1
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`MTX1055
`ModernaTX, Inc. v. CureVac AG
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`

`

`———1
`
`The Cover
`This illustration shows a portion of the inside of a cell nucleus, including some of the
`many proteins that copy, repair, and package DNA. DNA strands are shown in yel-
`low. Running through the center of the illustration, top to bottom, is a replication
`fork, showing DNA being copied by DNA polymerase. On the right and left sides of
`the illustration, RNA polymerase is 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 ribosomal units.
`(From N. Ban, P. Nissen, I. Hansen, P. B. Moore and T. A. Steitz, 2000. Science 289: 905.
`Courtesy of Thomas A. Steitz.)
`
`Part III opener image
`Probes to repeated sequences on chromosome 4 were hybridized to a human cell.
`The two copies of chromosome 4, identified by yellow fluorescence, occupy distinct
`territories in the nucleus.
`(From A. I. Lamond and W. C. Earnshaw, 1998. Science 280: 547.)
`
`|l
`|
`
`Part IV opener image
`Mitosis sequence: Telophase.
`(Conly L. Rieder / Biological Photo Service)
`
`
`
`
`
`a”'-.‘I-'n-'.:—__r317“)r59-_—-H-—.
`
`
`g,
`E
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`
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`
`
`
`The Cell: A Molecular Approach, Third Edition
`
`Copyright © 2004 by Geoffrey M. Cooper. All rights reserved.
`This book may not 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 USA.
`Address orders and requests for examination copies to Sinauer Associates, Inc.,
`PO. Box 407, 23 Plumtree Road, Sunderland, MA 01375 USA.
`Phone: 413-549-4300
`FAX: 413-549-1118
`email: orders@sinauer.com
`www.sinauer.com
`
`Library of Congress Cataloging-in-Publication Data
`
`Cooper, Geoffrey M.
`The cell : a molecular approach / Geoffrey M. Cooper, Robert E.
`Hausman.— 3rd ed.
`p. ; cm.
`Includes bibliographical references and index.
`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
`
`Printed in USA.
`5 4 3 2 1
`
`Steenbock Memorial Library
`University of Wisconsm — Madlsofl
`550 Babcock Drive
`Madison. WI 53706-1293
`
`
`
`2
`
`

`

`
`
`Chapter
`
`Fundamentals
`
`ofMolecular Biology
`
`Heredity, 6enes.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 IDS
`
`ONTEMPUIRARY MOLECULAR BIOLOGY is concerned principally with under—
`standing the mechanisms responsible for transmission and expression of
`the genetic information that governs cell structure and function. As
`reviewed in Chapter 1, all cells share a number ofbasic properties, and this
`underlying unity of cell biology is particularly apparent at the molecular level.
`Such unity has allowed scientists to choose simple organisms (such as bacteria}
`as models for many fundamental experiments, with the expectation that similar
`molecular mechanisms are operative in organisms as diverse as E. coii and
`humans. Numerous experiments have established the validity of this assump-
`tion, and it is now clear that the molecular biology of cells provides a unifying
`theme to understanding diverse aspects of cell behavior.
`Initial advances in molecular biology were made by taking advantage of the
`rapid growth and readily manipulable genetics of simple bacteria, such as E.
`coli, and their viruses. The development of recombinant DNA then allowed both
`the fundamental principles and many of the experimental approaches First
`developed in prokaryotes to be extended to eukaryotic cells. The application of
`recombinant DNA technology has had a tremendous impact, 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 things is the ability to
`reproduce. All organisms inherit 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 from parent to prog-
`eny cell at each cell division. How genetic information is replicated and trans-
`mitted from cell to cell and organism to 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 were dis-
`coveries that formed the foundation of our current understanding of biology at
`the molecular level.
`
`3
`
`

