1
`
`MTX1055
`ModernaTX, Inc. v. CureVac AG
`IPR2017-02194
`
`

`

`TS
`
`The Cover
`This illustration showsa portion of the insideof a cell nucleus, including some of the
`manyproteins that copy, repair, and package DNA. DNAstrandsare shownin yel-
`low. Running throughthe centerofthe illustration, top to bottom,is a replication
`fork, showing DNAbeing copied by DNApolymerase. Ontheright and left sides of
`theillustration, RNA polymeraseis synthesizing messenger RNA.Most of the DNA
`in the picture is wrapped around nucleosomes. Illustration by David S. Goodsell, The
`Scripps Research Institute.
`Part I opener image
`Microtubules and actin filaments are stained with red and green fluorescent dyes,
`respectively.
`(K. G. Murti/Visuals Unlimited)
`Part II opener image
`High resolution X-ray crystal structures of ribosomalunits.
`(From N.Ban,P. Nissen,J. Hansen, P. B. Moore andT. A.Steitz, 2000. Science 289: 905.
`Courtesy of ThomasA.Steitz.)
`Part III opener image
`Probesto repeated sequences on chromosome 4 were hybridized to a humancell.
`The twocopies of chromosome4,identified by yellow fluorescence, occupy distinct
`territories in the nucleus.
`(From A. I. Lamond and W.C. Earnshaw, 1998. Science 280: 547.)
`Part IV opener image
`Mitosis sequence: Telophase.
`(Conly L. Rieder/Biological Photo Service)
`
`Library of Congress Cataloging-in-Publication Data
`Cooper, Geoffrey M.
`Thecell :a molecular approach / Geoffrey M. Cooper, Robert E.
`Hausman.— 3rd ed.
`p.;cm.
`Includes bibliographical references andindex.
`ISBN 0-87893-214-3 (alk. paper)
`1. Cytology. 2. Molecular biology.
`[DNLM:1. Cytology. 2. Molecular Biology. QH 581.2 C776c 2004]I.
`Hausman, Robert E., 1947-II. Title.
`QH581.2.C66 2004
`571.6—dc21
`
`2003008953
`
`|
`
`er
`
`
`
`The Cell: A Molecular Approach, Third Edition
`Copyright © 2004 by Geoffrey M. Cooper. All rights reserved.
`This book maynot be reproduced in whole or in part without permission,
`Address editorial correspondence to ASM Press, c/o The American Society for
`Microbiology, 1752 N Street NW,Washington, DC 20036 U.S.A.
`Address orders and requests for examination copies to Sinauer Associates, Inc.,
`PO.Box 407, 23 Plumtree Road, Sunderland, MA 01375 U.S.A.
`Phone: 413-549-4300
`FAX: 413-549-1118
`email: orders@sinauer.com
`www.sinauer.com
`
`Madison, W1 53706-1293
`
`saat
`o_
`
`Steenbock Memorial Library
`University of Wisconsin - Madison
`550 Babcock Drive
`
`2
`
`

`

`
`
`Chapter
`
`Fundamentals
`ofMolecular Biology
`
`Heredity,Genes,and DNA 89
`Expression of Genetic
`Information 96
`
`Recombinant DNA 104
`
`Detection of Nucleic Acids and
`Proteins 117
`
`Gene Function in Eukaryotes 123
`KEY EXPERIMENT: The DNA
`Provirus Hypothesis 102
`MOLECULAR MEDICINE: HIV and
`AIDS 105
`
`ONTEMPORARY MOLECULAR BIOLOGYis concerned principally with under-
`standing the mechanismsresponsible for transmission and expression of
`the genetic information that governscell structure and function. As
`reviewed in Chapter1, all cells share a numberof basic properties, and this
`underlying unity ofcell biology is particularly apparent at the molecularlevel.
`Suchunity has allowed scientists to choose simple organisms(suchas bacteria)
`as models for many fundamental experiments, with the expectation that similar
`molecular mechanisms are operative in organisms as diverse as E. coli and
`humans. Numerousexperiments have established the validity of this assump-
`tion, andit is nowclearthat the molecular biology of cells provides a unifying
`theme to understanding diverse aspectsofcell behavior.
`Initial advances in molecular biology were made by taking advantageof the
`rapid growth andreadily manipulable genetics of simple bacteria, such as E.
`coli, and their viruses. The development of recombinant DNAthenallowed both
`the fundamental principles and many of the experimental approachesfirst
`developed in prokaryotes to be extended to eukaryotic cells. The application of
`recombinant DNA technology has had a tremendousimpact,initially allowing
`individual eukaryotic genes to be isolated and characterized in detail and more
`recently allowing the determination of the complete genome sequences of com-
`plex plants and animals, including humans.
`
`Heredity, Genes, and DNA
`Perhaps the most fundamental property of all living thingsis the ability to
`reproduce. All organismsinherit the genetic information specifying their struc-
`ture and function from their parents. Likewise,all cells arise from preexisting
`cells, so the genetic material must be replicated and passed fromparent to prog-
`enycell at each cell division. How genetic information is replicated and trans-
`mitted from cell to cell and organismto organism thus represents a question that
`is central to all of biology. Consequently, elucidation of the mechanisms of
`genetic transmission and identification of the genetic material as DNA weredis-
`coveries that formed the foundation of our current understanding of biology at
`the molecular level.
`
`3
`
`

