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`Cover Jmnge: Structure of the {3 subunit of E. coli DNA polymerase Ill holoenzyme, as revealed by
`x-ray crystallograph)•
`Cover Pltotograp!J: Courtesy of}. Kuriyan, Cell ( 1992) 69:42~37
`
`Copyright C 1996 by The Benjamin/Cummings Publishing Company, Inc.
`
`All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
`transmitted in any form or by any means, electronic, mechanical, photocopying. recording, or any
`other media embodiments now known or hereafter to becom(' known, without the prior written per(cid:173)
`mission of the publisher. Manufactured in the United States of America. Published simultaneously
`in Canada.
`
`Library of Congress Cataloging-in-Publication Data
`Mathews, Christopher K., 1937-
`Btochemistry I Christopher K. Mathews, K. E. van Holde.- 2nd ed.
`p. em.
`Lncludes bibliographical references and index.
`ISBN 0-8053-393 J ·0
`I. Biochemistry.
`I. van Holde, K. E. (Kensal Edward), 1928- .
`II. Title.
`QP514.2.M384
`574. 19'2-dc20
`
`95-18381
`
`1995
`
`2 3 4 5 6 7 8 9 10-VH-99 98 97 96 95
`
`The Benjamin/Cummings Publishing Company. Inc.
`2725 Sand !-till Road
`Menlo Purk, CA 94025
`
`2
`
`

`

`CHAPTER4
`
`NUCLEIC ACIDS
`
`n the next several chapters we introduce three major
`classes of biomolecules-nucleic acids, proteins, and carbohydrates. Together,
`they make up a large part of all living matter. As we saw in Chapter l , these sub(cid:173)
`stances exist as macromolecules, some of them giant. We shall find all of these
`macromolecules to be polymers; each type is made by the linking of a limited
`number of kinds of monomer units. However, these classes of macromolecules
`play entirely different roles in the life of the cell.
`
`THE NATURE OF NUCLEIC ACIDS
`
`It is appropriate to begin this section with the nucleic acids, for in a certain sense
`they are the most fundamental and important constituents of the living cell. It
`seems probable that life itself began its evolution with nucleic acids, for only they,
`of all biological substances, carry the remarkable potential for self-duplication.
`Today, nucleic acids act as the repositories and transmitters of genetic informa(cid:173)
`tion for every cell, tissue, and organism. The blueprint for an organism is encoded
`in its nucleic acid, in gigantic molecules like that shown in Figure 1.5 (page 9).
`Much of an organism's physical development throughout life is programmed in
`these molecules. The proteins that its cells will make and the functions that they
`will perform are all recorded on this molecular tape.
`In this chapter, and in the several that follow, we first describe the nucleic
`acids and then provide a brief introduction to the ways in which they preserve
`and transmit genetic information. The details of these processes will be covered
`in Part V of this book, but it is important that we consider, at the beginning, the
`role that nucleic acids play in the formation of proteins and cellular structure.
`
`T H E TWO T YPES OF NUCLEI C ACID: DNA AND R NA
`
`There are tvvo types of nucleic acid, ribonucleic acid (RNA) and deoxyribonu(cid:173)
`cleic acid (DNA). As Figure 4.1 shows, each is a polymer chain, with similar
`monomer units connected by covalent bonds. Structures of the monomer units
`are shown here:
`
`3
`
`

`

`THE NATURE OF NUCLEIC ACIDS
`
`87
`
`Phosphate
`
`Phosphate~
`
`0-P = O
`I
`0
`
`A•bose
`
`2'·deoxyribose
`
`sCHz
`
`I Bate I
`•. ~<O~c ,.
`H/ "6- 6/ '-H
`3'1
`12'
`0
`H
`I
`
`Repeating unit of
`ribonucleic acid (RNA)
`
`Repeating unit of
`deoxyribonucleic acid (DNA)
`
`In each case the monomer unit contains a five-carbon sugar, ribose in RNA and
`2' -deoxyribose in DNA (shown in blue in the structures). The difference between
`the two sugars lies solely in the 2' hydroxyl group on ribose. The connection be(cid:173)
`tween successive monomer units in nucleic acids is through a phosphate residue
`attached to the hydroxyl on the 5' carbon of one unit and the 3' hydroxyl of the
`next one. This forms a phosphodiester link between two sugar residues (Figure
`4.1 ). ln this way, long nucleic acid chains, sometimes containing hundreds of mil(cid:173)
`lions of units, are built up. The phosphate group is a strong acid, with a pK3 of
`about 1; this is why DNA and RNA are called nucleic acids. Every residue in a
`DNA or RNA molecule carries a negative charge at physiological pH.
`
`RNA
`
`I
`- o......_ /o
`p
`I'\
`0
`0
`
`Both DNA and RNA are polynucleo(cid:173)
`tides. RNA has the sugar ribose;
`DNA has deoxyribose. RNA contains
`the base uracil instead of thymine.
`
`FIGURE 4.1
`Chemical structures of ribonucleic
`add (RNA) and deoxyribonucleic add
`(DNA). The ribose-phosphate or deoxyribose(cid:173)
`phosphate backbone of each chain is shown in
`detail. The bases shown schematically here are
`detailed in Figure 4.2.
`
`0 Irving Geis.
`
`DNA
`
`Base
`
`H
`
`Base
`
`Phospho- [ - 0......_ /0
`diester
`,fp \
`link
`0
`0
`
`HO
`
`Phospho- [ - 0......_ /0
`diester
`,fp \
`0
`0
`link
`
`H
`
`H
`
`4
`
`

