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`Page 1
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`KASHIV EXHIBIT 1012
`IPR2019-00791
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`

`

`
`THE CELL
`
`MOLECULAR BIOLOGY
`
`fourth
`
`edition
`
`Bruce Alberts
`
`Alexander Johnson
`
`Julian Lewis
`
`Martin Raff
`
`Keith Roberts
`
`Peter Walter
`
`Garland Science
`Taylor & Francis Group
`
`Page 2
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`Page 2
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`

`

`
`
`;
`Garland
`Vice President: Denise Schanck
`Managing Editor: Sarah Gibbs
`SeniorEditorial Assistant: Kirsten Jenner
`Managing Production Editor: Emma Hunt
`Proofreader and Layout: Emma Hunt
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`Text Editors: Marjorie Singer Anderson andBetsy Dilernia
`Copy Editor: Bruce Goatly
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`Designer: Blink Studio, London
`Ilustrator: Nigel Orme
`Indexer: Janine Ross and Sherry Granuin
`Manufacturing: Nigel Eyre and Marion Morrow
`
`Bruce Alberts received his Ph.D. from Harvard University andis
`Presidentof the National Academyof Sciences and Professorof
`Biochemistry and Biophysicsat the University of California, San
`Francisco, Alexander Johnsonreceived his Ph.D. from Harvard
`University andis a Professor of Microbiology and Immunology
`and Co-Director ofthe Biochemistry and Molecular Biology
`Program 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 London Research Institute of
`Cancer Research UK. Martin Raff received his M.D. from McGill
`University and is at the Medical Research Council Laboratory for
`MolecularCell Biology and Cell Biology Unit andin the Biology
`Departmentat University College London. Keith Roberts received
`his Ph.D, from the University of Cambridge andis Associate
`ResearchDirector at the John Innes Centre, Norwich. Peter Walter
`received his Ph.D. from The Rockefeller University in New York and
`is Professor and Chairmanofthe Department of Biochemistry and
`Biophysics at the University of California, San Francisco, and an
`Investigator of the Howard Hughes MedicalInstitute.
`
`© 2002 by BruceAlberts, Alexander Johnson,Julian Lewis,
`Martin Raff, Keith Roberts, and Peter Walter.
`© 1983, 1989, 1994 by BruceAlberts, 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 maybe reproduced orused in any format in any form or
`by any means—graphic, electronic, or mechanical, including
`photocopying, recording, taping, orinformation storage and
`retrieval systems—without permission ofthe publisher,
`
`Library of Congress Cataloging-in-Publication Data
`Molecular biology ofthe cell / Bruce Alberts... fet al.].-- 4th ed.
`p.cm
`Includesbibliographical 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--de21
`
`200105447] CIP
`
`Published by GarlandScience, a memberof the Taylor & Francis Group,
`29 West35th Street, New York, NY 10001-2299
`
`Printed in the United States of America
`
`15 14 13 12 111098765 432
`
`Page 3
`
`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; showinga portion of Chromosome 1 from the
`initial sequencing of the human genome.
`
`Chapter opener Portion of chromosome 2 from the
`genomeofthefruit fly Drosophila melanogaster.
`(Reprinted with permission from M.D. Adams et al.,
`Science 287:2185-2195, 2000, © AAAS.)
`
`In 1967, the British artist Peter Blake
`Back cover
`created a design classic. Nearly 35 yearslater Nigel
`Orme(illustrator), Richard Denyer (photographer), and
`the authors have together producedanaffectionate
`tribute to Mr Blake’s image.Withits gallery of icons and
`influences, its assembly created almostas 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 guaranteedforall.”
`(Gunter Blobel, courtesy ofThe Rockefeller University; Marie
`Curie, Keystone Press AgencyInc; 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 Laze Corporation all
`rights reserved, used by permission;AlbertL. Lehninger,
`(unidentified photographer) courtesy ofThe 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, courtesyof ArtToday.com; Barbara McClintock,
`© David Micklos, 1983; Andrei Sakharov, courtesy of Elena
`Bonner; Frederick Sanger, © The Nobel Foundation, 1958.)
