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8
Nucleotides and Nucleic Acids
8.1 Some Basics 281 (rRNAs) are components of ribosomes, the complexes
that carry out the synthesis of proteins. Messenger
8.2 Nucleic Acid Structure 287 RNAs (mRNAs) are intermediaries, carrying genetic
8.3 Nucleic Acid Chemistry 297 information from one or a few genes to a ribosome,
where the corresponding proteins can be synthesized.
8.4 Other Functions of Nucleotides 306 Transfer RNAs (tRNAs) are adapter molecules that
faithfully translate the information in mRNA into a spe-

N
ucleotides have a variety of roles in cellular metabo- cific sequence of amino acids. In addition to these major
lism. They are the energy currency in metabolic classes there is a wide variety of RNAs with special
transactions, the essential chemical links in the functions, described in depth in Part III.
response of cells to hormones and other extracellular
stimuli, and the structural components of an array of Nucleotides and Nucleic Acids Have Characteristic
enzyme cofactors and metabolic intermediates. And, Bases and Pentoses
last but certainly not least, they are the constituents of
Nucleotides have three characteristic components: (1) a
nucleic acids: deoxyribonucleic acid (DNA) and ribo-
nitrogenous (nitrogen-containing) base, (2) a pentose,
nucleic acid (RNA), the molecular repositories of
and (3) one or more phosphates (Fig. 8–1). The mol-
genetic information. The structure of every protein, and
ecule without a phosphate group is called a nucleoside.
ultimately of every biomolecule and cellular component,
The nitrogenous bases are derivatives of two parent
is a product of information programmed into the nucle-
otide sequence of a cell’s nucleic acids. The ability to
store and transmit genetic information from one genera- Purine or
tion to the next is a fundamental condition for life. pyrimidine
base
This chapter provides an overview of the chemical O!
nature of the nucleotides and nucleic acids found in 5"
Phosphate
!
O P O CH2 O
most cells; a more detailed examination of the function #

of nucleic acids is the focus of Part III of this text. O 4"

H H 1"
Pentose
H H
3" 2"

8.1 Some Basics (a) OH OH

Nucleotides, Building Blocks of Nucleic Acids The amino

H H
acid sequence of every protein in a cell, and the nucleo- C
C 6 N
tide sequence of every RNA, is specified by a nucleo- N3
4 N1 5C
7
5 CH 8 CH
tide sequence in the cell’s DNA. A segment of a DNA HC 2 4
C 9
HC 2 6
CH 3
molecule that contains the information required for the 1
N N N
H
synthesis of a functional biological product, whether
(b) Pyrimidine Purine
protein or RNA, is referred to as a gene. A cell typi-
cally has many thousands of genes, and DNA molecules, FIGURE 8–1 Structure of nucleotides. (a) General structure showing
not surprisingly, tend to be very large. The storage the numbering convention for the pentose ring. This is a ribonucleotide.
and transmission of biological information are the only In deoxyribonucleotides the —OH group on the 29 carbon (in red) is
known functions of DNA. replaced with H. (b) The parent compounds of the pyrimidine and
RNAs have a broader range of functions, and purine bases of nucleotides and nucleic acids, showing the numbering
several classes are found in cells. Ribosomal RNAs conventions.

281
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282 Nucleotides and Nucleic Acids