`

`88
`
`C HAPT ER 4
`
`NUC~E I C AC IDS
`
`FIGURE 4 .2
`Purine and pyrimidine bases f o und In
`DNA and RNA. A major difference between
`the two types of nucleic acids is that RNA has
`uracil (U) instead of the thymine (1) found
`in DNA.
`
`Adenine (A)
`(DNA/ RNA)
`
`PURINES
`
`PYRIMIDINES
`
`Uracil (U)
`(RNA)
`
`The nucleic acid bases are of two
`kinds: the purines, adenine and gua(cid:173)
`nine, and the pyrimidines, cytosine,
`thymine, and uracil.
`
`The phosphodiester-linked sugar residues form the backbone of the nucleic
`acid molecule. By itself, the backbone is a repetitious structure, incapable of en(cid:173)
`coding information. The importance of the nucleic acids in information storage
`and transmission derives from their being heteropolymers. Each monomer in the
`chain carries a basic group, always attached to the 1' carbon of the sugar (see Fig(cid:173)
`ure 4.1). The structures of the major bases found in the nucleic acids are shown
`in Figure 4.2. There are two types of these basic substances, called purines and
`pyrimidines. DNA has two purines, adenine (A) and guanine (G), and two pyrim(cid:173)
`idines, cytosine (C) and thymine (T). RNA has the same bases except that uracil
`(U) replaces thymine. RNA and, to a lesser extent, DNA also contain a small frac(cid:173)
`tion of chemically modified bases. We will discuss these modified bases in later
`sections of this chapter.
`DNA and RNA can each be regarded as a polymer made from four kinds of
`monomers. The monomers are phosphorylated ribose or deoxyribose molecules
`with purine or pyrimidine bases attached to their 1' carbons. In purines the at(cid:173)
`tachment is at nitrogen 9, in pyrimidines at nitrogen l. These monomers are
`called nucleotides. Each nucleotide can be considered the 5'-monophosphory(cid:173)
`lated derivative of a sugar-base adduct called a nucleoside (Figure 4.3). Thus, the
`nucleotides could also be called nucleoside 5'-monophosphates. You have already
`encountered one of these molecules in Chapter 3: adenosine 5' -monophosphate,
`or AMP.
`
`5
`
`

`

`NUCLEOSIDES
`
`NUCLEOTIDES
`
`THE NATURE OP N UC L E IC AC IDS
`
`89
`
`HO,
`
`CWi<
`H n H
`
`OH
`
`OH
`
`Adenosine
`
`0
`
`Guanosine
`
`HO,CW-p(
`H n H
`
`OH
`
`OH
`
`HO,
`
`H
`
`NH2
`
`H
`
`c~
`
`0
`H Cytidine
`
`H
`
`NH2
`
`~t;J
`CH2 H 0 H \...lN~H
`
`- o,_f/o,
`~
`
`- 0 n-0-x Adenosine 5'-monophosphate
`.. n H
`
`(AMP)
`
`OH
`
`OH
`
`H
`
`NH2
`
`H
`
`- o 0 o
`-......./// 'cH
`- o
`
`H
`
`l~ 0
`
`H
`
`Cytidine 5' -monophosphate
`(CMP)
`
`OH
`
`OH
`
`OH
`
`OH
`
`H
`- o o o
`H J-.;o
`
`'~/ ' c>R<H2 H O H ~~-H
`
`- o
`
`o
`
`H
`
`H
`
`OH
`
`OH
`
`Uridine 5'-monophosphate
`(UMP)
`
`FIGURE 4 .3
`Nucleosldes and nucleotldes. The ribonucleosides and ribo·
`nucleotides are shown here; the deoxyribonucleosides and deoxy(cid:173)
`ribonucleotides are identical, except that they lack the 2' 0H. Each
`nucleoside is formed by coupling ribose or deoxyribose to a base;
`the nucleotides, which can be considered the monomer units of
`nucleic acids, are the 5' -monophosphates of the nucleosides.
`Nucleoside phosphates with phosphorylation at other hydroxyl
`groups exist, but they are not found in nucleic acids.
`
`6
`
`