`
`Page 3
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`
`% eeebeen ALAMEAORATATT HTT0aLTT ri
`
`305-
`663,
`Cific
`
`HOW CELLS READ THE
`GENOME: FROM
`DNA TO PROTEIN
`
`FROM DNATO RNA
`
`FROM RNA TO PROTEIN
`
`THE RNA WORLD AND THE
`ORIGINS OF LIFE
`
`Only when thestructure of DNA wasdiscoveredin theearly 1950sdid it become
`clear how the hereditary information in cells is encoded in DNA’s sequence of
`nucleotides. The progress since then has been astounding. Fifty years later, we
`have complete genome sequencesfor many organisms, including humans, and
`we therefore know the maximum amountof informationthatis required to pro-
`duce a complex organismlike ourselves. The limits on the hereditary informa-
`tion neededfor life constrain the biochemical andstructural features of cells
`and makeit clear that biology is not infinitely complex.
`In this chapter, we explain howcells decode anduse the informationin their
`genomes, We shall see that much has been learned about how the genetic
`instructions written in an alphabet of just four “letters’—the four different
`nucleotides in DNA—direct the formation of a bacterium,a fruitfly, or a human.
`Nevertheless, we still have a great deal to discover about how the information
`stored in an organism's genomeproduces eventhe simplestunicellular bacterium
`with 500 genes,
`let alone how it directs the development of a human with
`approximately 30,000 genes. An enormous amountof ignorance remains; many
`fascinating challenges therefore await the next generationofcell biologists.
`The problemscells face in decoding genomes can be appreciated by consid-
`ering a small portion of the genomeofthefruit fly Drosophila melanogaster(Fig-
`ure 6-1). Much of the DNA-encoded information present in this and other
`genomesis used to specify the linear order—the sequence—ofaminoacids for
`every protein the organism makes. As described in Chapter3, the amino acid
`sequence in turn dictates how each proteinfolds to give a molecule with a dis-
`tinctive shape and chemistry. Whenaparticular protein is made bythecell, the
`Corresponding region of the genome musttherefore be accurately decoded. Addi-
`tional information encoded in the DNA of the genomespecifies exactly when in
`thelife of an organism andin whichcell types each geneis to be expressed into
`Protein. Since proteins are the main constituents of cells, the decoding of the
`8enome determinesnotonly thesize, shape, biochemical properties, and behav-
`lor of cells, but also the distinctive features of each species on Earth,
`One might havepredictedthatthe information present in genomes would be
`atranged in an orderly fashion, resembling a dictionary or a telephonedirectory.
`
`299
`
`Page 4
`nn‘aiSSS
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`Page 4
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`

`

`| eh a
`im|
`
`a ww I wv
`
`color code for sequencesimilarity
`of genesidentified
`
`J
`
`ral
`
`tile M
`
`a ww i w
`MY
`Y
`no similarity
`;
`ge
`seer SON
`M = mammalian
`W = Celegans
`
`at .
`100,000 nucleotide pairs
`
`300
`
`Chapter 6 : HOW CELLS READ THE GENOME: FROM DNA TO PROTEIN
`
`Page 5
`
`M"ore- ! |
`oy|Q rr=tt
`
`wlthufI or fient rl
`ce |
`ANY Eg Ay tN
`ii aio ‘yh NN Mt |
`
`Y= 5S. cerevisiae
`
`%GC content=25
`Tstransposableelements
`110Se | MHWi 4fasntifadontopstrandofDNA
`lengthofbar iit yt peeand’prodiitsdwar
`
`5
`
`and predicted gene
`
`dentified on bottom straad of DNA
`
`i
`CDNAS|Menthe
`in databases
`
`Page 5
`
`

`

`=
`
`P ;
`
`= |
`
`Figure 6-1 (opposite page) Schematic depiction of a portion of chromosome 2 from the genome of
`the fruit fly Drosophila melanogaster.This figure represents approximately 3% of the total Drosophila genome,
`arranged as six contiguous segments. As summarized in the key, the symbolic representations are: rainbow-colored
`bar: G-C base-pair content; black vertical lines of various thicknesses:locations of transposable elements, with
`thicker bars indicating clusters of elements; colored boxes: genes (both known and predicted) coded on one strand
`of DNA (boxes above the midline) and genes coded on the other strand (boxes below the midline).The length of
`each predicted gene includes both its exons (protein-coding DNA)andits intrans (non-coding DNA) (see Figure
`4-25). As indicated in the key, the height of each gene box is proportional to the number of cDNAsin various
`databases that match the gene.As described in Chapter 8, cDNAs are DNA copies of mRNA molecules, and
`large collections of the nucleotide sequences of cDNAshave been deposited in a variety of databases.The higher
`the number of matches between the nucleotide sequences of cDNAsandthat of a particular predicted gene, the
`higher the confidence that the predicted geneis transcribed into RNA andis thus a genuine gene.The colorof
`each gene box (see color cade in the key) indicates whethera closely related gene is known to occurin other
`organisms. For example, MWY means the gene hasclose relatives in mammals,in the nematode worm
`Caenorhabditis elegans, and in the yeast Saccharomyces cerevisiae, MW indicates the gene has closerelatives in
`mammals and the worm but not in yeast, (From Mark D. Adamsetal., Science 287:2185-2195, 2000. © AAAS.)