`

`=Chapter 3
`
`Figure 3.1 Inheritance of dominant
`and recessive genes
`
` 90
`
`
`(green) phenatypes,
`
`
`The parental strains of peas each contain
`two copies(alleles) of the gene for either
`yellow(Y) or green (vi seeds.
`
`
`
`
`
`The parents produce germ cells (gametes),
`nee
`
`each conlaining one of these genes, that
`give rise to hybrid F, progeny.
`
`
`Since Y is dominant, all the F, planis have
`yellowseeds.
`
` A cross between (wo F, plants yields an
`
`Fa generation, with a characteristic
`4:1 ratio of dominant(yellow) to recessive
`
`i
`
`@)
`
`()
`
`()
`
`2
`
`Gameles
`F\ peneration
`
`Gametes
`
`F, generation
`
`Genes and Chromosomes
`Theclassical principles of genetics were deduced by Gregor Mendel in
`1865, on the basis of the results of breeding experiments with peas. Mendel
`studied the inheritance of a numberof well-defined traits, such as seed
`color, and wasable to deduce general rules for their transmission. In all
`cases, he could correctly interpret the observed patterns of inheritance by
`assuming that eachtrait is determined bya pairofinherited factors, which
`are now called genes. One gene copy (called anallele) specifying each trait
`is inherited from each parent. For example, breeding twostrains of peas—
`one having yellow seeds, and the other green seeds—yields the following
`results (Figure 3.1). The parental strains each have two identical copies of
`the gene specifying yellow (Y)or green (y) seeds, respectively. The progeny
`plants are therefore hybrids, having inherited one gene for yellow seeds(Y)
`and onefor green seeds (y), All these progenyplants(thefirstfilial, or Fi;
`generation) have yellow seeds, so yellow (Y) is said to be dominant and
`green(y) recessive. The genotype(genetic composition) of the F, peasis
`thus Yy, and their phenotype (physical appearance)is yellow.If one F, off-
`spring is bred with another, giving rise to F, progeny, the genesfor yellow
`and green seeds segregate in a characteristic manner such that the ratio
`betweenF, plants with yellow seeds and those with green seedsis 3;1.
`Mendel’s findings, apparently ahead oftheir time, were largely ignored
`until 1900, when Mendel’s laws were rediscovered andtheir importance
`recognized. Shortly thereafter, the role of chromosomesas the carriers of
`genes was proposed.It was realized that mostcells of higher plants and ani-
`mals are diploid—containing two copies of each chromosome. Formation
`of the germ cells (the sperm and egg), however, involves a unique type of
`cell division (meiosis) in which only one memberof each chromosome pair
`is transmitted to each progeny cell (Figure 3.2). Consequently, the sperm
`and egg are haploid, containing only one copy of each chromosome.The
`union of these two haploid cellsatfertilization creates a new diploid organ-
`
`4
`
`