`

`90
`
`Chapter 3
`
`Figure 3.1 Inheritance of dominant
`and recessive genes
`
`
`
`
`
`The parental strains of peas each conlaln
`two copies (alleles! or the gene tor either
`
`\ ellow t 'r't or green [vr seeds.
`
`
`a
`Lamolos
`
`.
`®
`
`.
`_
`The patenls protlui e germ [ ells leanielesl.
`® Udl'h t'nnlalnlng one {Ii these genes, lltal
`
`give rise to hybrid Fl progeny.
`
`
`
`
`Fl generation
`
`®
`
`Since t‘ is tlominanl, all the F1 plants have
`vi-l low seeds.
`
`
`
`Comoros
`
`F3 generation
`
`
`
`:greem phenotypes.
`
`f_. generation, with a charatterislit
`t:l ratio of dominant [yellowt to retessive
`
` A cross between two Fl plants yields an
`
`Genes and Chromosomes
`
`The classical 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 number of well-defined traits, such as seed
`color, and was able to deduce general rules for their transmission. In all
`cases, he could correctly interpret the observed patterns of inheritance by
`assuming that each trait is determined by a pair of inherited factors, which
`are now called genes. One gene copy (called an allele) specifying each trait
`is inherited from each parent. For example, breeding two strains 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 (3;) seeds, respectively. The progeny
`plants are therefore hybrids, having inherited one gene for yellow seeds (Y)
`and one for green seeds (5;). All these progeny plants (the first filial, or F1,
`generation) have yellow seeds, so yellow (Y) is said to be dominant and
`green (y) recessive. The genotype (genetic composition) of the F] peas is
`thus Yy, and their phenotype (physical appearance) is yellow. if one 131 off-
`spring is bred with another, giving rise to I32 progeny, the genes for yellow
`and green seeds segregate in a characteristic manner such that the ratio
`between 132 plants with yellow seeds and those with green seeds is 3:1.
`Mendel’s findings, apparently ahead of their time, were largely ignored
`until 1900, when Mendel’s laws Were rediscovered and their importance
`recognized. Shortly thereafter, the role of chromosomes as the carriers of
`genes was proposed. It was realized that most cells of higher plants and ani-
`mals are diploidbcontaining 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 member of 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 cells at fertilization creates a new diploid organ-
`
`4
`
`

`

`Male parenl
`
`Female parent
`
`Fundamentals ofMoleculm' Biology
`
`91
`
`
`
`Diploirl cells contain two copies or
`each chromosome.
`
`
`
`Figure 3.2 Chromosomes at meiosis
`and fertilization
`
`Two chromosome pairs of a hypotheti—
`cal organism are illustrated.
`
`Meiosis
`
`l
`
`has
`
`
`
`Meiosis gives rise [a Imploiil
`
`gametes t‘onlaining imlv one
`
`
`
`member of eat l1 chromosome pair.
`
`
`Diplold
`
`
`
`I
`
`_
`
`
`
`Fertilization C“)I)
`l
`Fi-.-rti|i.ralion results- in ll‘It' turmalion '
`
`
`Embryo
`
`of a diploid embryo, containing
`climmust Imes tontriliulml by
`both parents.
`
`‘7‘.
`
`ism, now containing one member of each chromosome pair 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 chromosomes were largely established by experiments
`performed with the fruit fly, Drosoplo'ia melanogostcr. Drosophilo 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 Drosoplrilrr an organism of choice for genetic stud-
`ies of animals, particularly the genetic analysis of development and differ-
`entiation.
`
`In the early 19005, a number of genetic alterations (mutations) were iden-
`tified in Drosoplriln, usually affecting readily observable characteristics such
`as eye color or wing shape. Breeding experiments indicated that some of the
`genes governing these traits are inherited independently of each other, sug—
`gesting that these genes are located on different chromosomes that segre-
`gate independently during meiosis (Figure 3.3). Other genes, however, are
`frequently inherited together as paired characteristics. Such genes are said
`to be linked to each other by virtue of being located on the same chromo-
`some. The number of groups of linked genes is the same as the number of
`chromosomes (four in Dmsophilo), supporting the idea that chromosomes
`are carriers of the genes. By 1915, nearly a hundred genes had been defined
`and mapped onto the four chromosomes of Drosopliilo, leading to general
`acceptance of the chromosomal basis of heredity.
`
`Genes and Enzymes
`Early genetic studies focused on the identification and chromosomal local-
`ization of genes that control readily observable characteristics, such as the
`eye color of Drosophflo. How these genes lead to the observed phenotypes,
`however, was unclear. The first insight into the relationship between genes
`and enzymes came in 1909, when it was realized that the inherited human
`
`
`
`5
`
`