`
`DNA replication
`
`DNA repair~aoe DNA
`
`Although the genomesof somebacteria seemfairly well organized, the genomes
`of most multicellular organisms, such as our Drosophila example, are surpris-
`ingly disorderly. Small bits of coding DNA(thatis, DNAthat codes for protein)
`are interspersed with large blocks of seemingly meaningless DNA. Somesections
`of the genome contain many genes and others lack genes altogether. Proteins
`that work closely with one anotherin the cell often have their genes located on
`different chromosomes,and adjacentgenes typically encode proteins that have
`{ittle to do with eachotherin the cell. Decoding genomesis therefore no simple
`matter. Even with the aid of powerful computers,it is still difficult for researchers
`to locate definitively the beginning and endof genes in the DNA sequences of
`complex genomes, muchless to predict when eachgeneis expressed in thelife
`of the organism. Although the DNA sequenceof the human genomeis known,it
`will probably take at least a decade for humansto identify every gene and deter-
`minethe precise aminoacid sequenceofthe protein it produces,Yet the cells in
`our body do this thousands oftimes a second.
`The DNA in genomes does not direct protein synthesis itself, but instead
`uses RNA as an intermediary molecule. When the cell needs a particularprotein,
`the nucleotide sequenceofthe appropriate portion of the immensely long DNA
`molecule in a chromosomeisfirst copied into RNA (a processcalled transcrip-
`tion). It is these RNA copies of segments of the DNAthat are used directly as
`templates to direct the synthesis of the protein (a process called translation).
`Theflow of genetic informationincells is therefore from DNA to RNAto protein
`(Figure 6-2). All cells, from bacteria to humans, express their genetic informa-
`tion in this way—a principle so fundamental thatit is termed the central dogma
`of molecular biology.
`Despite the universality of the central dogma, there are important variations
`in the way informationflows from DNAtoprotein. Principal amongtheseis that
`RNAtranscripts in eucaryotic cells are subject to a series of processing steps in
`the nucleus, including RNAsplicing, before they are permitted to exit from the
`nucleus and be translated into protein, These processing steps can critically
`RNA
`changethe “meaning” of an RNA molecule andare therefore crucial for under-
`standing how eucaryotic cells read the genome.Finally, although we focus on
`
`6WNTTETEY©
`the production of the proteins encoded by the genomein this chapter, we see
`protein synthesis
`(translation)
`that for some genes RNAis thefinal product. Like proteins, many of these RNAs
`fold into precise three-dimensionalstructuresthat have structural and catalytic
`roles in the cell.
`We begin this chapterwith thefirst step in decoding a genome:the process
`of transcription by which an RNA moleculeis produced from the DNAofa gene.
`Wethenfollow the fate of this RNA molecule throughthecell, finishing when a
`correctly folded protein molecule has been formed,At the endof the chapter, we
`consider how the present, quite complex, schemeof informationstorage, tran-
`scription, and translation mighthavearisen from simpler systemsin the earliest
`Stages of cellular evolution.
`
`
`
`3
`
`5
`
`RNA synthesis
`(transeription)
`
`PROTEIN
`
`amino acids
`
`Figure 6-2 The pathway from DNA
`to protein. Theflow of genetic
`information from DNA to RNA
`(transcription) and from RNA to protein
`(translation) occursin all living cells.
