MOLECULAR BIOLOGY OF
`
`f o u r t h
`
`e d
`
`t
`
`o n
`
`Bruce Alberts
`
`Alexander Johnson
`
`Julian Lewis
`
`Martin Raff
`
`Keith Roberts
`
`Peter Walter
`
`GS Garland Science
`
`Taylor & Francis Group
`
`Genzyme Ex. 1031, pg 850
`
`

`
`Garland
`Vice President: Denise Schanck
`Managing Editor: Sarah Gibbs
`Senior Editorial Assistant: Kirsten Jenner
`Managing Production Editor: Emma Hunt
`Proofreader and Layout: Emma Hunt
`Production Assistant: Angela Bennett
`Text Editors: Marjorie Singer Anderson and Betsy Dilernia
`Copy Editor: Bruce Goatly
`Word Processors: Fran Dependahl, Misty Landers and Carol Winter
`Designer: Blink Studio, London
`Illustrator: Nigel Orme
`Indexer: Janine Ross and Sherry Granum
`Manufacturing: Nigel Eyre and Marion Morrow
`
`Bruce Alberts received his Ph.D. from Harvard University and is
`President of the National Academy of Sciences and Professor of
`Biochemistry and Biophysics at the University of California, San
`Francisco. Alexander Johnson received his Ph.D. from Harvard
`University and is a Professor of Microbiology and Immunology at
`the University of California, San Francisco. Julian Lewis received
`his D.Phil. from the University of Oxford and is a Principal
`Scientist at the Imperial Cancer Research Fund, London.
`Martin Raff received his M.D. from McGill University and is at the
`Medical Research Council Laboratory for Molecular Cell Biology
`and Cell Biology Unit and in the Biology Department at University
`College London. Keith Roberts received his Ph.D. from the
`University of Cambridge and is Associate Research Director at the
`John Innes Centre, Norwich. Peter Walter received his Ph.D. from
`The Rockefeller University in New York and is Professor and
`Chairman of the Department of Biochemistry and Biophysics at
`the University of California, San Francisco, and an Investigator of
`the Howard Hughes Medical Institute.
`
`© 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis,
`Martin Raff, Keith Roberts, and Peter Walter.
`© 1983, 1989, 1994 by Bruce Alberts, Dennis Bray, Julian Lewis,
`Martin Raff, Keith Roberts, and James D. Watson.
`
`All rights reserved. No part of this book covered by the copyright
`hereon may be reproduced or used in any format in any form or
`by any means-graphic, electronic, or mechanical, including
`photocopying, recording, taping, or information storage and
`retrieval systems-without permission of the publisher.
`
`Library of Congress Cataloging-in-Publicaton Data
`Molecular biology of the cell/ Bruce Alberts ... [et al.].-- 4th ed.
`p.cm
`Includes bibliographical references and index.
`ISBN 0-8153-3218-1 (hardbound)-- ISBN 0-8153-4072-9 (pbk.)
`1. Cytology. 2. Molecular biology. I. Alberts, Bruce.
`[DNLM: 1. Cells. 2. Molecular Biology. ]
`QH581.2 .M64 2002
`571.6--dc21
`
`2001054471 CIP
`
`Published by Garland Science, a member of the Taylor & Francis Group,
`29 West 35th Street, New York, NY 10001-2299
`
`Printed in the United States of America
`
`15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
`
`Cell Biology Interactive
`Artistic and Scientific Direction: Peter Walter
`Narrated by: Julie Theriot
`Production, Design, and Development: Mike Morales
`
`Front cover Human Genome: Reprinted by permission
`from Nature, International Human Genome Sequencing
`Consortium, 409:860-921, 2001 ©Macmillan Magazines
`Ltd. Adapted from an image by Francis Collins, NHGRI;
`Jim Kent, UCSC; Ewan Birney, EBI; and Darryl Leja,
`NHGRI; showing a portion of Chromosome 1 from the
`initial sequencing of the human genome.