`

`
`
`92
`
`Chapter 3
`
`[Al Segregation of two hypothetical genes located on different
`chromosomes {Ara = square-hound and Bio = redlhiuet
`
` Pare ntai
`
`(B) Linkage of two genes located on the same chromosome
`
`strains
`
`
`
`gt
`
`
`Because both genes
`
`Since Ihe chromosomes
`are carried on the
`same chromosome.
`segregate indepenclenlly al
`
`
`meiosis, the F1 generation
`_
`they do not separate
`
`-.
`'
`gives rise to four different
`from each other at
`'
`types of gametes.
`meiosis. Consequently,
`
`the F, generation
`.
`
`
`produces only two
`
`types of gametes.
`'
`
` l
`
`Parenlal
`strains
`
`Gameles
`
`g
`
`s
`
`A
`
`Fl generation
`
`Gametes
`
`
`
`
`
`The F; generation displays only two phenotypes—
`
`
`squarefred and roundfblue—uin the 3:1 ratio Ihat is
`
`characteristic of inheritance of a single gene.
`
`
`
`
`
`The F_, generation therefore
`displays four distinct
`phenotypes—squareired.
`
`squareihlue. roundfred,
`
`and roundrhluo—in a
`
`9:3:3:T ralio.
`
`
`F3 generation
`
`
`
`Figure 3.3 Gene segregation
`and linkage
`(A) Segregation of two hypothetical
`genes for shape (A In : square/round)
`and color (B/b = red/blue) located on
`different chromosomes. (B) Linkage of
`two genes located 0“ the same Chm'
`mosorne.
`
`disease phenylketonuria (see Molecular Medicine in Chapter 2) results from
`a genetic defect in metabolism of the amino acid phenylalanine. This defect
`was hypothesized to result from a deficiency in the enzyme needed to eat
`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
`
`

`

`
`
`Fm-rdamenteis of'Moiecnlnr Biology
`
`93
`
`the fungus Nem'ospora crassa. in the laboratory, Neurospora can be grown on
`minimal or rich media similar to those discussed in Chapter 1 for the growth
`of. E, coli. For Neurosporu, minimal media consist only of salts, glucose, and
`biotin; rich media are supplemented with amino acids, vitamins, purines,
`and pyrimidines. Beadle and Tatum isolated mutants of Neurospom that
`grew normally on rich media but could not grow on minimal media. Each
`mutant was found to require a specific nutritional supplement, such as a
`particular amino acid, for growth. Furthermore, the requirement for a spe—
`cific nutritional Supplement correlated with the failure of the mutant to syn—
`thesize that particular compound. Thus, each mutation resulted in a defi—
`ciency in a specific metabolic pathway. Since such metabolic pathways were
`known to 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 known to consist of
`mulb'ple polypeptides, so the currently accepted statement of this hypothe-
`sis is that each gene specifies the structure of a single polypeptide chain.
`
`identification of DNA as the Genetic Materiai
`
`Understanding the chromosomal basis of heredity and the relationship
`between genes and enzymes did not in itself provide a molecular explana-
`tion of the gene. Chromosomes contain proteins as well as DNA, and it was
`initially thought that genes were proteins. The first evidence leading to the
`identification of DNA as the genetic material came from studies in bacteria.
`These experiments represent a prototype for current approaches to defining
`the function of genes by introducing new DNA sequences into cells, 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 (Puciunoceccns). Virulent strains of
`Partiiiiococcus are surrounded by a polysaccharide capsule that protects the
`bacteria from attack by the immune system of the host. Because the capsule
`gives bacterial colonies a smooth appearance in culture, encapsulated
`strains are denoted S. Mutant strains that have lost 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 (S)
`bacteria developed pneumonia and died. lmportantly, the bacteria that
`were then isolated from these mice were of the 5 type. Subsequent experi—
`ments showed that a cell-free extract of S bacteria was similarly capable of
`converting (or transforming) R bacteria to the S state. Thus, a substance in
`theS extract (called the transforming principle) was responsible for induc-
`ing the genetic transformation of R to S bacteria.
`In 1944 Oswald Avery, Colin Macl..eod, and Maclyn McCarty established
`that the transforming principle was DNA, both by purifying it from bacter-
`ial extracts and by demonstrating that the activity of the transforming prin-
`ciple is abolished by enzymatic digestion of DNA but not by 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 than the viral
`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 of these results with continuing studies of the
`activity of DNA in bacterial transformation led to acceptance of the idea
`that DNA is the genetic material.
`
`7
`
`