`

`90
`
`CHAPTER 4
`
`NUCLE I C AC I DS
`
`TABlE 4.1 Ionization constants of ribonucleotides expressed as
`pK .. values
`
`Phosphate
`
`Primary Constant
`
`Secondary Constant
`
`0
`II
`HO-P-R =
`I
`OH
`
`0
`II
`HO-P-R
`I
`o-
`+ H•
`
`0
`0
`II
`II
`HO-P-R === -o -P-R
`I
`I
`o
`o-
`+ W
`
`5' AMP
`5'GMP
`
`5' UMP
`5'CMP
`
`0.9
`0.7
`
`1.0
`0.8
`
`6.1
`6.1
`
`6.4
`6.3
`
`Base
`
`Constant
`
`Reaction (as Change
`from Neutral Form)
`
`3.8
`2.4
`9.4
`9.5
`4.5
`
`Protonation at N-1
`Protonation at N-7
`Loss of proton at N -I
`Loss of proton at N-3
`Protonation at N-3
`
`CYTOSINE
`
`Amino
`
`Imino
`
`Since all of the nucleic acids may be regarded as polymers of nucleotides,
`they are often referred to by the generic name polynucleotides. Small polymers,
`containing only a few residues, are called oligonucleotides.
`
`P ROP ERTIES O F T H E NUCLEOTIDES
`
`Nudeotides are quite strong acids; the primary ionization of the phosphate oc(cid:173)
`curs with a pi<;, of approximately 1.0. Both secondary ionization of the phosphate
`and protonatjon of some of the groups on the bases within the nucleotides can
`be observed at pH values quite close to neutrality (Table 4.1.) The bases are also
`capable of conversion between tautomeric forms. The major forms are those
`shown in Figure 4.2, but G, T, and U can isomerize slightly to enol forms, A and
`C to imjno forms, as shown in Figure 4.4.
`As a consequence of the conjugated double-bond systems in the purine and
`pyrimidine rings, the bases and all of their derivatives (nucleosides, nucleotides,
`and nucleic acids) absorb light strongly in the near-ultraviolet region of the spec(cid:173)
`trum. This absorption depends somewhat on pH, because of the ionization re(cid:173)
`actions in the bases; representative spectra at neutral pH for ribonucleotides
`are depicted in Figure 4.5. This strong absorbance is often used for quantitative
`determination of nucleic acids; it makes it possible to measure nucleic acid con(cid:173)
`centrations at the microgram/mL level by spectrophotometry (see Tools of Bio(cid:173)
`chemistry 6A).
`
`THYMINE
`
`STABILI T Y AND FORMATION O F THE
`
`PHOSPHODI EST ER LINKAG E
`
`Keto
`
`Enol
`
`FIGURE 4 .4
`Tautomerlzatlon of the bases. The most
`stable (and therefore common) forms are
`shown on the left. The less common imino
`and enol forms, shown on the right, are found
`in some special base interactions. Still other
`tautomers (not shown here) are possible.
`
`If we compare the structures of the nucleotides shown in Figure 4.3 with the
`polynucleotide chains depicted in Figure 4.1, we see that, in principle, a polynu(cid:173)
`cleotide could be generated from its nucleotide monomers by elimination of a
`water molecule between each pair of monomers. That is, we might imagine
`adding another nucleotide residue to a polynucleotide chain by the dehydration
`reaction shown in Figure 4.6. However, the free energy change in this dehydration
`reaction is quite positive, about +25 kJ/mol; therefore equilibrium lies far to the
`side of hydrolysis of the phosphodiester bond in the aqueous environment of the
`cell. Hydrolysis of polynucleotides to nucleotides is the thermodynamically fa(cid:173)
`vored process.
`We encounter here the first of many examples of the metastability (see Chap(cid:173)
`ter 3) of biologically important polymers. Although polynucleotides are thermo-
`
`7
`
`