`

`Fundamentals of Molecular Biology
`
`91
`
`Figure 3.2 Chromosomesat meiosis
`and fertilization
`Two chromosomepairs of a hypotheti-
`cal organism areillustrated.
`
`
`
`Diploid cells contain two capies of
`each chromosome.
`
`|
`
`Eee
`
`
`
`Melasis gives rise to haploid
`gatnetes containing only one
`
`
`
`member of each chromosomepair.
`
`
`
`
`of a diploid embryo, containing
`chromosomes contributed by
`both parents.
`
`Male parent
`
`Female parent
`
`Meiosis
`
`Embryo
`
`;
`
`
`Diploid
`
`Fertilization AW/)
`|
`Fertilizationresultsintheformation|
`
`
`Se"
`
`ism, now containing one memberof each chromosomepair derived from
`the male and one from the female parent. The behavior of chromosome
`pairs thus parallels that of genes, leading to the conclusion that genes are
`carried on chromosomes.
`The fundamentals of mutation, genetic linkage, and the relationships
`between genes and chromosomeswerelargely established by experiments
`performed with thefruit fly, Drosophila melanogaster. Drosophila can be easily
`maintained in the laboratory, and they reproduce about every two weeks,
`which is a considerable advantage for genetic experiments. Indeed, these
`features continue to make Drosophila an organism of choice for genetic stud-
`ies of animals, particularly the genetic analysis of development and differ-
`entiation.
`In the early 1900s, a numberof genetic alterations (mutations) were iden-
`tified in Drosophila, usually affecting readily observable characteristics such
`as eye color or wing shape. Breeding experiments indicated that someof the
`genes governingthesetraits are inherited independently of each other, sug-
`gesting that these genes are located on different chromosomesthat segre-
`gate independently during meiosis (Figure 3.3). Other genes, however, are
`frequently inherited together as paired characteristics. Such genesare said
`to be linked to each other by virtue of being located on the same chromo-
`some. The numberof groupsof linked genes is the same as the numberof
`chromosomes(four in Drosophila), supporting the idea that chromosomes
`are carriers of the genes. By 1915, nearly a hundred genes had been defined
`and mapped onto the four chromosomesof Drosophila, leading to general
`acceptance of the chromosomalbasis ofheredity.
`
`Genes and Enzymes
`Early genetic studies focused onthe identification and chromosomallocal-
`ization of genes that control readily observable characteristics, such as the
`eye color of Drosophila. How these genes lead to the observed phenotypes,
`however, was unclear. Thefirst insight into the relationship between genes
`and enzymes camein 1909, whenit wasrealized that the inherited human
`
`
`
`5
`
`

`

`
`
`92
`
`Chapter 3
`
`(A) Segregation of two hypothetical genes located on different
`chromosomes (A/a = square/round and B/b = red/blue)
`
`(B) Linkage of two genes located on the same chromosome
`
`Parental
`strains
`
`Gametes
`
`\
`
`/
`
`Parental
`strains
`
`t t
`
`J
`
`(.|a
`
`\@ 1a =
`
`Gametes
`
`8
`
`A
`
`|
`
`b
`
`a
`
`|
`
`
`
`
`
`
`
`Since the chromosomes
`segregate independently at
`meiosis, the F, generation
`gives rise to four different
`types of gametes.
`
`
`
`F, generation
`
`Because both genes
`are carried on the
`same chromosome,
`they do not separate
`irom each other at
`meiosis. Consequently,
`the F, generation
`produces only two
`types of gametes.
`
`| Gametes
`
`Fs generation
`
`
`
`
`The F; generation displays only two phenotypes—
`square/red and round/blue—in the 3:1 ratio that is
`
`characteristic of inheritance ofa single gene.
`
`F, generation
`
`Gametes
`
`
`
`F, generation
`
`
`
`The F, generation therefore
`displays four distinct
`phenotypes—square/red,
`
`square/blue, round/red,
`
`and round/blue—in a
`9:3:3:1 ratio.
`
`
`
`Figure 3.3 Gene segregation
`and linkage
`(A) Segregation of two hypothetical
`genesfor shape (A/a = square/round)
`and color (B/b = red/blue) located on
`different chromosomes. (B) Linkage of
`twogeneslocated on the samechro-
`mosome.
`
`disease phenylketonuria (see Molecular Medicine in Chapter2) results from
`a genetic defect in metabolism of the amino acid phenylalanine. This defect
`was hypothesized to result from a deficiency in the enzyme neededtocat-
`alyze the relevant metabolic reaction, leading to the general suggestion that
`genes specify the synthesis of enzymes.
`Clearer evidence linking genes with the synthesis of enzymes came from
`experiments of George Beadle and Edward Tatum, performed in 1941 with
`
`6
`
`