`

`
`
`94
`
`Chapter 3
`
`
`
` Pathogenic
`
`S liacleria
`
`Nonpalhogonir
`
`R bacteria
`
` Capsule
`Purify DNA 1
`
`seem
`9%me mum
`awesome
`«mm
`
`Add purified
`S lIZJNA to
`R )aclerla
`
`5 bacteria
`
`Figure 3.4 Transfer of genetic information by DNA
`DNA is extracted from a pathogenic strain of Pnertmococcus, which is Surrounded
`by a capsule and forms smooth colonies (‘3). Addition of the purified S DNA to a
`culture of nonpa thogenic, nor-[encapsulated bacteria (R for “rough” colonies) ren
`Stilts in the formation of S colonies. The purified DNA therefore contains the genet-
`ic information responsible for transformation of R 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
`known to be a polymer composed of four nucleic acid bases—-—two purines
`(adenine [A] and guanine [G]) and two pyrimidines (cytosine [C] and
`thymine [TD—linked to phosphorylated sugars. Given the central role of
`DNA as the genetic material, elucidation of its three~dimensional structure
`appeared critical to understanding its function. Watson and Crick’s consid—
`eration of the problem was heavily influenced by Linus Pauling’s descrip-
`tion of hydrogen bonding and the o: helix, 3 common element of the sec-
`ondary structure of proteins (see Chapter 2). Moreover, experimental data
`on the structure of DNA were available from X-ray crystallography studies
`by Maurice Wilkins and Rosalind Franklin. Analysis of these data revealed
`that the DNA molecule is a helix that turns every 3.4 nm. In addition, the
`data showed that the distance between adjacent bases is 0.34 nm, so there
`are ten bases per turn of the helix. An important finding was that the diam-
`eter of the helix is approximately 2 nm, suggesting that it is composed of
`not one but two DNA chains.
`From these data, Watson and Crick built their model of DNA (Figure 3.5).
`The central features of the model are that DNA is a double helix with the
`sugar—phosphate backbones on the outside of the molecule. The bases are
`
`8g
`
`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 always pairs with T and G with C. This specificity accounts for the
`earlier results of Erwin Charge ff, who had analyzed the base composition of
`various DNAs and found that the amount of adenine was always equal to
`that of thymine, and the amount of guanine to that of cytosine. Because of
`this specific base pairing, the two strands of a DNA molecule are comple-
`mentary: Each strand contains all the information required to specify the
`sequences of bases on the other.
`
`Replication ofDNA
`
`The discovery of complementary base pairing between DNA strands imme-
`diately suggested a molecular solution to the question of how the genetic
`material could direct its own replication—a process that is required each time
`a cell divides. It was proposed that the two strands 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
`
`directions, defined by the 3’ and i' groups of tleoxyribose.
`
`
`
`DNA is a double helix with the bases on the
`
`inside and the sugar-phosphate backbones on
`
`the outside of the molecule.
`
`
`
`
`
`
`
` Bases on opposite strands are paired In hydrogen bonds. between adenine [At and
`thymine (Ti, and between guanine 10.! and cytosine EC}. The two DNA strands run in opposite
`
`Figure 3.5 The structure of DNA
`
`
`
`9
`
`

`

`
`
`96
`
`Chapter 3
`
`(Figure 3.6). The process is called semiconservative replication because one
`strand of parental DNA is 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 Stah], in which DNA was labeled with isotopes that altered its
`density (Figure 3.7). E. coli were first grown in media containing the heavy
`isotope of nitrogen (lsN) in place of the normal light isotope (”N}. The
`DNA of these bacteria consequently contained 15N and was heavier than
`that of bacteria groWn in MN. Such heavy DNA could be separated From
`DNA containing 14N by equilibrium centrifugation in a density gradient of
`CsCl. This ability to separate heavy (13M) DNA from light (”N) DNA
`enabled the study of DNA synthesis. E. coti that had been grown in 15N
`were transferred to media centaining I“IN and allowed to replicate one more
`time. Their DNA was then extracted and analyzed by CsCl density gradient
`centrifugation. The results of this analysis indicated that all of the heavy
`DNA had been replaced by newly synthesized DNA with a density inter-
`mediate between that of heavy (15N) and that of light (”N) DNA molecules.
`The implication was that during replication, the two parental strands of
`heavy DNA separated and served as templates for newly synthesized prog—
`eny strands of 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 strands of the double helix.
`The ability of DNA to serve as a template for its own replication was fur-
`ther established with the demonstration that an enzyme purified from E.
`coli [DNA polymerase) could catalyze DNA replication in vitro. In the pres-
`ence of DNA to act as a template, DNA polymerase was able to direct the
`incorporation of nucleotides into a complementary DNA molecule.
`
`.3’
`
`
`
`
`
`
`
`
`
`
`
`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 as the genetic material and the elucidation of its structure
`
`Old DNA strand
`
`New DNA strand
`
`5i
`
`3'
`
`Figure 3.6 Semiconservative replication of DNA
`The two strands of parental DNA separate, and each serves as a
`template for synthesis of a new daughter strand by complemen—
`tary base pairing.
`
`
`
`10
`
`