`

`THll NATURE OF NU C LEI C AC I OS
`
`91
`
`14,000
`
`E
`0
`
`12.000
`
`8000
`
`6000
`
`10,000
`~
`:!-
`~ a.
`0
`"' .0
`Cll m
`0
`~
`
`4000
`
`2000
`
`230
`
`240
`
`270
`260
`250
`Wavelength (A.), nm
`
`280
`
`290
`
`FIGURE 4 .5
`Ultraviolet spectra of rlbonucleotldes. The dimensions of
`the absorption coefficients are M - 1cm- •. Thus a 1o- 4 M solution
`of UMP would have an absorbance of 0.95 at 260 nm in a 1-cm(cid:173)
`thick cuvette. (Absorbance = e x cuvette thickness x concentra(cid:173)
`tion; see Tools of Biochemistry 6A.)
`
`Oa~ from A. L Lehninger, D. L N~100, and M. M. Cox, Principles of Biochemistty. 2nd
`Kl. (N<!W Yotk: Worth, 1993), p. 330, 12.10.
`
`dynamically unstable in vivo, their hydrolysis is exceedingly slow unless catalyzed.
`This characteristic is of the greatest importance, for it ensures that the DNA in
`cells is sufficiently stable to serve as a useful repository of genetic information. In(cid:173)
`deed, DNA is so stable that it has even been possible to recover fragments of DNA
`molecules from some ancient fossils. When catalysts are present, however, hydrol(cid:173)
`ysis can be exceedingly rapid. Acid catalysis leads to hydrolysis of the phosphodi(cid:173)
`ester bonds in both DNA and RNA, yielding a mixture of nucleotides. In extremely
`acid conditions (e.g., 12M perchloric acid at l00°C), the bond between the base
`and the sugar is also hydrolyzed; a mix1ure of bases, phosphoric acid, and ribose
`(or deoxyribose) is produced. RNA, but not DNA, is also labile in alkaline solu(cid:173)
`tion; treatment with 0.1 M alkali yields a mixture of2' and 3' nucleoside phos(cid:173)
`phates. Finally, and most important biologically, the enzymes called nudeases
`catalyze the hydrolysis of phosphodiester bonds in both RNA and DNA. Your
`body is able to break down polynucleotides in the foodstuffs you consume be(cid:173)
`cause your digestive system contains nucleases. Examples of such enzymes are de(cid:173)
`scribed in Chapter 11.
`The unfavorable thermodynamics of the hypothetical dehydration reaction
`shown in Figure 4.6 leads us to ask, If polynucleotides cannot be synthesized in
`vivo by the direct elimination of water, how are they actually made? The answer is
`that their synthesis involves the high-energy nucleoside triphosphates. Although
`the process as it occurs in cells is quite complex, the basic reaction is simple. In-
`
`FIGURE 4 .6
`Formation of a polynucleotide by a hypothetical dehydra(cid:173)
`tion reaction. We might imagine that a polynucleotide could be
`formed d irectly from nucleoside monophosphates by removal of
`water, as shown here, but this dehydration reaction is thermody(cid:173)
`namically unfavorable. The reverse reaction, hydrolysis, is favored.
`Note that in this and subsequent figures we adopt a somewhat
`more compact way of representing the sugar-phosphate backbone.
`
`I
`0
`I
`-o-P=O
`I
`0
`I
`CH2
`
`HO
`Polynucleotide with N residues
`
`+
`
`9~ .. ,
`
`OH
`Deoxynucleoside
`monophosphate
`
`Hydrolysis
`
`I
`0
`I
`-o-P=O
`I
`0
`I
`CH2
`
`CH2 q~ .. ,
`
`OH
`Polynucleotide with N + 1 nucleoside residues
`
`8
`
`