`
`Back cover In 1967, the British artist Peter Blake created
`a design classic. Nearly 35 years later Nigel Orme
`(illustrator), Richard Denyer (photographer), and the
`authors have together produced an affectionate tribute
`to Mr Blake's image. With its gallery of icons and
`influences, its assembly created almost as much
`complexity, intrigue and mystery as the original.
`Drosophila, Arabidopsis, Dolly and the assembled
`company tempt you to dip inside where, as in the
`original, "a splendid time is guaranteed for all."
`(Gunter Blobel, courtesy of The Rockefeller University; Marie
`Curie, Keystone Press Agency Inc; Darwin bust, by permission
`of the President and Council of the Royal Society; Rosalind
`Franklin, courtesy of Cold Spring Harbor Laboratory Archives;
`Dorothy Hodgkin, ©The Nobel Foundation, 1964; James Joyce,
`etching by Peter Blake; Robert Johnson, photo booth
`self-portrait early 1930s, © 1986 Delta Haze Corporation all
`rights reserved, used by permission; Albert L. Lehninger,
`(unidentified photographer) courtesy of The Alan Mason
`Chesney Medical Archives of The Johns Hopkins Medical
`Institutions; Linus Pauling, from Ava Helen and Linus Pauling
`Papers, Special Collections, Oregon State University; Nicholas
`Poussin, courtesy of ArtToday.com; Barbara McClintock,
`©David Micklos, 1983; Andrei Sakl1arov, courtesy of Elena
`Bonner; Frederick Sanger,© The Nobel Foundation, 1958.)
`
`Genzyme Ex. 1031, pg 851
`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`eve stripe 2
`forms here
`
`.....
`0
`" 0
`·.;::; g
`
`" Q) " " 0 "
`
`-
`
`anterior
`
`position along embryo
`
`posterior-
`
`We have already discussed two mechanisms of combinatorial control of
`gene expression-heterodimerization of gene regulatory proteins in solution
`(see Figure 7-22) and the assembly of combinations of gene regulatory proteins
`into small complexes on DNA (see Figure 7-50). It is likely that both mechanisms
`participate in the complex regulation of eve expression. In addition, the regula(cid:173)
`tion of stripe 2 just described illustrates a third type of combinatorial control.
`Because the individual regulatory sequences in the eve stripe 2 module are
`strung out along the DNA, many sets of gene regulatory proteins can be bound
`simultaneously and influence the promoter of a gene. The promoter integrates
`the transcriptional cues provided by all of the bound proteins (Figure 7-57).
`The regulation of eve expression is an impressive example of combinatorial
`control. Seven combinations of gene regulatory proteins-one combination for
`each stripe-activate eve expression, while many other combinations (all those
`found in the interstripe regions of the embryo) keep the stripe elements silent.
`The other stripe regulatory modules are thought to be constructed along lines
`similar to those described for stripe 2, being designed to read positional infor(cid:173)
`mation provided by other combinations of gene regulatory proteins. The entire
`gene control region, strung out over 20,000 nucleotide pairs of DNA, binds more
`than 20 different proteins. A large and complex control region is thereby built
`from a series of smaller modules, each of which consists of a unique arrange(cid:173)
`ment of short DNA sequences recognized by specific gene regulatory proteins.
`Although the details are not yet understood, these gene regulatory proteins are
`thought to employ a number of the mechanisms previously described for acti(cid:173)
`vators and repressors. In this way, a single gene can respond to an enormous
`number of combinatorial inputs.
`
`Complex Mammalian Gene Control Regions Are Also
`Constructed from Simple Regulatory Modules
`It has been estimated that 5-10% of the coding capacity of a mammalian
`genome is devoted to the synthesis of proteins that serve as regulators of gene
`
`strongly activating
`assembly
`
`neutral assembly of
`regulatory p,roteins
`
`strongly
`
`ir~ h lbil~ng a
`
`protern ~
`
`spacer /II
`DNA--rJ
`
`weakly
`activati ng
`protein
`assembly
`
`------.....