`
`HOW CELLS READ THE GENOME: FROM DNATO PROTEIN
`
`301
`
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`Page 6
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`Page 6
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`

`

`Figure 6-3 Genes can be expressed
`with different efficiencies. Gene A js
`transcribed and translated much more
`efficiently than gene B. This allows the
`amount ofprotein A in the cell to be
`Ee RNA =a RNA
`much greater than that of protein B.
`ESSSe
`LSS
`
`
`
`gene A
`gene B
`reen
`| rrawscriprion
`| TRANSCRIPTION
`
`[SRANSRATON
`
`TRANSLATION
`
`FROM DNA TO RNA
`
`Transcription and translation are the means by whichcells read out, or express,
`the genetic instructionsin their genes. Because many identical RNA copies can
`be made from the same gene, and each RNA molecule can direct the synthesis
`of many identical protein molecules, cells can synthesize a large amount of
`protein rapidly when necessary. But each gene can also be transcribed and
`translated with a differentefficiency, allowingthe cell to make vast quantities of
`someproteins and tiny quantities of others (Figure 6-3). Moreover, as wesee in
`the next chapter, a cell can change (or regulate) the expression of each ofits
`genes according to the needs of the moment—mostobviously by controlling
`the productionof its RNA,
`.
`
`Portions of DNA Sequence Are Transcribed into RNA
`Thefirst step a cell takes in reading out a needed partofits genetic instructions
`is to copy a particular portion of its DNA nucleotide sequence—a gene—into an
`RNA nucleotide sequence.The information in RNA, althoughcopied into another
`chemical form,is still written in essentially the same language asit is in DNA—
`the language of a nucleotide sequence. Hence the nametranscription.
`Like DNA, RNAisalinear polymer made offourdifferenttypes of nucleotide
`subunits linked together by phosphodiester bonds (Figure 6-4). It differs from
`DNA chemically in two respects:
`(1)
`the nucleotides
`in RNA are
`ribonucleotides—thatis, they contain the sugar ribose (hence the nameribonu-
`cleic acid) rather than deoxyribose; (2) although, like DNA, RNA contains the
`bases adenine(A), guanine (G), and cytosine(C), it contains the base uracil (U)
`instead of the thymine (T) in DNA. Since U,like T, can base-pair by hydrogen-
`bonding with A (Figure 6-5),
`the complementary base-pairing properties
`described for DNA in Chapters 4 and 5 apply also to RNA(in RNA,G pairs with
`C, and A pairs with U). It is not uncommon, however,to find other types of base
`pairs in RNA:for example, G pairing with U occasionally.
`Despite these small chemical differences, DNA and RNAdiffer quite dra-
`matically in overall structure. Whereas DNA always occursin cells as a double-
`stranded helix, RNA is single-stranded. RNA chains therefore fold up into a
`variety of shapes, just as a polypeptide chain folds up to form the final shape of
`a protein (Figure 6-6), As wesee later in this chapter, the ability to fold into com-
`plex three-dimensional shapes allows some RNA molecules to have structural
`and catalytic functions.
`
`Transcription Produces RNA Complementary to
`One Strand of DNA
`
`All of the RNAina cell is made by DNAtranscription, a process that has cer-
`tain similarities to the process of DNA replication discussed in Chapter 5.
`
`302
`
`Chapter 6 : HOW CELLS READ THE GENOME: FROM DNA TO PROTEIN
`
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`Page 7
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`Page 7
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`

`5’ and
`
`(C)
`
`H |C
`
`5
`
`uracil
`
`H
`
`c
`ee
`NOSE
`|
`|
`Cc
`Cc
`of bees ae So
`|
`=
`H
`H
`
`7
`
`(A)
`
`HOCH, O.
`OH
`
`
`H
`H
`OH QOH
`7
`'
`_fibose
`used in ribonucleic
`acid (RNA)
`
`HOCH;
`
`OL
`
`OF
`
`H
`
`H
`
`OH H
`ae
`|
`‘deoxyribose
`used in deoxyribonucleic
`acid (DNA)
`
`(B)
`
`sy
`
`Oo
`oO
`1
`wet
`HC~ “NH
`~~ ~NH
`He.