`

`densily
`
`Extract DNA
`
`Fxtract DNA
`
`C. entriluge in
`CsCl solulinn
`
`EC?""‘9
`5‘15:
`
`Cenlrifuge in
`CsCl solution
`
`Extract DNA
`
`Crlniril'uge in
`CsL'l solution
`
`Bacteria grown in I4N media
`
`Bacleria grown in 1’N media
`
`Transfer to 1'lN media I'or one division
`
`Fundamentals ofMolecular Biology
`
`97
`
`5. {viiQ —" ‘EK/fl
`
`C: fibril. .
`
`a; '5
`
`Light DNA
`
`Increasing
`
`‘
`
`density
`
`Heavy DNA
`
`Increasing
`density
`
`thritl DNA
`
`Increasing
`
`Figure 3.7 Experimental demonstra-
`tion of semiconservative replication
`BacteIIa grown in medium containing
`the normal isotope of nitrogen (HN)
`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 "1N and grown for
`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
`where their density is equal to that of
`the CsCl solution DNA of the bacteria
`transferred from 11N to ”N medium
`for a single generation bands at a den—
`sity intermediate between that of I:‘N
`DNA and that of MN DNA. indicating
`that it represents a hybrid molecule
`with one heavy and one light strand.
`
`revealed that genetic information must be specified by the order of the four
`bases (A, C, G, and T) that make up the DNA molecule. Proteins, in turn,
`are polymers of 20 amino acids, 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,
`when it was found that patients with the inherited disease sickle-cell ane—
`mia had hemoglobin molecules that differed from normal ones by a single
`amino acid substitution. Deeper understanding of the molecular relation—
`ship between DNA and proteins came, however, from a series of experi~
`ments that took advantage of E. coii and its viruses as genetic models.
`
`Colineariry of Genes and Proteins
`
`The simplest hypothesis to account for the relationship between genes and
`enzymes was that the order of nucleotides in DNA specified the order of
`amino acids in a protein. Mutations in a gene would correspond to alter—
`ations in the sequence of DNA, which might result 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 then lead to cor—
`responding changes in the amino acid sequence of the protein encoded by
`the gene in question. This hypothesis predicted that different mutations
`within a single gene could alter different amino acids in the encoded pro-
`tein, and that the positions of mutations in a gene should reflect the posi—
`tions of amino acid alterations in its protein product.
`The rapid replication and the simplicity of the genetic system of E. coli
`were of major help in addressing these questions. A variety of mutants of E.
`coil could be isolated, including nutritional mutants that (like the Neurospom
`mutants discussed earlier) require particular amino acids for growth.
`Importantly, the rapid growth of 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 mapped a series of mutations in
`the gene that encodes an enzyme required for synthesis of the amino acid
`tryptophan. Analysis of the enzymes encoded by the mutant genes indi-
`cated that the relative positions of the amino acid alterations were the same
`as those of the corresponding mutations {Figure 3.8). Thus, the sequence of
`amino acids in the protein was colinear with that of mutations in the gene,
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`Chapter 3
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`Mutations
`DNA
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`Normal prolei n
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`Amino acid substitutions
`resulting from mutations
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`Figure 3.8 Colinearity of genes and proteins
`A series of mutations (arrowheads) were mapped in the E. coff 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 rnu~
`tantbacteria (bottom line). These studies revealed that the order of mutations in
`DNA was the same as the order of amino acid substitutions in the encoded protein.
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`Figure 3.9 Synthesis of RNA from
`DNA
`The two strands of DNA unwind, and
`one is used as a template for synthesis
`of a complementary strand of RNA.
`
`as expected if the order of nucleotides in DNA specifies the order of amino
`acids in proteins.
`
`The Role ofMessenger RNA
`Although the sequence of nucleotides in DNA appeared to specify the order
`of amino acids in proteins, it did not necessarily follow that DNA itself
`directs protein synthesis. Indeed, this appeared not to be the case, since
`DNA is 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 to the sites of protein synthesis (the
`ribosomes).
`