`

`92
`
`C HAPTER 4
`
`NUCLEIC ACIDS
`
`I
`0
`I
`0-P=O
`I
`0
`I
`
`~0~ ;_;·
`
`stead of the dehydration reaction of Figure 4.6, what happens in living cells is the
`reaction shown in Figure 4.7. The nucleoside monophospbate being added to the
`growing chain is presented as a nucleoside triphosphate, like ATP or deoxy ATP
`(dATP), and pyrophosphate is released in the reaction. We can calculate the free
`energy change for this reaction by noting that it can be considered the sum of two
`reactions-hydrolysis of a nucleoside triphosphate and formation of a phospho(cid:173)
`diester link by elimination of water:
`
`Nucleoside triphosphate ~ nucleoside monophosphate
`+ H20
`+ pyrophosphate (PP;)
`
`~
`
`~a ··
`
`- 31 kJ/mol
`
`(Polynucleotide chain)N + ~ (polynucleotide chain)N + 1
`nucleoside monophosphate
`+ H20
`
`+25 kJ/mol
`
`Sum:
`
`(Polynucleotide chain)N + ~ (polynucleotide chain)N + 1
`nucleoside triphosphate
`+ pyrophosphate (PP1)
`
`- 6 kJ/mol
`
`HO
`
`Polynucleotide
`with N residues
`
`+
`
`HO
`
`Deoxynucleoside
`triphosphate
`
`Pyrophosphate
`
`I
`0
`I
`- o-P = O
`I
`0
`I
`CHz
`
`;_;···
`
`~0~
`
`The coupled reaction is favorable because the net 6.G 01 is negative. The reac(cid:173)
`tion is further favored because the hydrolysis of the pyrophosphate product (PPi)
`' = -33 kJ/mol. Thus, the pyrophosphate is
`to orthophosphate (PJ has a 6.G 0
`readily removed, driving the synthesis reaction even further to the right. Poly(cid:173)
`nucleotide synthesis is an example of a principle we emphasized in Chapter 3-
`the use of favorable reactions to drive thermodynamically unfavorable ones.
`It is important to appreciate bow the energetics of such processes fit into the
`overall scheme of life. An organism obtains energy-either from photosynthesis,
`if it is a plant, or from metabolism of foodstuffs-and stores part of this energy
`by generating ATP, GTP, dATP, dGTP, and so forth. It uses these compounds in
`turn as energy sources to drive the synthesis of macromolecules like DNA, RNA,
`and proteins. This use of triphosphates as the energy currency of the cell is a
`theme that you will see repeated again and again throughout this book.
`
`PRIMARY STRUCTURE OF NUCLEIC ACIDS
`
`THE NATURE AND SIGNIFICANCE OF PRIMARY STRUCTURE
`
`A closer examination of Figure 4.1 reveals two important features of all
`polynudeotides:
`l. A polynucleotide chain has a sense or directionality. The phosphodiester
`linkage between monomer units is between the 3' carbon of one monomer
`and the 5' carbon of the next. Thus, the two ends of a linear polynucleotide
`chain are distinguishable. One end normally carries an unreacted 5' phos(cid:173)
`phate, the other end an unreacted 3' hydroxyl group.
`2. A polynucleotide chain has individuality, determined by the sequence of its
`bases-that is, the nucleotide sequence. This sequence is called the primary
`structure of that particular nucleic acid.
`
`FIGURE 4 .7
`How polynucleotldes are actually formed. In this reaction,
`each monomer is presented as a nucleoside triphosphate to be
`added to the chain. Cleavage of the nucleoside triphosphate pro(cid:173)
`vides the free energy that makes the reaction thermodynamically
`favorable.
`
`Polynucleotide with
`N + 1 residu es
`
`9
`
`

`

`PRIMARY STRUCTURE OP NUCL E IC ACIDS
`
`If we want to describe a particular polynucleotide sequence (either DNA or
`RNA), it is exceedingly awkward to draw the molecule in its entirety as in Figure
`4.1. Accordingly, some compact nomenclatures have been devised. If we state that
`we are describing a DNA molecule or an RNA molecule, then most of the struc(cid:173)
`ture is understood. We can then abbreviate a small DNA as follows:
`
`93
`..-------------~~
`Every naturally occurring poly(cid:173)
`nucleotide has a defined sequence,
`its primary structure.
`
`I
`
`A
`
`C
`
`G
`
`T
`
`T
`
`k k k k k
`
`P~ P~ P~ P~ P~ OH
`5'
`5'
`5'
`5'
`
`5'
`
`This notation shows ( 1) the sequence of nucleotides, by their letter abbreviations
`(A, C, G, T); (2) that all phosphodiester links are 3' -
`5'; and (3) that this par(cid:173)
`ticular molecule has a phosphate group at its 5' end and an unreacted 3' hydroxyl
`at its 3' end.
`5' (as is usu(cid:173)
`If all of the phosphodiester links can be assumed to be 3' -
`ally the case), a more compact notation is possible for the same molecule:
`
`pApCpGpTpT
`
`The 3' - OH group is understood to be present and unreacted. Were there a
`phosphate on the 3' end and an unreacted hydroxyl on the 5' end, we would write
`
`ApCpGpTpTp
`
`Finally, if we are concerned only with the sequence of bases in the molecule, as
`will often be the case, we can write it still more compactly as
`
`ACGTI
`
`Note that the sequence of a polynucleotide chain is usually written, by conven(cid:173)
`tion, with the 5' end to the left and the 3' end to the right.
`The main importance of primary structure, or sequence, is that genetic infor(cid:173)
`mation is stored in the primary structure of DNA. A gene is nothing more than a
`particular DNA sequence, encoding information in a four-letter language in which
`each "letter" is one of the bases.
`
`DNA AS THE GENETIC SUBSTANCE: EARLY EVIDENCE
`
`The search for the substance of which genes are made has a long history. In the
`late 1800s, shortly after the German biochemist Friedrich Miescher had first iso(cid:173)
`lated DNA from salmon sperm, some scientists suspected that DNA might be the
`genetic material. But subsequent studies showing that DNA contained only four
`kinds of monomers seemed to deny that it could have such a complicated role.
`Early researchers thought it more likely that genes were made of proteins, for pro(cid:173)
`teins were beginning to be recognized as much more complex molecules. For
`most of the first half of the twentieth century, nucleic acids were considered to be
`merely some kind of structural material in the cell nucleus.
`Between 1944 and 1952 a series of crucial experiments clearly pointed to DNA
`as the genetic material. In 1944 Oswald Avery, Colin MacLeod, and Maclyn Mc(cid:173)
`Carty found that the DNA from pathogenic strains of the bacterium Pneumococ(cid:173)
`cus could be transferred into nonpathogenic strains, making them pathogenic
`
`The primary structure of DNA
`encodes genetic information.
`
`10
`
`