`

`
`
`Fundamentals of MolecularBiology
`
`93
`
`the fungus Newrospora crassa. In the laboratory, Neurospora can be grown on
`minimalor rich media similar to those discussed in Chapter1 for the growth
`of E.coli. For Neurospora, minimal media consist only ofsalts, glucose, and
`biotin; rich media are supplemented with aminoacids, vitamins, purines,
`and pyrimidines. Beadle and Tatum isolated mutants of Neurospora that
`grew normally on rich media but could not growon minimal media. Each
`mutant was found to require a specific nutritional supplement, such as a
`particular amino acid, for growth. Furthermore, the requirementfor a spe-
`cific nutritional supplementcorrelated with the failure of the mutantto syn-
`thesize that particular compound. Thus, each mutation resultedin a defi-
`ciency in a specific metabolic pathway. Since such metabolic pathways were
`knownto be governed by enzymes, the conclusion from these experiments
`was that each gene specified the structure of a single enzyme—the one
`gene-one enzyme hypothesis. Many enzymes are now knownto consist of
`multiple polypeptides, so the currently accepted statementof this hypothe-
`sis is that each genespecifies the structure of a single polypeptide chain.
`
`Identification of DNA asthe Genetic Material
`Understanding the chromosomal basis of heredity and the relationship
`between genes and enzymesdid notinitself provide a molecular explana-
`tion of the gene. Chromosomescontain proteins as well as DNA,andit was
`initially thought that genes wereproteins. Thefirst evidence leading to the
`identification of DNA as the genetic material camefromstudiesin bacteria.
`These experiments represent a prototypefor current approachesto defining
`the function of genes by introducing new DNA sequencesintocells, as dis-
`cussed later in this chapter.
`The experiments that defined the role of DNA were derived from studies
`of the bacterium that causes pneumonia (Prewmococcus). Virulent strains of
`Pneumococcus are surrounded by a polysaccharide capsule that protects the
`bacteria from attack by the immunesystem ofthe host. Because the capsule
`gives bacterial colonies a smooth appearancein culture, encapsulated
`strains are denoted S. Mutant strains that havelost the ability to make a
`capsule (denoted R) form rough-edged colonies in culture and are no longer
`lethal when inoculated into mice. In 1928 it was observed that mice inocu-
`lated with nonencapsulated (R) bacteria plus heat-killed encapsulated (5)
`bacteria developed pneumonia and died. Importantly, the bacteria that
`were then isolated from these mice wereof the S type. Subsequent experi-
`ments showedthata cell-free extract of S bacteria was similarly capable of
`converting (or transforming) R bacteria to the S state. Thus, a substancein
`the S extract (called the transforming principle) was responsible for induc-
`ing the genetic transformation of R to S bacteria.
`In 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty established
`that the transforming principle was DNA,bothby purifyingit from bacter-
`ial extracts and by demonstratingthat the activity of the transformingprin-
`ciple is abolished by enzymatic digestion of DNA but notby digestion of
`proteins (Figure 3.4). Although these studies did not immediately lead to
`the acceptance of DNA as the genetic material, they were extended within a
`few years by experiments with bacterial viruses.In particular, it was shown
`that, when a bacterial virus infects a cell, the viral DNA rather thantheviral
`protein must enter the cell in order for the virus to replicate. Moreover, the
`parental viral DNA (but not the protein) is transmitted to progeny virus
`particles. The concurrence ofthese results with continuing studiesof the
`activity of DNA in bacterial transformation led to acceptanceof the idea
`that DNA is the genetic material.
`
`7
`
`