`Le
`He.
`Le
`Sy So
`Sy7 So
`|
`|
`H
`H
`uracil
`thymine
`used in RNA
`used in DNA
`Figure 6-4 The chemicalstructure of RNA.(A) RNAcontains the
`sugar ribose, which differs from deoxyribose, the sugar used in DNA,by the
`presence of an additional -OH group. (B) RNA contains the base uracil,
`which differs from thymine, the equivalent base in DNA,by the absence of a
`—CH3 group. (C) A short length of RNA.The phosphodiester chemical
`linkage between nucleotides in RNA is the sameas thatin DNA.
`
`
`
`Transcription begins with the opening and unwinding of a small portion of the
`DNA double helix to expose the bases on each DNAstrand, One of the two
`strands of the DNA double helix thenacts as a template for the synthesis of an
`
`RNA molecule. As in DNAreplication, the nucleotide sequence of the RNA chain
`
`is determined by the complementary base-pairing between incoming
`
`nucleotides and the DNA template. When a good matchis made, the incoming
`
`ribonucleotide is covalently linked to the growing RNA chain in an enzymati-
`
`cally catalyzed reaction, The RNA chain produced by transcription—the fran-
`
`script—is therefore elongated one nucleotide at a time, and it has a nucleotide
`
`sequencethat is exactly complementary to the strand of DNA used as the tem-
`
`plate (Figure 6-7).
`
`Transcription, however, differs from DNAreplication in several crucial ways.
`
`Unlike a newly formed DNAstrand,the RNAstrand does not remain hydrogen-
`
`bondedto the DNAtemplate strand.Instead, just behind the region where the
`
`ribonucleotides are being added, the RNA chainis displaced and the DNAhelix
`
`re-forms, Thus, the RNA molecules produced bytranscription are released from
`
`the DNA templateas single strands. In addition, because they are copied from
`
`only a limited region of the DNA, RNA molecules are muchshorter than DNA
`
`molecules. A DNA molecule in a human chromosomecanbe up to 250 million
`
`nucleotide-pairs long; in contrast, most RNAs are no more than a few thousand
`
`nucleotides long, and manyare considerably shorter.
`
`The enzymesthat perform transcription are called RNA polymerases.Like
`
`the DNA polymerase that catalyzes DNAreplication (discussed in Chapter5),
`
`RNApolymerasescatalyze the formation of the phosphodiester bondsthat link
`
`the nucleotides together to form a linear chain, The RNA polymerase moves
`
`stepwise along the DNA, unwinding the DNA helix just ahead of the activesite
`
`for polymerization to expose a new region of the template strand for comple-
`
`mentary base-pairing. In this way, the growing RNAchainis extended by one
`
`nucleotide at a time in the 5’-to-3’ direction (Figure 6-8), The substrates are
`
`nucleoside triphosphates (ATP, CTR UTP, and GTP); as for DNAreplication, a
`
`hydrolysis of high-energy bonds provides the energy needed to drive the reac-
`
`tion forward(see Figure 5-4).
`The almost immediate release of the RNA strand from the DNAasit is syn-
`thesized means that many RNAcopies can be madefrom the same genein a
`Figure 6-5 Uracil forms base pairs with adenine. The absence of a
`methyl group in U has no effect on base-pairing; thus, U-A base pairs closely
`resemble T—A basepairs (see Figure 44).
`
`
`
`Fl
`
`N
`
`SH
`
`N
`
`adenine
`Bs H
`
`5!
`
`sugar-phasphate backbone
`
`3
`
`303
`
`FROM DNATO RNA
`
`
`
`Page 8
`aRS|
`
`Page 8
`
`

`

`
`
`Figure 6-6 RNA canfold into specific structures. RNAis largely single-stranded, but it often contains short
`stretches of nucleotides that can form conventional base-pairs with complementary sequences found elsewhere
`on the same molecule. These interactions, along with additional “nonconyentional” base-pair interactions, allow an
`RNA molecule to fold into a three-dimensional structure that is determined by its sequence of nucleotides.
