig Peterson, Mark Ptashne, Uttam RajBhandary, and Bruce Stillman. In addition, Craig Hunter drafted the section on the worm for Chapter 21. We also thank those who provided us with figures, or the wherewithall to create them, including: Sean Carroll, Seth Darst, Edward Egelman, Georg Halder, Stuart Kim, Bill McGinnis, Steve Paddock, Phoebe Rice, Matt Scott, Peter Sorger, Andrzej Stasiak, Tom Steitz, Dan Voytas, and Steve West. We are most grateful to Leemor Joshua-Tor who rendered all the structure figures, often producing multiple versions and patiently helping us see which best �()'fe��'gA(_� needed. We are also grateful to those who provided their software1: Per Kraulis, Robert Esnouf, Ethan Merritt, and Barry Honig. Coordinates were obtained from the Protein Data Bank (www.rcsb.org/pdb/); and citations to those who solved each structure are included in the figure legends. Our art program was developed and rendered by a talented and enthusiastic team from the Dragonfly Media Group, led by Mike Demaray and Craig Durant. Renate Hellmiss helped to develop some of our initial sketches and provided early renderings of a number of figures. The cover image was rendered by Torno Narashima from an author concept sketch by Erica Beade (MBC Graphics). We thank those at Cold Spring Harbor Laboratory Press who handled development of this book. Jan Argentine, despite having to enforce the deadlines, was throughout less cajoling than she was tirelessly engaged in helping us solve the problems these presented. Maryliz Dickerson kept organized the mass of material we generated and Nora Rice helped coordinate author meetings and other aspects of the project. Denise Weiss and Ed Atkeson produced the cover design; and John Inglis, who initiated this collaboration, was on hand with advice at critical points in the process. Most of all, Kaaren Janssen, our editor, kept everything afloat with an energy, enthusiasm, and activity far beyond anything we could reasonably have asked for; things simply would not have got to this point without her. We also wish to acknowledge the work of those at Benjamin Cummings who coordinated production of the book. Frank Ruggirello oversaw the process carried out by Jim Smith, Kay Ueno. Corinne Benson, Alexandra Fellowes, Jeanne Zalesky, and Donna Kalal. Ingrid Mount at Elm Street Publishing Services coped cheerfully with the many rounds of changes to art and text even very late in the process. Michele Sordi, while part of the Benjamin Cummings team, helped bring us all together in the first place. And finally we gratefully acknowledge our families and friends who, throughout this period, provided such strong support, despite having to put up with our frequent absences and distractions. James D. Watson Tania A. Baker Stephen P. Bell Alexander Gann Michael Levine Richard Losick 1 Per Kraulis granted permission to use MolScript (Kraulis P. }. 1991. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. Journal of Applied Crystallography 24: 946-950). Robert Esnouf gave permission to use BobScript (Esnouf R.M. 1997. Journal of Molecular Graphics 15: 132-134). In addition, Ethan Merritt gave us use of Raster3D (Merritt E.A. and Bacon D.J. 1997. Raster3D: Photorealistic Molecular Graphics. Methods in Enzymology 277: 505-524), and Barry Honig granted permission to use GRASP (Nicolls A., Sharp K.A., and Honig B. 1991. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11: 281-296). NOT FOR SALE About the Authors JAMES D. WATSON was Director of Cold Spring Harbor Laboratory from 1968 to 1993 and is now its President. He spent his undergraduate years at the University of Chicago and received his Ph.D. in 1950 from Indiana University. Between 1950 and 1953, he did postdoctoral research in Copenhagen and Cambridge, England. While at Cambridge, he began the collaboration that resulted in the elucidation of the double-helical structure of DNA in 1953. (For this discovery, Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in 1962.) Later in 1953, he went to the California Institute of Technology. He moved to Harvard in 1955, where he taught and did research on RNA synthesis and protein synthesis until 1976. He was the first Director of the National Center for Genome Research of the National Institutes of Health from 1989 to 1992. Dr. Watson was sole author of the first, second, and third editions of Molecular Biology of the Gene, and a co-author of the fourth edition. These were published in 1965, 1970, 1976, and 1987 respectively. Watson has also been involved in two other textbooks: he was one of the original authors of Molecular Biology of the Cell, and is also an author of Recombinant DNA: a Short Course. TANIA A. BAKER is the Whitehead Professor of Biology at the Massachusetts Institute of Technology and an Investigator of the Howard Hughes Medical Institute. She received a B.S. in biochemistry from the University of Wisconsin, Madison, and a Ph.D. in biochemistry from Stanford University in 1988. Her graduate research was carried out in the laboratory of Professor Arthur Kornberg and focused on mechanisms of initiation of DNA replication. She did postdoctoral research in the laboratory of