compounds, pyrimidine and purine. The bases and NH2 O

pentoses of the common nucleotides are heterocyclic
C N C N
compounds. N C HN C
CH CH
HC C C C
KEY CONVENTION: The carbon and nitrogen atoms in the N N
N H2N N
parent structures are conventionally numbered to facili- H H
tate the naming and identification of the many deriva- Adenine Guanine
tive compounds. The convention for the pentose ring Purines
follows rules outlined in Chapter 7, but in the pentoses
of nucleotides and nucleosides the carbon numbers are O
NH2 O
given a prime (9) designation to distinguish them from
the numbered atoms of the nitrogenous bases. ■ C C CH3 C
N CH HN C HN CH
The base of a nucleotide is joined covalently (at C CH C CH C CH
N-1 of pyrimidines and N-9 of purines) in an N-!- O N O N O N
H H H
glycosyl bond to the 19 carbon of the pentose, and the
Cytosine Thymine Uracil
phosphate is esterified to the 59 carbon. The N-!- (DNA) (RNA)
glycosyl bond is formed by removal of the elements of Pyrimidines
water (a hydroxyl group from the pentose and hydro-
gen from the base), as in O-glycosidic bond formation FIGURE 8–2 Major purine and pyrimidine bases of nucleic acids. Some
of the common names of these bases reflect the circumstances of their
(see Fig. 7–30).
discovery. Guanine, for example, was first isolated from guano (bird
Both DNA and RNA contain two major purine
manure), and thymine was first isolated from thymus tissue.
bases, adenine (A) and guanine (G), and two major
pyrimidines. In both DNA and RNA one of the pyrimi-
dines is cytosine (C), but the second common pyrimi- form. As Figure 8–3 shows, the pentose ring is not
dine is not the same in both: it is thymine (T) in DNA planar but occurs in one of a variety of conformations
and uracil (U) in RNA. Only occasionally does thy- generally described as “puckered.”
mine occur in RNA or uracil in DNA. The structures of
the five major bases are shown in Figure 8–2, and the KEY CONVENTION: Although DNA and RNA seem to have
nomenclature of their corresponding nucleotides and two distinctions—different pentoses and the presence
nucleosides is summarized in Table 8–1. of uracil in RNA and thymine in DNA—it is the pentoses
Nucleic acids have two kinds of pentoses. The that define the identity of a nucleic acid. If the nucleic
recurring deoxyribonucleotide units of DNA contain acid contains 29-deoxy-D-ribose, it is DNA by defini-
29-deoxy-D-ribose, and the ribonucleotide units of RNA tion even though it may contain uracil. Similarly, if the
contain D-ribose. In nucleotides, both types of pentoses nucleic acid contains D-ribose, it is RNA regardless of its
are in their !-furanose (closed five-membered ring) base composition. ■

TABLE 8–1 Nucleotide and Nucleic Acid Nomenclature

Base Nucleoside Nucleotide Nucleic acid
Purines
Adenine Adenosine Adenylate RNA
Deoxyadenosine Deoxyadenylate DNA
Guanine Guanosine Guanylate RNA
Deoxyguanosine Deoxyguanylate DNA
Pyrimidines
Cytosine Cytidine Cytidylate RNA
Deoxycytidine Deoxycytidylate DNA
Thymine Thymidine or deoxythymidine Thymidylate or deoxythymidylate DNA
Uracil Uridine Uridylate RNA
Note: “Nucleoside” and “nucleotide” are generic terms that include both ribo- and deoxyribo- forms. Also, ribonucleosides and ribonucleotides are
here designated simply as nucleosides and nucleotides (e.g., riboadenosine as adenosine), and deoxyribonucleosides and deoxyribonucleotides as
deoxynucleosides and deoxynucleotides (e.g., deoxyriboadenosine as deoxyadenosine). Both forms of naming are acceptable, but the shortened
names are more commonly used. Thymine is an exception; “ribothymidine” is used to describe its unusual occurrence in RNA.
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8.1 Some Basics 283

H 5" 5"
OH
C O
CH 2 O OH 4" C-3" endo 4"
H C OH
H H
H C OH H H C-2" endo Base Base
H C OH 3"
OH OH
1" C-3" exo 1"
CH2OH
(a) Aldehyde # -Furanose (b) C-2" exo 2"

FIGURE 8–3 Conformations of ribose. (a) In solution, the straight-chain exist in four different puckered conformations. In all cases, four of the
(aldehyde) and ring (!-furanose) forms of free ribose are in equilibrium. five atoms are nearly in a single plane. The fifth atom (C-29 or C-39) is
RNA contains only the ring form, !-D-ribofuranose. Deoxyribose under- on either the same (endo) or the opposite (exo) side of the plane
goes a similar interconversion in solution, but in DNA exists solely as relative to the C-59 atom.
!-29-deoxy-D-ribofuranose. (b) Ribofuranose rings in nucleotides can

Figure 8–4 gives the structures and names of the DNAs, and the four major ribonucleotides (ribonu-
four major deoxyribonucleotides (deoxyribonucleo- cleoside 59-monophosphates), the structural units of
side 59-monophosphates), the structural units of RNAs.