`

`94
`
`CHAPTER 4
`
`NUCLEIC ACIDS
`
`FIGURE 4.8
`Experfment.s that showed
`DNA to be the genetic
`substance. (a) Avery et al.
`showed that nonpathogenic
`pneumococci could be made
`pathogenic by transfer of
`DNA from a pathogenic
`strain. (b) Hershey and
`Chase showed that it is the
`transfer of viral DNA from a
`virus to a bacterium that
`gives rise to new viruses.
`
`Pathogenic
`S (smooth) cell
`
`32P-Iabeled - - : ; - JSS-Iabeled
`viral DNA
`protein coat
`
`Bacterial DNA
`
`Heat to kill:
`S fragment
`released
`
`1
`
`Nonpathogenic
`R (rough) cell
`
`S (g)~ Entry of chromosome
`
`fragment bearing
`S gene into R cell
`
`and cell division
`
`! Recombination
`© S cell
`
`Some daughter cells
`areS-type
`
`(a) Bacterial DNA alters phenotype
`(Avery et al.)
`
`I
`
`Mixing of virus
`with host cells
`results in
`adsorption of
`virus to host cell
`
`E. coli
`cell
`
`Violent agitation
`in a mixer
`
`Protein "ghost"
`labeled with 35S
`
`Viral DNA
`labeled with 32p
`I
`
`(""'=====~~)
`l
`
`Multiplication of
`viral DNA in the
`absence of the
`original protein coat
`
`Release of new
`progeny viruses, some
`of which contain 32p
`in their DNA and
`none of which
`contains 35S
`in its coat
`
`(b) Phage DNA gives rise to phages
`(Hershey and Chase)
`
`(Figure 4.8a). The transformation was genetically stable; succeeding generations
`of bacteria retained the new characteristics. However, it was an elegant experi(cid:173)
`ment by Alfred Hershey and Martha Chase that finally convinced many scientists.
`Hershey and Chase studied the infection of the bacterium Escherichia coli by a
`bacterial virus, the bacteriophage T2. Making use of the fact that the bacterio(cid:173)
`phage proteins contain sulfur but little phosphorus and that the DNA contains
`phosphorus but no sulfur, they labeled T2 bacteriophage with the radioisotopes
`
`11
`
`