`

`
`
`94
`
`Chapter 3
`
`
`Nonpathogenic
`
` Pathogenic
`
`5 bacteria
`
`R bacteria
`
` Capsule
`Purity DNA |
`DRG
`CRNA NUNUNN
`Bune
`ROWUN
`
`Add puritied
`S oe to
`R bacteria
`
`§ bacteria
`
`Figure 3.4 Transferof genetic information by DNA
`DNA is extracted from a pathogenicstrain of Pneuniococcus, which is surrounded
`by a capsule and forms smooth colonies (S). Addition of the purified S DNA toa
`culture of nonpathogenic, nonencapsulated bacteria (R for “rough”colonies) re-
`sults in the formation of S colonies. The purified DNAtherefore contains the genet-
`ic information responsible for transformationofR to S bacteria.
`
`The Structure of DNA
`Our understanding of the three-dimensional structure of DNA, deduced in
`1953 by James Watson and Francis Crick, has been the basis for present-day
`molecular biology. At the time of Watson and Crick’s work, DNA was
`knownto be a polymer composed of four nucleic acid bases—two purines
`(adenine [A] and guanine [G]) and two pyrimidines (cytosine [C] and
`thymine [T])—linked to phosphorylated sugars. Given the central role of
`DNAasthe genetic material, elucidation ofits three-dimensional structure
`appearedcritical to understanding its function. Watson and Crick’s consid-
`eration of the problemwasheavily influenced by Linus Pauling’s descrip-
`tion of hydrogen bonding and the @ helix, a common elementof the sec-
`ondary structure of proteins (see Chapter 2). Moreover, experimental data
`on the structure of DNA wereavailable from X-ray crystallography studies
`by Maurice Wilkins and Rosalind Franklin. Analysis of these data revealed
`that the DNA moleculeis a helix that turns every 3.4 nm. In addition, the
`data showedthatthe distance between adjacentbasesis 0.34 nm, so there
`are ten bases per turn ofthe helix. An importantfinding wasthat the diam-
`eter of the helix is approximately 2 nm, suggesting thatit is composedof
`not one but two DNA chains.
`From these data, Watson and Crick built their model of DNA (Figure3.5).
`The central features of the model are that DNAis a double helix with the
`sugar—phosphate backbones onthe outside of the molecule. The bases are
`
`8m
`
`eee
`
`8
`
`

`

`
`
`Fundamentals of Molecular Biology
`
`95
`
`on the inside, oriented such that hydrogen bonds are formed between
`purines and pyrimidines on opposite chains. The base pairing is very spe-
`cific: A alwayspairs with T and G with C.This specificity accounts for the
`earlier results of Erwin Chargaff, who had analyzed the base composition of
`various DNAsand foundthat the amount of adenine was always equal to
`that of thymine, and the amountof guanineto that of cytosine. Because of
`this specific base pairing, the two strands of a DNA molecule are comple-
`mentary: Each strand containsall the information required to specify the
`sequencesof bases on the other.
`
`
`
`Replication ofDNA
`The discovery of complementary base pairing between DNAstrands imme-
`diately suggested a molecular solution to the question of how the genetic
`material could direct its own replication—aprocessthatis required each time
`a cell divides. It was proposed that the twostrands of a DNA molecule could
`separate and serve as templates for synthesis of new complementary strands,
`the sequence of which would be dictated by the specificity of base pairing
`
`
`
`DNA is a double helix with the bases on the
`inside and the sugar—-phosphate backbones on
`the outside of the molecule.
`
`Figure 3.5 The structure of DNA
` Bases on opposite strands are paired by hydrogen bonds between adenine (A) and
`thymine (T), and between guanine (G) andcytosine (C), The two DNAstrands run in opposite
`directions, detined bythe 5° and 3° groups of deoxyribose.
`
`3’ end
`
`9
`
`