NH2 O O NH2
CH3
N N HN N
N HN

N N H2N N N O N O N
O! O! O! O!
O P O CH2 O !
O P O CH2 O !
O P O CH2 O !
O P O CH2 O
O H H O H H O H H O H H
H H H H H H H H

OH H OH H OH H OH H
Nucleotide: Deoxyadenylate Deoxyguanylate Deoxythymidylate Deoxycytidylate
(deoxyadenosine (deoxyguanosine (deoxythymidine (deoxycytidine
5"-monophosphate) 5"-monophosphate) 5"-monophosphate) 5"-monophosphate)
Symbols: A, dA, dAMP G, dG, dGMP T, dT, dTMP C, dC, dCMP
Nucleoside: Deoxyadenosine Deoxyguanosine Deoxythymidine Deoxycytidine

(a) Deoxyribonucleotides

NH2 O O NH2

N N HN N
N HN

N N H2N N N O N O N
O! O! O! O!
!
O P O CH2 O !
O P O CH2 O !
O P O CH2 O !
O P O CH2 O
O H H O H H O H H O H H
H H H H H H H H

OH OH OH OH OH OH OH OH
Nucleotide: Adenylate (adenosine Guanylate (guanosine Uridylate (uridine Cytidylate (cytidine
5"-monophosphate) 5"-monophosphate) 5"-monophosphate) 5"-monophosphate)
Symbols: A, AMP G, GMP U, UMP C, CMP
Nucleoside: Adenosine Guanosine Uridine Cytidine

(b) Ribonucleotides

FIGURE 8–4 Deoxyribonucleotides and ribonucleotides of nucleic UMP, and CMP. For each nucleotide in the figure, the more common
acids. All nucleotides are shown in their free form at pH 7.0. The name is followed by the complete name in parentheses. All abbrevia-
nucleotide units of DNA (a) are usually symbolized as A, G, T, and C, tions assume that the phosphate group is at the 59 position. The nucleo-
sometimes as dA, dG, dT, and dC; those of RNA (b) as A, G, U, and C. side portion of each molecule is shaded in light red. In this and the
In their free form the deoxyribonucleotides are commonly abbreviated following illustrations, the ring carbons are not shown.
dAMP, dGMP, dTMP, and dCMP; the ribonucleotides, AMP, GMP,
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284 Nucleotides and Nucleic Acids

CH3 Adenine
NH2 NH HO CH2 O
CH3 N O! H H
N N 6 Adenine
5 5" H H
!
O P O CH2 O 2"
O N N N 4" 1" OH O
O H H
Ribose H H !
O P O!
Ribose 3" 2"

5-Methylcytidine N6-Methyladenosine OH OH O
Adenosine 5"-monophosphate Adenosine 2"-monophosphate
O NH2
N CH2OH
HN N 5 Adenine
2 Adenine
N HO CH2 O
H3C N N O N HO CH2 O
H H H
Ribose Ribose H H H H
3" H H
2
(a) N -Methylguanosine 5-Hydroxymethylcytidine O OH 3" 2"
O O
!
O P O! P
O O
Ribose O O O!
N
HN HN 5
Adenosine 3"-monophosphate Adenosine 2",3"-cyclic
monophosphate
N N O N
H FIGURE 8–6 Some adenosine monophosphates. Adenosine 29-mono-
Ribose
phosphate, 39-monophosphate, and 29,39-cyclic monophosphate are formed
Inosine Pseudouridine by enzymatic and alkaline hydrolysis of RNA.