Dr. Kiyoshi Mizuuchi at the National Institutes of Health, studying the mechanism and regulation of DNA transposition. Her current research explores mechanisms and regulation of genetic recombination, enzyme-catalyzed protein unfolding, and ATP-dependent protein degradation. Professor Baker received the 2001 Eli Lilly Research Award from the American Society of Microbiology and the 2000 MIT School of Science Teaching Prize for Undergraduate Education. She is co-author (with Arthur Kornberg) of the book DNA Replication, Second Edition. STEPHEN P. BELL is a Professor of Biology at the Massachusetts Institute of Technology and an Assistant Investigator of the Howard Hughes Medical Institute. He received B.A. degrees from the Department of Biochemistry, Molecular Biology, and Cell Biology and the Integrated Sciences Program at Northwestern University and a Ph.D. in biochemistry at the University of California, Berkeley in 1991. His graduate research was carried out in the laboratory of Dr. Robert Tjian and focused on eukaryotic transcription. He did postdoctoral research in the laboratory of Dr. Bruce Stillman at Cold Spring Harbor Laboratory, working on the initiation of eukaryotic DNA replication. His current research focuses on the mechanisms controlling the duplication of eukaryotic chromosomes. Professor Bell received the 2001 ASBMB-Schering Plough Scientific Achievement Award and the Everett Moore Baker Memorial Award for Excellence in Undergraduate Teaching at MIT in 1998. NOT FOR SALE ALEXANDER GANN is Editorial Director of Cold Spring Harbor Laboratory Press, and a faculty member of the Watson School of Biological Sciences at Cold Spring Harbor Laboratory. He received his B.Sc in microbiology from University College London and a Ph.D. in molecular biology from The University of Edinburgh in 1989. His graduate research was carried out in the laboratory of Noreen Murray and focused on DNA recognition by restriction enzymes. He did postdoctoral research in the laboratory of Mark Ptashne at Harvard, working on transcriptional regulation, and that of Jeremy Brockes at the Ludwig Institute of Cancer Research at University College London, where he .worked on newt limb regeneration. He was a Lecturer at Lancaster University, England, from 1996 to 1999, before moving to Cold Spring Harbor Laboratory. He is co-author (with Mark Ptashne) of the book Genes & Signals (2002). MICHAEL LEVINE is a Professor of Molecular and Cell Biology at the University of California, Berkeley, and is also Co-Director of the Center for Integrative Genomics. He received his B.A. from the Department of Genetics at the University of California, Berkeley, and his Ph.D. with Alan Garen in the Department of Molecular Biophysics and Biochemistry from Yale University in 1981. As a postdoctoral fellow with Walter Gehring and Gerry Rubin from 1982 - 1984, he studied the molecular genetics of Drosophila development. Professor Levine's research group currently studies the gene networks responsible for the gastrulation of the Drosophila and Ciona (sea squirt) embryos. He holds the F. Williams Chair in Genetics and Development at the University of California, Berkeley. He was awarded the Monsanto Prize in Molecular Biology from the National Academy of Sciences in 1996, and was elected to the American Academy of Arts and Sciences in 1996 and the National Academy of Sciences in 1998. RICHARD M. LOSICK is the Maria Moors Cabot Professor of Biology, a Harvard College Professor, and a Howard Hughes Medical Institute Professor in the Faculty of Arts & Sciences at Harvard University. He received his A.B. in chemistry at Princeton University and his Ph.D. in biochemistry at the Massachusetts Institute of Technology. Upon completion of his graduate work, Professor Losick was named a Junior Fellow of the Harvard Society of Fellows when he began his studies on RNA polymerase and the regulation of gene transcription in bacteria. Professor Losick is a past Chairman of the Departments of Cellular and Developmental Biology and Molecular and Cellular Biology at Harvard University. He received the Camille and Henry Dreyfuss Teacher-Scholar Award, is a member of the National Academy of Sciences, a Fellow of the American Academy of Arts and Sciences, a Fellow of the American Association for the Advancement of Science, a Fellow of the American Academy of Microbiology, and a former Visiting Scholar of the Phi Beta Kappa Society. NOT FOR SALE Brief Contents PAR T 1 PART 2 PART 3 CHEMISTRY AND GENETICS 1 2 l The Mendelian View of the World 5 Nucleic Acids Convey Genetic Information 19 41 55 3 The Importance of Weak Chemical Interactions 4 The Importance of High-Energy Bonds s Weak and Strong Bonds Determine Macromolecular Structure MAINTENANCE OF THE GENOME 6 69 93 The Structures of DNA and RNA 97 129 181 235 259 7 Chromosomes, Chromatin, and the Nucleosome o The Replication of DNA 9 The Mutability and Repair of DNA to Homologous Recombination at the Molecular Level 11 Site-Specific Recombination and Transposition of DNA EXPRESSION OF THE GENOME 343 12 Mechanisms of Transcription 293 347 379 411 13 RNA Splicing 14 Translation 15 The Genetic Code 461 ix NOT FOR SALE