`RNA appeared a 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 component is
`ribose instead of deoxyribose, and it contains the pyrimidine base uracil (U)
`instead of thymine (T) (see Figure 2.10}. However, neither the change in
`sugar nor the substitution of U for T alters base pairing, so the synthesis of
`RNA can be readily directed by a DNA template. Moreover, since RNA is
`located primarily in the cytoplasm, it appeared a logical intermediate to
`convey information from DNA to the ribosomes. These characteristics of
`RNA suggested a pathway for the flow of genetic information that is known
`as the central dogma of molecular biology:
`DNA —> RNA —} Protein
`
`According to this concept, RNA molecules are synthesized from DNA tem—
`plates (a process called transcription), and proteins are synthesized from
`RNA templates (a process called translation).
`Experimental evidence for the RNA intermediates postulated by the cen—
`tral dogma was obtained by Sidney Brenner, Francois Jacob, and Matthew
`Meselson in studies of E. cofi infected with the bacteriophage T4. The syn—
`thesis of E. ceti RNA stops following infection by T4, and the only new RNA
`synthesized in infected bacteria is transcribed from T4 DNA. This T4 RNA
`becomes associated with bacterial ribosomes, thus conveying the in forma-
`tion from DNA to the site 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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`Fundiimentrtls ofMoiecular 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, two other types of RNA molecules are important
`inprotein synthesis. Ribosomal RNA (rRNAl is a component of ribosomes,
`and transfer RNAS HRNAs) serve as adaptor molecules that align amino
`acids along the mRNA template. The structures and functions of these mol—
`ecules are discussed in the following section and in more detail in Chapters
`6 and 7.
`
`The Genetic Code
`
`How is the nucleotide sequence of mRNA translated into the amino acid
`sequence of a protein? In this step of gene expression genetic information is
`transferred between chemically unrelated types of macromolecules——
`nucleic acids and proteins—~raising two new types of problems in under-
`standing the action of genes.
`First, since amino acids are structurally unrelated to the nucleic acid
`bases, direct complementary pairing between mRNA and amino acids dur-
`ing the incorporation of amino acids into proteins seemed impossible. How
`then could amino acids align on an mRNA template during protein synthe—
`sis? This question was solved by the discovery that tRNAs serve as adaptors
`between amino acids and mRNA during translation (Figure 3.10). Prior to its
`use in protein synthesis, each amino acid is attached by a specific enzyme to
`its appropriate tRNA. Base pairing between a recognition sequence on each
`tRNA and a complementary sequence on the mRNA then directs the
`attached amino acid to its correct position on the mRNA template.
`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 four different 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 amino acid. Used singly, four nucleotides
`could encode only four amino acids and, used in pairs, four nucleotides
`could encode only sixteen (42) amino acids. Used as triplets, however, four
`nucleotides could encode 64 (43) different amino acids—more than enough
`to account for the 20 amino acids actually found in proteins.
`Direct experimental evidence for the triplet code was obtained by studies
`of bacteriophage T4 bearing mutations in an extensively studied gene
`called rll. Phages with mutations in this gene form abnormally large
`plaques, which can be clearly distinguished from those formed by wild-
`type phages. Hence, isolating and mapping a number of rll mutants was
`easy and led to the establishment of a detailed genetic map of this locus.
`Study of recombinants between rll mutants that had arisen by additions or
`deletions of nucleotides revealed that phages containing additions or 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 gene is read in groups of three nucleotides, starting from a
`
`Figure 3.10 Function of transfer RNA
`Transfer RNA serves as an adaptor during protein synthesis. Each amino acid {e.g.,
`histidine) is attached to the 3' end of a specific tRNA by an appropriate enzyme (an
`aminoacyl tRNA synthetase}. The charged tRNAs then align on an mRNA template
`by complementary base pairing.
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`Ilislirlyl [RNA
`synlhetase
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`Complementarv
`base pairin