O CH3 S

HN N$ HN 4 convention (used here) is simply to indicate the ring

7
position of the substituent by its number—for example,
H2N N N O N 5-methylcytosine, 7-methylguanine, and 5-hydroxymeth-
Ribose
ylcytosine (shown as the nucleosides in Fig. 8–5). The
Ribose
element to which the substituent is attached (N, C, O) is
(b) 7-Methylguanosine 4-Thiouridine not identified. The convention changes when the substi-
FIGURE 8–5 Some minor purine and pyrimidine bases, shown as the tuted atom is exocyclic (not within the ring structure),
nucleosides. (a) Minor bases of DNA. 5-Methylcytidine occurs in the
in which case the type of atom is identified and the ring
DNA of animals and higher plants, N6-methyladenosine in bacterial position to which it is attached is denoted with a super-
DNA, and 5-hydroxymethylcytidine in the DNA of animals and of bacte- script. The amino nitrogen attached to C-6 of adenine
ria infected with certain bacteriophages. (b) Some minor bases of is N 6; similarly, the carbonyl oxygen and amino nitrogen
tRNAs. Inosine contains the base hypoxanthine. Note that pseudouri- at C-6 and C-2 of guanine are O6 and N 2, respectively.
dine, like uridine, contains uracil; they are distinct in the point of attach- Examples of this nomenclature are N 6-methyladenosine
ment to the ribose—in uridine, uracil is attached through N-1, the usual and N2-methylguanosine (Fig. 8–5). ■
attachment point for pyrimidines; in pseudouridine, through C-5.
Cells also contain nucleotides with phosphate
Although nucleotides bearing the major purines and groups in positions other than on the 59 carbon (Fig.
pyrimidines are most common, both DNA and RNA also 8–6). Ribonucleoside 2!,3!-cyclic monophosphates
contain some minor bases (Fig. 8–5). In DNA the most are isolatable intermediates, and ribonucleoside
common of these are methylated forms of the major 3!-monophosphates are end products of the hydroly-
bases; in some viral DNAs, certain bases may be sis of RNA by certain ribonucleases. Other variations
hydroxymethylated or glucosylated. Altered or unusual are adenosine 39,59-cyclic monophosphate (cAMP) and
bases in DNA molecules often have roles in regulating or guanosine 39,59-cyclic monophosphate (cGMP), consid-
protecting the genetic information. Minor bases of many ered at the end of this chapter.
types are also found in RNAs, especially in tRNAs (see
Fig. 8–25 and Fig. 26–22).
Phosphodiester Bonds Link Successive
KEY CONVENTION: The nomenclature for the minor bases Nucleotides in Nucleic Acids
can be confusing. Like the major bases, many have The successive nucleotides of both DNA and RNA are
common names—hypoxanthine, for example, shown as covalently linked through phosphate-group “bridges,” in
its nucleoside inosine in Figure 8–5. When an atom in which the 59-phosphate group of one nucleotide unit is
the purine or pyrimidine ring is substituted, the usual joined to the 39-hydroxyl group of the next nucleotide,
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8.1 Some Basics 285

creating a phosphodiester linkage (Fig. 8–7). Thus DNA RNA

the covalent backbones of nucleic acids consist of alter- 5" End 5" End
nating phosphate and pentose residues, and the nitro- O! O!
genous bases may be regarded as side groups joined to !
O P O O
!
P O
the backbone at regular intervals. The backbones of
both DNA and RNA are hydrophilic. The hydroxyl O A O U
groups of the sugar residues form hydrogen bonds with 5" CH2 O 5" CH2 O
water. The phosphate groups, with a pKa near 0, are
H H H H
completely ionized and negatively charged at pH 7, and H H H H
the negative charges are generally neutralized by ionic 3" 3"
interactions with positive charges on proteins, metal O H O OH
Phospho-
ions, and polyamines. diester
!
O P O !
O P O
linkage
O O
KEY CONVENTION: All the phosphodiester linkages in DNA T G
and RNA have the same orientation along the chain 5!
5" CH2 O 5" CH2 O
(Fig. 8–7), giving each linear nucleic acid strand a spe- H H H H
cific polarity and distinct 59 and 39 ends. By definition, H H H H
3!
the 5! end lacks a nucleotide at the 59 position and 3" 3"
O H O OH
the 3! end lacks a nucleotide at the 39 position. Other
groups (most often one or more phosphates) may be
!
O P O !
O P O
present on one or both ends. The 59 to 39 orientation of O G O C
a strand of nucleic acid refers to the ends of the strand, O
5" CH2 5" CH2 O
not the orientation of the individual phosphodiester
bonds linking its constituent nucleotides. ■ H H H H
H H H H
3" 3"
The covalent backbone of DNA and RNA is subject O H O OH
to slow, nonenzymatic hydrolysis of the phosphodiester H H
bonds. In the test tube, RNA is hydrolyzed rapidly under
3" End 3" End
alkaline conditions, but DNA is not; the 29-hydroxyl
groups in RNA (absent in DNA) are directly involved in FIGURE 8–7 Phosphodiester linkages in the covalent backbone of DNA
the process. Cyclic 29,39-monophosphate nucleotides and RNA. The phosphodiester bonds (one of which is shaded in the
are the first products of the action of alkali on RNA and DNA) link successive nucleotide units. The backbone of alternating pen-
are rapidly hydrolyzed further to yield a mixture of 29- tose and phosphate groups in both types of nucleic acid is highly polar.
and 39-nucleoside monophosphates (Fig. 8–8). The 59 and 39 ends of the macromolecule may be free or may have an
The nucleotide sequences of nucleic acids can be attached phosphoryl group.
represented schematically, as illustrated below by a seg-
ment of DNA with five nucleotide units. The phosphate