`(A) Diagram of a folded RNA structure showing only conventional base-pair interactions; (B) structure with both
`conventional (red) and nonconventional(green) base-pair interactions; (C) structure of an actual RNA,a portion of
`a group |
`intron (see Figure 6-36). Each conventional base-pairinteraction is indicated by a “rung” in the double
`helix, Bases in other configurations are indicated by broken rungs.
`relatively short time, the synthesis of additional RNA molecules being started
`beforethe first RNA is completed (Figure 6-9). When RNA polymerase molecules
`follow hard on each other's heels in this way, each moving at about 20
`nucleotides per second (the speed in eucaryotes), over a thousand transcripts
`can be synthesized in an hour from a single gene.
`Although RNA polymerase catalyzes essentially the same chemicalreaction
`as DNA polymerase, there are some important differences between the two
`enzymes, First, and most obvious, RNA polymerase catalyzes the linkage of
`ribonucleotides, not deoxyribonucleotides. Second,unlike the DNA polymerases
`involved in DNAreplication, RNA polymerases can start an RNA chain without
`a primer. This difference may exist because transcription need notbe as accu-
`rate as DNAreplication (see Table 5-1, p. 243). Unlike DNA, RNA does notper-
`manently store genetic information in cells. RNA polymerases make about one
`mistake for every 10* nucleotides copied into RNA (compared with anerror rate
`for direct copying by DNA polymeraseof about onein 107 nucleotides), and the
`consequences of an error in RNAtranscription are muchless significant than
`that in DNAreplication.
`Although RNA polymerases are not nearly as accurate as the DNA poly-
`merases that replicate DNA, they nonetheless have a modest proofreading
`mechanism.If the incorrect ribonucleotide is added to the growing RNA chain,
`the polymerase can back up, and the active site of the enzyme can perform an
`excision reaction that mimics the reverse of the polymerization reaction, except
`that water instead of pyrophosphate is used (see Figure 5-4). RNA polymerase
`hovers around a misincorporated ribonucleotide longer than it does for a cor-
`
`Cem.
`template strand
`
`
`|reanscrion
`
`RNA
`Figure ict DNA trenseripdén
`Ccells Produce Several Types of RNA
`The majority of genescarried in a cell’s DNA specify the amino acid sequence of_producesa single-stranded RNA
`proteins; the RNA molecules that are copied from these genes (which ultimately
`Molecule that is complementary
`direct the synthesis of proteins) are called messenger RNA (mRNA) molecules.
`_—_*® one strand of DNA,
`
`rect addition, causingexcisionto be favoredforincorrectnucleotides. However,
`
`RNA polymerasealso excises many correct basesas part of the cost for improved
`accuracy.
`
`304
`
`Chapter 6 : HOW CELLS READ THE GENOME: FROM DNA TO PROTEIN
`
`Page 9
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`3 5
`
`
`
`jawsin closed
`configuration
`
`
`
`DNA double
`helix
`
`RNA polymerase
`
`rewinding
`/ triphosphates
`
`
`DNA
`\
`
`ribonucleotide
`
`direction of
`transcription
`
`Figure 6-8 DNAis transcribed by
`the enzyme RNApolymerase. The
`RNApolymerase (pale blue) moves
`stepwise along the DNA, unwinding the
`DINAhelix atits active site. Asit
`progresses, the polymerase adds
`nucleotides (here, small “T” shapes) one by
`one to the RNA chain at the
`polymerization site using an exposed
`DNAstrand as a template. The RNA
`transcript is thus a single-stranded
`;
`4
`flap in
`complementary copy of one of the two
`ribonucleotide
`;
`alased
`:
`:
`
`
`DNAstrands. The polymerase has a
`
`position active site_triphosphateRNA exit
`
`5’ channel
`tunnel
`
`rudder (see Figure 6-| |) that displaces
`newly synthesized
`short region of
`the newly formed RNA,allowing the two
`RNA transcript
`DNA/RNA helix
`strands of DNA behind the polymerase to
`rewind.A short region of DNA/RNAhelix
`(approximately nine nucleotides in length)
`is therefore formed only transiently, and a
`“window” of DNA/RNA helix therefore
`moves along the DNA with the
`polymerase.The incoming nucleotides are
`in the form of ribonucleoside
`triphosphates (ATR UTP. CTP, and GTP),
`and the energy storedin their
`phosphate-phosphate bonds provides the
`driving force for the polymerization
`reaction (see Figure 5-4). (Adapted from a
`figure kindly supplied by Robert Landick.)