`
`PROBABILITY OF
`INITIATING
`TRANSCRIPTION
`
`TATA
`
`41 0
`
`Chapter 7 :CONTROL OF GENE EXPRESSION
`
`Figure 7-56 Distribution of the gen
`e
`.
`'b
`I
`regu atory protems respons1 le for
`ensuring that eve is expressed in
`stripe 2. The distributions of these
`proteins were visualized by staining a
`developing Drosophila embryo with
`antibodies directed against each of the
`four proteins (see Figures 7-52 and 7-5-J .
`The expression of eve in stripe 2 occurs
`only at the position where the two
`activators (Bicoid and Hunchback) are
`present and the two repressors (Giant
`and Krllppel) are absent. In fly embryos
`that lack Krllppel, for example, stripe 2
`expands posteriorly. Likewise, stripe 2
`expands posteriorly if the DNA-binding
`sites for Krllppel in the stripe 2 module
`(see Figure 7-55) are inactivated by
`mutation and this regulatory region is
`reintroduced into the genome.
`The eve gene itself encodes a gene
`regulatory protein, which, after its pattern
`of expression is set up in seven stripes,
`regulates the expression of other
`Drosophila genes. As development
`proceeds, the embryo is thus subdivided
`into finer and finer regions that eventu~lly.
`give rise to the different body parts of llle
`adult fly, as discussed in Chapter 21.
`This example from Drosophila embryos
`is unusual in that the nuclei are exposed
`directly to positional cues in the form of
`concentrations of gene regulatory
`proteins. In embryos of most other
`organisms, individual nuclei are in separate
`cells, and extracellular positional
`information must either pass across the
`plasma membrane or, more usually,
`generate signals in the cytosol in order t~
`influence the genome.
`
`Figure 7-57 Integration at a
`promoter. Multiple sets of gene
`regulatory proteins can work together co
`influence transcription initiation at a
`promoter, as they do in the eve stripe 2
`module illustrated previously in Figure
`7-55. It is not yet Llr1de rs'tood in deta il
`·s
`how the integratio n of multiple Inputs 1
`achieved, but it is likely that the final
`transcriptional activity of the gene resU 1~'
`from a competition between activators
`and repressors that act by the
`-4~
`1
`mechanisms summarized in Figures 7
`7-44, 7-45, 7-46, and 7-49.
`
`Genzyme Ex. 1031, pg 852
`
`

`
`LIGAND
`BINDING
`
`PROTEIN
`PHOSPHORYLATION
`
`(B)
`
`If I
`
`(C)
`
`ADDITION OF
`SECOND SUBUNIT
`
`e
`
`subunit
`activation
`subunit
`
`(D)
`
`PROTEIN
`SYNTHESIS
`
`tl CTIVE
`
`;A-CTIIf
`
`,,
`
`(A)
`
`UNMASKING
`
`~
`
`CT(VE
`
`'
`r-· t ~C) NA-bindi ng
`.... ,~
`!
`- t=
`6 ,,,,
`
`STIMULATION OF
`NUCLEAR ENTRY
`
`RELEASE FROM
`MEMBRANE
`
`inhibitory
`protein
`
`(E)
`
`(F)
`
`(G)
`
`transcription. This large number of genes reflects the exceedingly complex
`oel:\·vork of controls governing expression of mammalian genes. Each gene is
`regulated by a set of gene regulatory proteins; each of those proteins is the prod(cid:173)
`uct of a gene that is in turn regulated by a whole set of other proteins, and so on.