`

`SECONDARY AND TERTIARY STRUCTURE OF NUCLEI C AC IDS
`
`95
`
`35S and 32P (Figure 4.8b). They then showed that when the bacteriophage at(cid:173)
`tached to E. coli, it was mainly the 32P (and hence the DNA) that was transferred
`to the bacteria. Even if the residual protein part of the bacteriophage was shaken
`off the bacteria, the inserted DNA was sufficient to direct the formation of new
`bacteriophage.
`Through these and similar experiments, it was generally recognized by 1952
`that DNA must be the genetic substance. But how could it carry the enormous
`amount of information that a cell needed, how could it transmit that information
`to the cell, and, above all, how could it be accurately replicated in cell division?
`The answers to these questions came only after one of the most momentous dis(cid:173)
`coveries in the history of science. In 1953 James Watson and Francis Crick pro(cid:173)
`posed a structure for DNA that opened a whole new world of molecular biology.
`
`SECONDARY AND TERTIARY STRUCTURE
`
`OF NUCLEIC ACIDS
`
`THE DOUBLE HELIX
`
`Watson and Crick sought the answers to the questions posed above in the three(cid:173)
`dimensional structure of DNA. For some time, a number of laboratories had been
`investigating fibers drawn from concentrated DNA solutions, using the technique
`of x-ray diffraction (see Tools of Biochemistry 4C). Watson and Crick, working
`at Cambridge University in England, had access to DNA diffraction patterns photo(cid:173)
`graphed by Rosalind Franklin, a researcher in the laboratory of Maurice Wllki.ns at
`King's College, London. The critical photographs were some of the best patterns
`from wet DNA fibers that had yet been obtained. They clearly showed that the
`DNA in the fibers must have some kind of regular, repetitive three-dimensional
`structure. We refer to such regular folding in polymers as secondary structure, as
`distinguished from the primary structure, which is simply the sequence of residues.
`Watson and Crick quickly recognized that the DNA fiber diffraction exhibited
`a cross pattern typical of a helical secondary structure (Figure 4.9). They noted
`that since the layer line spacing was one-tenth of the pattern repeat, there must be
`10 residues per turn (see Tools of Biochemistry 4C). Data on the density of the
`fibers suggested that there must be two DNA strands in each helical molecule. So
`far, only direct scientific deductions had been made from the data. The great leap
`of intuition that Watson and Crick made was the realization that a tvvo-strand
`helix could be stabilized by hydrogen bonding between bases on opposite strands
`if the bases were paired in one particular way-the A-T and G-C pairs shown in
`Figure 4.10. With this pairing, strong hydrogen bonds are formed between the
`bases. Furthermore, distances between the 1' carbons of the deoxyribose moieties
`of A-T and of G-C are the same-about 1.1 nm in each case (Figure 4.10a.). This
`pairing arrangement meant that the double helix could be regular in diameter, an
`impossibility if purines paired with purines or pyrimidines with pyrimidines.
`In the Watson- Crick model, the hydrophilic phosphate-deoxyribose back(cid:173)
`bones of the helix were on the outside, in contact with the aqueous environment,
`and the base pairs were stacked on one another with their planes perpendicular to
`the helix axis. Two views of such a structure are shown in Figure 4.10b and c. (The
`figure shows a recent, refined model, based on better data than Watson and Crick
`had available: The bases are not exactly perpendicular to the helix axis, and the
`sugar conformation is slightly different from that proposed by Watson and Crick.)
`Stacking of the bases, as shown in Figure 4.10b, allows strong van der Waals in(cid:173)
`teraction between them. Each base pair is rotated by 36° with respect to the next
`to accommodate 10 base pairs in each turn of the helix. The diffraction pattern
`showed the repeat distance to be about 3.4 nm, so the helix rise, that is, the dis-
`
`FIGURE 4 .9
`Evidence for the structure of DNA. This
`photograph, taken by Rosalind Franklin, shows
`the x-ray diffraction pattern produced QY wet
`DNA fibers. It played a key role in the elucida(cid:173)
`tion of DNA structure. The cross pattern indi(cid:173)
`cates a helical structure, and the strong spots
`at top and bottom correspond to a helical rise
`of 0.34 nm. The layer line spacing is one-tenth
`of the distance from the center to either of
`these spots, showing that there are 1 0 base
`pairs per repeat.
`
`Reprinted by permission from R. E. Franklin and R. Gosling,
`Nature (1953) 171:740; Q 1953 MacMillan Magazines, ltd.
`
`12
`
`

`

`96
`
`C HAP TI!R 4
`
`NU C LBIC AC IDS
`
`C-1'
`
`' ' ' ' ' '
`mr~ .... ·· / '
`\ , /\·
`
`(§>('~
`
`.,.--
`
`---------
`
`-.._...,
`.........
`
`........... '
`
`S'end
`0
`0
`
`~
`0
`Cytosine
`
`0
`
`Double
`
`, , -----------
`
`(b)
`
`(c)
`
`FIGURE 4 .10
`Fundamental elements of structure In the DNA double helix. (a) Base
`pairing. A-T and G-C are the base pairs in the Watson-Crick model of DNA. This
`pairing allows the Cl ' carbons on the two strands to be exactly the same distance
`apart for both pairs. (b) Stacking of the base pairs. This view down the helix axis
`shows how the base pairs stack on one another, with each pair rotated 36° with
`respect to the next. (c) Distance between the base pairs. A side view of the base
`pairs shows the 0.34-nm distance between them. This distance is called the rise of
`the helix.
`
`0 Irving Geis
`
`Key:
`
`C in phosphate
`ester chain
`
`C and N in bases
`
`FIGURE 4 .11
`A space-filling model of DNA. The DNA
`molecule as modeled by Watson and Crick is
`shown here with each atom given its van der
`Waals radius. This model shows more clearly
`than Figure 4.10 how closely the bases are
`packed within the helix. The major and minor
`grooves are indicated.
`
`13
`
`