`
`
`
`Jort
`
`“an
`
`yth
`yof
`le
`
`The final product of a minority of genes, however, is the RNA itself. Careful
`analysis of the complete DNA sequenceofthe genomeofthe yeastS. cerevisiae
`
`has uncoveredwell over 750 genes (somewhat more than 10% of the total num-
`
`berof yeast genes) that produce RNAastheirfinal product, although this num-
`
`ber includes multiple copies of some highly repeated genes. These RNAs,like
`
`proteins, serve as enzymatic and structural components for a wide variety of
`
`processes in thecell. In Chapter 5 we encountered oneof those RNAs, the tem-
`
`plate carried by the enzyme telomerase. Although notall of their functions are
`
`known, wesee in this chapter that some small nuclear RNA (snRNA) molecules
`
`direct the splicing of pre-mRNA to form mRNA, that ribosomal RNA (rRNA)
`
`molecules form the core of ribosomes, andthat éransfer RNA (tRNA) molecules
`
`formthe adaptors that select amino acids and hold them inplace on a ribosome
`
`for incorporation into protein (Table 6-1).
`
`Each transcribed segment of DNAis called a transcription unit. In eucary-
`
`otes, a transcriptionunit typically carries the informationof just one gene, and
`therefore codesfor either a single RNA molecule ora single protein (or group of
`
`related proteinsif the initial RNA transcriptis spliced in more than one way to
`
`producedifferent mRNAs). In bacteria, a set of adjacent genes is often trans-
`
`cribed as a unit; the resulting mRNA molecule therefore carries the information
`
`for several distinct proteins.
`
`Overall, RNA makes up a few percentofa cell's dry weight. Most of the RNA
`
`in cells is rRNA; mRNA comprises only 3-5% of the total RNA in a typical mam-
`
`malian cell. The mRNA population is made upof tens of thousands ofdifferent
`
`species, and there are on average only 10-15 molecules of each species of mRNA
`present in each cell.
`Pad
`ais
`
`
`
`
`
`Ee
`
`Tum
`
`Figure 6-9 Transcription of two genes as observed under the
`electron microscope. The micrograph shows many molecules of RNA
`polymerase simultaneously transcribing each of two adjacent genes.
`Molecules of RNA polymerase are visible as a series of dots along the DNA
`with the newly synthesized transcripts (fine threads) attached to them.The
`RNA molecules (ribosomal RNAs) shownin this example are not translated
`into protein but are instead used directly as components of ribosomes, the
`machines on which translation,takes place.The particles at the 5’ end (the
`free end) of each rRNA transcript are believed to reflect the beginnings of
`ribosome assembly. From the lengths of the newly synthesized transcripts,it
`can be deduced that the RNA polymerase molecules are transcribing from
`left to right. (Courtesy of Ulrich Scheer.)
`
`FROM DNA TO RNA
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`Gt—_—_—_——
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`TABLE 6—! Principal Types of RNAs Producedin Cellseese
`
` TYPE OF RNA
`FUNCTION
`
`
`
`mRNAs
`TRNAS
`
`tRNAs
`
`snRNAs
`
`snoRNAs
`
`messenger RNAs, code for proteins
`ribosomal RNAs,form the basic structure of the
`ribosomeand catalyze protein synthesis
`transfer RNAs, central to protein synthesis as adaptors
`between mRNA and amino acids
`small nuclear RNAs, function in a variety of nuclear
`processes, including the splicing of pre-mRNA
`small nucleolar RNAs, used to process and chemically
`modify rRNAs
`function in diverse cellular processes, including
`Other noncoding
`telomere synthesis, X-chromosomeinactivation,
`RNAs
`and the transportofproteins into the ER—_—_Lr————————————————
`
`
`
`Signals Encoded in DNA Tell RNA Polymerase Where
`to Start and Stop
`To transcribe a gene accurately, RNA polymerase must recognize where on the
`genome to start and where to finish, The way in which RNA polymerases per-
`form these tasks differs somewhat between bacteria and eucaryotes, Because
`the processin bacteria is simpler, we look therefirst.