`

`
`
`96
`
`Chapter 3
`
`(Figure 3.6). The processis called semiconservative replication because one
`strand of parental DNAis conserved in each progeny DNA molecule.
`Direct support for semiconservative DNA replication was obtained in
`1958 as a result of elegant experiments, performed by Matthew Meselson
`and Frank Stahl, in which DNA waslabeled with isotopesthat altered its
`density (Figure 3.7). E. coli were first grown in media containing the heavy
`isotope of nitrogen ('5N) in place of the normal light isotope ('*N). The
`DNAofthese bacteria consequently contained '°N and washeavier than
`that of bacteria grown in 4N. Such heavy DNAcould be separated from
`DNAcontaining 'N by equilibrium centrifugation in a density gradient of
`CsCl. This ability to separate heavy (SN) DNA from light ('4"N) DNA
`enabled the study of DNA synthesis. E. coli that had been grown in '°N
`were transferred to media containing ''N and allowedto replicate one more
`time. Their DNA wasthen extracted and analyzed by CsCl density gradient
`centrifugation. The results of this analysis indicated that all of the heavy
`DNAhad been replaced by newly synthesized DNA with a density inter-
`mediate betweenthat of heavy ('°N) andthatoflight ("N) DNA molecules,
`The implication wasthat during replication, the two parentalstrands of
`heavy DNAseparated and served as templates for newly synthesized prog-
`eny strandsof light DNA,yielding double-stranded molecules of interme-
`diate density. This experiment thus provided direct evidence for semicon-
`servative DNA replication, clearly underscoring the importance of
`complementary base pairing between strandsof the double helix.
`The ability of DNA to serve as a template for its own replication wasfur-
`ther established with the demonstration that an enzymepurified from E.
`coli (DNA polymerase) could catalyze DNAreplication invitro. In the pres-
`ence of DNAto act as a template, DNA polymerase was able to direct the
`incorporation of nucleotides into a complementary DNA molecule.
`
`
`
`
`
`
`
`
`
`
`
`
`Expression of Genetic Information
`Genes act by determining the structure of proteins, which are responsible
`
`for directing cell metabolism through their activity as enzymes. The identi-
`
`fication of DNA asthe genetic material and the elucidationof its structure
`
`Old DNA strand
`
`New DNA strand
`
`Figure 3.6 Semiconservative replication of DNA
`The twostrands of parental DNA separate, and each serves as a
`template for synthesis of a new daughter strand by complemen-
`tary base pairing.
`
`3
`
`
`
`10
`
`

`

`density
`
`Extract DNA
`Centrifuge in
`CsCl solution
`
`—=>
`——=>
`
`Light DNA
`
`—
`
`i
`a:
`:
`>
`
`Heavy DNA
`
`Bacteria grown in '4N media
`
`Bacteria grown in 1°N media
`
`Ecolt Gomee®) —S== oe
`
`Fundamentals of Molecular Biology
`
`97
`
`Transfer to '4N media for onedivision
`
`Extract DNA
`
`Centrifugein
`CsCl solution
`
`Extract DNA
`
`Centrifuge in
`CsC] solution
`
`Increasing
`density
`
`Increasing
`density
`
`Hybrid DNA
`
`Increasing
`
`Figure 3.7 Experimental demonstra-
`tion of semiconservative replication
`Bacteria grown in medium containing
`the normal isotope of nitrogen (!4N)
`are transferred into medium contain-
`ing the heavy isotope (°N) and grown
`in this medium for several generations.
`They are then transferred back to
`medium containing ''N and grownfor
`one additional generation. DNA is ex-
`tracted from these bacteria and ana-
`lyzed by equilibrium ultracentrifuga-
`tion in a CsCl solution. The CsCl sedi-
`ments to form a density gradient, and
`the DNA molecules band at a position
`wheretheir density is equal to that of
`the CsCl solution. DNA of the bacteria
`transferred from N to *N medium
`for a single generation bands at a den-
`sity intermediate betweenthat of °N
`DNAandthat of '4N DNA, indicating
`that it represents a hybrid molecule
`with one heavy and onelight strand.
`
`revealed that genetic information mustbe specified by the orderof the four
`bases (A, C, G, and T) that make up the DNA molecule. Proteins, in turn,
`are polymers of 20 aminoacids, the sequence of which determines their
`structure and function. The first direct link between a genetic mutation and
`an alteration in the amino acid sequence of a protein was made in 1957,
`whenit was found that patients withthe inherited disease sickle-cell ane-
`mia had hemoglobin moleculesthat differed from normalones bya single
`amino acid substitution. Deeper understanding of the molecularrelation-
`ship between DNAandproteins came, however, from a series of experi-
`ments that took advantageof E,coli andits viruses as genetic models.
`
`Colinearity of Genes and Proteins
`The simplest hypothesis to accountforthe relationship between genes and
`enzymes wasthat the orderof nucleotides in DNAspecified the order of
`aminoacids in a protein. Mutations in a gene would correspond to alter-
`ations in the sequence of DNA, which mightresult from the substitution of
`one nucleotide for another or from the addition or deletion of nucleotides.
`These changes in the nucleotide sequence of DNA would thenlead to cor-
`responding changes in the amino acid sequenceofthe protein encoded by
`the gene in question. This hypothesis predicted that different mutations
`within a single gene could alter different amino acidsin the encoded pro-
`tein, and that the positions of mutations in a gene shouldreflect the posi-
`tions of aminoacid alterationsin its protein product.
`The rapid replication and the simplicity of the genetic system ofE. coli
`were of major help in addressing these questions. A variety of mutants ofE.
`coli could be isolated, including nutritional mutantsthat (like the Newrospora
`mutants discussed earlier) require particular amino acids for growth.
`Importantly, the rapid growthof E. coli made feasible the isolation and
`mapping of multiple mutants in a single gene, leading to the first demon-
`stration of the linear relationship between genes and proteins. In these stud-
`ies, Charles Yanofsky and his colleagues mappeda series of mutations in
`the gene that encodes an enzymerequired for synthesis of the amino acid
`tryptophan, Analysis of the enzymes encoded by the mutant genesindi-
`cated that the relative positions of the aminoacid alterations were the same
`as those of the corresponding mutations (Figure 3.8). Thus, the sequence of
`aminoacids in the protein was colinear with that of mutationsin the gene,
`
`11
`
`11
`
`€
`