`

`SECONDARY AND TERTIARY STRUCTURE OF NUCLEIC ACIDS
`
`97
`
`TABLE 4.2 Base compositions of DNAs from various organisms
`
`Source
`
`Bacteriophage ~X-174
`Bacteriophage T7
`Escherichia coli B
`Neurospora
`Corn (maize)
`Tetrahymena
`Octopus
`Drosophila
`Starfish
`Salmon
`Frog
`Chicken
`Rat
`Calf
`Human
`
`Mol % of Bases
`
`Ratios
`
`Adenine
`(A)
`
`Guanine
`(G)
`
`Cytosine"
`(C)
`
`Thymine
`(T)
`
`(G + C)
`
`AIT
`
`G/C
`
`24.0
`26.0
`23.8
`23.0
`26.8
`35.4
`33.2
`30.7
`29.8
`28.0
`26.3
`28.0
`28.6
`27.3
`29.3
`
`23.3
`23.8
`26.8
`27.1
`22.8
`14.5
`17.6
`19.6
`20.7
`22.0
`23.5
`22.0
`21.4
`22.5
`20.7
`
`21.5
`23.6
`26.3
`26.6
`23.2
`14.7
`17.6
`20.2
`20.7
`21.8
`23.8
`21.6
`21.6
`22.5
`20.0
`
`31.2
`26.6
`23.1
`23.3
`27.2
`35.4
`31.6
`29.5
`28.8
`27.8
`26.8
`28.4
`28.4
`27.7
`30.0
`
`44.8
`47.4
`53.2
`53.8
`46.1
`29.2
`35.2
`39.8
`41.3
`44.1
`47.4
`43.7
`42.9
`45.0
`40.7
`
`0.77b
`0.98
`1.03
`0.99
`0.99
`1.00
`1.05
`1.03
`1.03
`1.01
`1.00
`0.99
`1.01
`0.99
`0.98
`
`1.08b
`1.01
`1.02
`1.02
`0.98
`0.99
`1.00
`0.97
`1.00
`1.01
`0.99
`1.02
`1.00
`1.00
`1.04
`
`The Watson-Crick model for DNA
`was a two-strand, antiparallel dou(cid:173)
`ble helix with 1 0 base pairs per turn.
`Pairing was A-T and G-C.
`
`Source: Data taken from H. E. Sober (ed.), Handbook of Biochemistry, 2nd ed. (Chemical Rubber Pub(cid:173)
`lishing Co., 1970). Values for higher organisms vary slightly from one tissue to another, probably as a
`result of experimental error.
`"Amount includes, for some organisms, a few percent of a modified base, 5-methylcytosine.
`bThis bacteriophage has a single-strand DNA, which need not follow Chargaff's rule.
`
`tance between base pairs, had to be about 0.34 nm (Figure 4.10c). This distance
`is just twice the van der Waals thickness of a planar ring (see Table 2.2 on page
`30), so the bases are closely packed within the helix, as shown in a space-filling
`model (Figure 4.11 ). The model also shows that although the bases are inside, they
`can be approached through two deep spiral grooves called the major and minor
`grooves. Building molecular models of two-strand DNA structures soon con(cid:173)
`vinced Watson and Crick that the DNA strands must run in opposite directions.
`This arrangement can be seen clearly in Figure 4.10c. The model Watson and Crick
`presented was for a right-hand helix, although at that time evidence for the sense
`(direction of the turn) of the helix was weak.
`As is often the case with a good theory or model, the Watson-Crick structure
`also explained other data that had not been understood until then. The biochemist
`Erwin Chargaff, who had measured the relative amounts of A, T, G, and C in
`DNAs from many organisms, had noted the perplexing fact that A and T were al(cid:173)
`most always present in nearly equal quantities, as were G and C (Table 4.2). If
`most DNA in cells was double-stranded, with the Watson-Crick base pairing,
`then Chargaff's rule followed as a natural consequence.
`The Watson-Crick model not only explained the structure of DNA and Char(cid:173)
`gaff's rule but also carried implications that went to the very heart of biology.
`Since A always pairs with T, and G always pairs with C, the two strands are com(cid:173)
`plementary. If the strands could be separated and new DNA synthesized along each,
`following the same base-pairing rule, two double-strand DNA molecules w