`Mpreover, the regulatory protein molecules are themselves influenced by signals
`from outside the cell, which can make them active or inactive in a whole variety
`of ways (Figure 7-58). Thus, pattern of gene expression in a cell can be viewed as
`rhe result of a complicated molecular computation that the intracellular gene
`control network performs in response to information from the cell's surround(cid:173)
`lllg&. We shall discuss this further in Chapter 21, dealing with multicellular devel(cid:173)
`op·ment, but the complexity is remarkable even at· the level of the individual
`g~ne tic switch, regulating activity of a single gene. It is not unusual, for example,
`to find a mammalian gene with a control region that is 50,000 nucleotide pairs
`in length, in which many modules, each containing a number of regulatory
`sequences that bind gene regulatory proteins, are interspersed with long
`Stretches of spacer DNA.
`One of the best-understood examples of a complex mammalian regulatory
`region is found in the human ~-globin gene, which is expressed exclusively in
`rea blood cells and at a specific time in their development. A complex array of
`gene regulatory proteins controls the expression of the gene, some acting as
`1\GLivators and others as repressors (Figure 7-59). The concentrations (or activi(cid:173)
`ties) of many of these gene regulatory proteins are thought to change during
`d.evelopme.tl , and only a particular combination of all the proteins triggers tran(cid:173)
`scription of the gene. The human ~-globin gene is part of a cluster of globin
`&!3ne (Figure 7-60A). The five genes of the cluster are transcribed exclusively in
`~l'~throid cells, that is, cells of the red blood cell lineage. Moreover, each gene is
`Urt1ed on at a different stage of development (see Figure 7-60B) and in different
`:~ans: the £-globin gene is expressed in the embryonic yolk sac, yin the yolk sac
`tl ~e fetal liver, and 8 and ~ primarily in the adult bone marrow. Each of the
`~ obm genes has its own set of regulatory proteins that are necessary to turn the
`~elle on at the appropriate time and tissue. In addition to the individual regula(cid:173)
`J~t~. of each of the globin genes, the entire cluster appears to be subject to a
`ated control region called a locus control region (LCR). The LCR lies far
`Haw
`
`GENETIC SWITCHES WORK
`
`Figure 7-58 Some ways in which the
`activity of gene regulatory proteins
`is regulated in eucaryotic cells.
`(A) The protein is synthesized only when
`needed and is rapidly degraded by
`proteolysis so that it does not accumulate.
`(B) Activation by ligand binding. (C)
`Activation by phosphorylation. (D)
`Formation of a complex between a
`DNA·binding protein and a separate
`protein with a transcription-activating
`domain. (E) Unmasking of an activation
`domain by the phosphorylation of an
`inhibitor protein. (F) Stimulation of nuclear
`entry by removal of an inhibitory protein
`that otherwise keeps the regulatory
`protein from entering the nucleus. (G)
`Release of a gene regulatory protein from
`a membrane bilayer by regulated
`proteolysis.
`Each of these mechanisms is typically
`controlled by extracellular signals which
`are communicated across the plasma
`membrane to the gene regulatory
`proteins in the cell. The ways in which this
`signaling occurs is discussed in Chapter
`15. Mechanisms (A)-(F) are readily
`reversible and therefore also provide the
`means to selectively inactivate gene
`regulatory proteins.
`
`411
`
`Genzyme Ex. 1031, pg 853
`
`

`
`
`
`
`
`
`
`
`
`
`
` 1
`
`
`
`
`
`
`
`
`
`
`
` 1
`
` 1
`
`
`
`.--- - - -- - --
`
`gene control regions - - - -- - ---,
`
`RNA start
`
`exons
`I
`\
`
`poly-A addition site
`
`DNA
`
`0
`
`CP1 \
`
`NF1
`-220
`
`EKLF
`
`GATA-1
`
`+2200
`
`-30
`
`GATA-1
`
`GATA-1 GATA-1
`
`+2400
`
`upstream from the gene cluster (see Figure 7-60A), and we shall discuss its func(cid:173)
`tion next.