`

`98
`
`Chapter 3
`
`Mutations
`DNA
`
`Normal protein
`
`Amino acid substitutions
`resulting from mutations
`
`
`
`
`
`
`
`
`
`Figure 3.9 Synthesis of RNA from
`DNA
`The twostrands of DNA unwind, and
`one is used as a template for synthesis
`of a complementary strand of RNA.
`
`Figure 3.8 Colinearity of genes and proteins
`Aseries of mutations (arrowheads) were mapped inthe E. coli gene encoding tryp-
`tophan synthetase (top line). The amino acid substitutions resulting from each of
`the mutations were then determined by sequence analysis of the proteins of mu-
`tant bacteria (bottom line). These studies revealed that the order of mutations in
`DNAwasthe sameas the order of amino acid substitutions in the encoded protein.
`
`as expectedif the order of nucleotides in DNAspecifies the order of amino
`acidsin proteins.
`
`The Role ofMessenger RNA
`Although the sequence of nucleotides in DNA appearedto specify the order
`of amino acidsin proteins,it did not necessarily follow that DNAitself
`directs protein synthesis. Indeed, this appeared notto be the case, since
`DNAis located in the nucleus of eukaryotic cells, whereas protein synthesis
`takes place in the cytoplasm. Some other molecule was therefore needed to
`convey genetic information from DNA tothesites of protein synthesis (the
`ribosomes).
`RNA appeareda likely candidate for such an intermediate because the
`similarity of its structure to that of DNA suggested that RNA could be syn-
`thesized from a DNA template (Figure 3.9). RNA differs from DNA in that
`it is single-stranded rather than double-stranded,its sugar componentis
`ribose instead of deoxyribose, and it contains the pyrimidine baseuracil (U)
`instead of thymine (T) (see Figure 2.10). However, neither the changein
`sugarnorthe substitution of U for T alters base pairing, so the synthesis of
`RNA canbereadily directed by a DNA template. Moreover, since RNA is
`located primarily in the cytoplasm,it appeared a logical intermediate to
`convey information from DNAto the ribosomes. These characteristicsof
`RNAsuggested a pathwayfor the flow of genetic information that is known
`as the central dogma of molecularbiology:
`
`DNA — RNA = Protein
`
`According to this concept, RNA molecules are synthesized from DNA tem-
`plates (a process called transcription), and proteins are synthesized from
`RNAtemplates (a process called translation).
`Experimental evidence for the RNA intermediates postulated by the cen-
`tral dogma wasobtained by Sidney Brenner, Francois Jacob, and Matthew
`Meselsonin studies of E, coli infected with the bacteriophage T4. The syn-
`thesis of E. coli RNA stops following infection by T4, and the only new RNA
`synthesized in infected bacteria is transcribed from T4 DNA. This T4 RNA
`becomesassociated with bacterial ribosomes, thus conveying the informa-
`tion from DNAto thesite of protein synthesis. Because of their role as inter-
`mediates in the flow of genetic information, RNA molecules that serve as
`templates for protein synthesis are called messenger RNAs (mRNAs). They
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`Fundamentals ofMolecular Biology
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`99
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`are transcribed by an enzyme (RNA polymerase)that catalyzes the synthe-
`sis of RNA from a DNA template.
`In addition to mRNA, twoother types of RNA molecules are important
`in protein synthesis. Ribosomal RNA (rRNA)is a component of ribosomes,
`and transfer RNAs(tRNAs)serve as adaptor molecules that align amino
`acids along the mRNAtemplate. The structures and functions of these mol-
`ecules are discussedin the following section and in moredetail in Chapters
`6and7.
`
`The Genetic Code
`Howis the nucleotide sequence of mRNAtranslated into the aminoacid
`sequence of a protein? In this step of gene expression genetic informationis