`Theinitiationof transcription is an especially important step in gene expres-
`sion becauseit is the main point at whichthe cell regulates which proteins are
`to be produced and at what rate. Bacterial RNA polymerase is a multisubunit
`complex, A detachable subunit, called sigma(o) factor, is largely responsible for
`its ability to read the signals in the DNAthattell it where to begin transcribing
`(Figure 6-10), RNA polymerase molecules adhere only weakly to the bacterial
`DNA when they collide with it, and a polymerase moleculetypically slides
`rapidly along the long DNA molecule until it dissociates again.However, when
`the polymeraseslides into a region on the DNA doublehelix called a promoter,
`a special sequence ofnucleotides indicating the starting point for RNA synthe-
`sis, it binds tightly to it. The polymerase,usingits o factor, recognizes this DNA
`sequence by making specific contacts with the portions of the bases that are
`exposed on the outside of the helix (Step J in Figure 6-10).
`After the RNA polymerase bindstightly to the promoter DNAin this way,it
`opens up the double helix to expose a shortstretch of nucleotides on each
`strand (Step 2 in Figure 6-10). Unlike a DNAhelicase reaction (see Figure 5-15),
`this limited openingof the helix does not require the energy of ATP hydrolysis.
`Instead, the polymerase and DNA both undergoreversible structural changes
`that result in a more energetically favorable state, With the DNA unwound,one
`of the two exposed DNAstrands acts as a template for complementary base-
`pairing with incoming ribonucleotides(see Figure 6-7), two of which are joined
`together by the polymerase to begin an RNA chain. After the first ten or so
`nucleotides of RNA have been synthesized(a relatively inefficient process dur-
`ing which polymerase synthesizes and discards short nucleotide oligomers), the
`o factorrelaxesits tight hold on the polymerase and evenutally dissociates from
`it. During this process, the polymerase undergoesadditional structural changes
`that enable it to move forward rapidly, transcribing without the o factor(Step 4
`in Figure 6-10). Chain elongation continues (at a speed of approximately 50
`nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a
`secondsignal in the DNA, the terminator (described below), where the poly-
`merase halts and releases both the DNA template and the newly made RNA
`chain (Step 7 in Figure 6-10), After the polymerase has been releasedat a termi-
`nator,it reassociates witha free o factor and searchesfor a new promoter, where
`it can begin the process of transcription again.
`Several structural features of bacterial RNA polymerase makeit particularly
`adept at performing the transcription cycle just described. Once the o factor
`
`306
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`Chapter 6 : HOW CELLS READ THE GENOME: FROM DNA TO PROTEIN
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`o factor
`
`OS
`
`RNA
`
`7
`
`x
`|
`
`es
`
`DNA
`
`2
`
`\:
`
`RNA
`
`4
`
`OS
`
`a}
`
`RNA
`
`promoter
`st
`RNApolymerase a= polymerase has managed to synthesize
`grip,andthepolymeraseundergoesa
`
`Figure 6-10 The transcription cycle
`
`ofbacterial RNA polymerase.In step
`
`|, the RNA polymerase holoenzyme (core
`polymerase plus o factor) forms and then
`locates a promoter (see Figure 6-12). The
`polymerase unwinds the DNA at the
`position at which transcription is to begin
`(step 2) and begins transcribing (step 3),
`This initial RNA synthesis (sometimes
`called “abortive initiation”) is relatively
`inefficient. However, once RNA
`
`about 10 nucleotides of RNA, o relaxes its
`
`series of conformational changes (which
`probably includes a tightening ofits jaws
`and the placementof RNAin the exit
`channel [see Figure 6-1 1]).The
`polymerase now shifts to the elongation
`mode of RNA synthesis (step 4), moving
`rightwards along the DNAin this diagram.
`During the elongation mode (step 5)
`
`transcriptionishighlyprocessive,withthe
`
`polymerase leaving the DNA template and
`releasing the newly transcribed RNAonly
`whenit encounters a termination signal
`(step 6). Termination signals are encoded
`in DNA and many function by forming an
`RNAstructure that destabilizes the
`polymerase’s hold on th