`In cells where the globin genes are not expressed (such as brain or skin cells),
`the whole gene cluster appears tightly packaged into chromatin. In erythroid
`cells, by contrast, the entire gene cluster is still folded into nucleosomes, but the
`higher-order packing of the chromatin has become decondensed This change
`occurs even before the individual globin genes are transcribed, suggesting that
`there are two steps of regulation. In the first, the chromatin of the entire globin
`locus becomes decondensed, which is presumed to allow additional gene regu(cid:173)
`latory proteins access to the DNA. In the second step, the remaining gene regu(cid:173)
`latory proteins assemble on the DNA and direct the expression of individual
`genes.
`The LCR appears to act by controlling chromatin condensation, and its
`importance can be seen in patients with a certain type of thalassemia, a severe
`inherited form of anemia. In these patients, the P-globin locus is found to have
`undergone deletions that remove all or part of the LCR, and although the p-globin
`gene and its nearby regulatory regions are intact, the gene remains transcrip(cid:173)
`tionally silent even in erythroid cells. Moreover, the P-globin gene in the ery(cid:173)
`throid cells fails to undergo the normal chromatin decondensation step that
`occurs during erythroid cell development.
`Many LCRs (that is, DNA regulatory sequences that regulate the accessibili(cid:173)
`ty and expression of distant genes or gene clusters) are present in the human
`genome, and they regulate a wide variety of cell-type specific genes. The way in
`which they function is not understood in detail, but several models have been
`proposed. The simplest is based on principles we have already discussed in this
`chapter: the gene regulatory proteins that bind to the LCR interact through DNA
`
`Figure 7-59 Model for the control ol
`the human ~-globin gene. The diagr~m
`shows some of the gene regulatory
`proteins thought to control expression of
`the gene during red blood cell
`development (see Figure 7-60). Some of
`the gene regulatory proteins shown, such
`as CP I, are found in many types of cells,
`while others, such as GAT A-I, are present
`in only a few types of cells-including red
`blood cells-and therefore are thought to
`contribute to the cell-type specificity of
`~-globin gene expression. As indicated by
`the double-headed arrows, several of the
`binding sites for GATA-1 overlap those 6!
`other gene regulatory proteins; it is
`thought that occupancy of these sites by
`GAT A-I excludes binding of other
`proteins. Once bound to DNA. the gene
`regulatory proteins recruit chromatin
`remodeling complexes, histone modifying
`enzymes, the general transcription factoJ~
`and RNA polymerase to the promoter.
`(Adapted from B. Emerson, in Gene
`Expression: General and Cell-Type Spec1Rc
`[M. Karin, ed.], pp. I 16-161. Boston:
`Birkhauser, 1993.)
`
`100,000 nucleotide pairs
`
`locus
`control
`region
`I
`
`cluster of globin genes
`
`€ •
`
`0
`
`fl
`
`p-globin gene
`
`"'
`'iii
`Q) .s
`c
`"' c
`:0
`0 c;,
`Q)
`>
`~
`Qi
`~0
`
`(A)
`
`(B)
`
`-X p
`>-C""
`
`12
`
`24
`
`36 t
`
`BIRTH
`
`12
`
`24
`36
`age in weeks
`
`48
`
`Figure 7-60 The cluster of ~-like globin genes in humans. (A) The large chromosomal region shown
`spans I 00,000 nucleotide pairs and contains the five globin genes and a locus control region (LCR).
`h
`
`(B) Changes in the expression of the ~-like globin genes at various stages of human development. Each oft;
`
`globin chains encoded by these genes combines with an a-globin chain to form the hemoglobin in red biOO
`cells (see Figure 7-1 15). (A. after F. Grosveld, G.B. van Assendelft, D.R. Greaves, and G. Kollias, Cell
`51 :975-985, 1987. © Elsevier.)
`
`412
`
`Chapter 7 : CONTROL OF GENE EXPRESSION
`
`Genzyme Ex. 1031, pg 854