`transferred between chemically unrelated types of macromolecules—
`nucleic acids and proteins—raising two new typesof problemsin under-
`standing the action of genes.
`First, since amino acids are structurally unrelated to the nucleic acid
`bases, direct complementary pairing between mRNAand aminoacids dur-
`ing the incorporation of aminoacids into proteins seemed impossible. How
`then could amino acids align on an mRNAtemplate during protein synthe-
`sis? This question wassolved by the discovery that tRNAsserve as adaptors
`between amino acids and mRNAduring translation (Figure 3.10). Prior to its
`use in protein synthesis, each aminoacid is attached by a specific enzymeto
`its appropriate tRNA. Base pairing between a recognition sequence on each
`tRNA and a complementary sequence on the mRNA thendirects the
`attached aminoacidto its correct position on the mRNAtemplate.
`The second problem in the translation of nucleotide sequence to amino
`acid sequence was determination of the genetic code. How could the infor-
`mation contained in the sequence of fourdifferent nucleotides be converted
`to the sequences of 20 different amino acids in proteins? Because 20 amino
`acids must be specified by only four nucleotides, at least three nucleotides
`must be used to encode each aminoacid. Used singly, four nucleotides
`could encode only four aminoacids and, used in pairs, four nucleotides
`could encode only sixteen (4°) amino acids. Used as triplets, however, four
`nucleotides could encode 64 (4°) different amino acids—morethan enough
`to account for the 20 aminoacidsactually found in proteins.
`Direct experimental evidencefor the triplet code was obtained by studies
`of bacteriophage T4 bearing mutations in an extensively studied gene
`called rlJ. Phages with mutations in this gene form abnormally large
`plaques, which can beclearly distinguished from those formed by wild-
`type phages. Hence,isolating and mapping a numberof rll mutants was
`easy and led to the establishment of a detailed genetic mapof this locus.
`Study of recombinants betweenr/] mutants that had arisen by additions or
`deletions of nucleotides revealed that phages containing additionsor dele-
`tions of one or two nucleotides always exhibited the mutant phenotype.
`Phages containing additions or deletions of three nucleotides, however,
`were frequently wild-type in function (Figure 3.11). These findings sug-
`gested that the geneis read in groupsof three nucleotides, starting from a
`
`Figure 3.10 Function of transfer RNA
`Transfer RNA serves as an adaptor during protein synthesis. Each aminoacid (e.g.,
`histidine) is attached to the 3’ end of a specific tRNA byan appropriate enzyme(an
`aminoacyl tRNA synthetase). The charged tRNAsthen align on an mRNAtemplate
`by complementary basepairing.
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`Histidy] tRNA
`synthetase
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`Complementary
`base pairing
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`A mRNA
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`Amino acids(The)Ser (yn) Pro His G Glu
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`Active protein’ —*» WTphage
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`DNA ACG TCA TAT CCG CAT ACC GAG...
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`Normal gene
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`Amino acids (Th) ‘Asp His (ite)Arg, (ite)Pro
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` 100=Chapter3
`
`fixed point. Additions or deletions of one or two nucleotides would then
`alter the reading frameof the entire gene, leading to the coding of abnormal
`aminoacids throughoutthe encoded protein. In contrast, additions or dele-
`tions of three nucleotides would lead to the addition or deletion of only a
`single aminoacid; the rest of the amino acid sequence would remain un