!
O P O
O
!
O P O 2",3"-Cyclic CH2 O Base1
Mixture of 2"- and
H2O
monophosphate 3"-monophosphate
O H H
derivative H H derivatives
CH2 O Base1
O O
H H
H H P
!
O O
O O H !
OH
$
O
!
P O
O OH
CH2 O Base2 CH2 O Base2
FIGURE 8–8 Hydrolysis of RNA under alkaline conditions.
H H H H The 29 hydroxyl acts as a nucleophile in an intramolecular
H H H H
displacement. The 29,39-cyclic monophosphate derivative
O OH O OH is further hydrolyzed to a mixture of 29- and 39-mono-
RNA Shortened
O
!
P O O
!
P O phosphates. DNA, which lacks 29 hydroxyls, is stable
RNA
under similar conditions.
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286 Nucleotides and Nucleic Acids

groups are symbolized by P , and each deoxyribose is O OH OH

symbolized by a vertical line, from C-19 at the top to
HN N N
C-59 at the bottom (but keep in mind that the sugar is
always in its closed-ring !-furanose form in nucleic O N O N HO N
acids). The connecting lines between nucleotides H H
(which pass through P ) are drawn diagonally from the Lactam Lactim Double lactim
middle (C-39) of the deoxyribose of one nucleotide to Uracil
the bottom (C-59) of the next.
FIGURE 8–9 Tautomeric forms of uracil. The lactam form predominates
A C G T A at pH 7.0; the other forms become more prominent as pH decreases.
The other free pyrimidines and the free purines also have tautomeric
5" End 3" End forms, but they are more rarely encountered.

P P P P P OH

Some simpler representations of this pentadeoxyribo- may exist in two or more tautomeric forms depending
nucleotide are pA-C-G-T-AOH, pApCpGpTpA, and on the pH. Uracil, for example, occurs in lactam, lactim,
pACGTA. and double lactim forms (Fig. 8–9). The structures
shown in Figure 8–2 are the tautomers that predomi-
KEY CONVENTION: The sequence of a single strand of nu- nate at pH 7.0. All nucleotide bases absorb UV light, and
cleic acid is always written with the 59 end at the left and nucleic acids are characterized by a strong absorption at
the 39 end at the right—that is, in the 59S 39 direction. ■ wavelengths near 260 nm (Fig. 8–10).
The purine and pyrimidine bases are hydrophobic
A short nucleic acid is referred to as an oligonucle- and relatively insoluble in water at the near-neutral pH
otide. The definition of “short” is somewhat arbitrary, of the cell. At acidic or alkaline pH the bases become
but polymers containing 50 or fewer nucleotides are charged and their solubility in water increases. Hydro-
generally called oligonucleotides. A longer nucleic acid phobic stacking interactions in which two or more bases
is called a polynucleotide. are positioned with the planes of their rings parallel
(like a stack of coins) are one of two important modes
of interaction between bases in nucleic acids. The stack-
The Properties of Nucleotide Bases Affect the ing also involves a combination of van der Waals and
Three-Dimensional Structure of Nucleic Acids dipole-dipole interactions between the bases. Base
Free pyrimidines and purines are weakly basic com- stacking helps to minimize contact of the bases with
pounds and thus are called bases. The purines and water, and base-stacking interactions are very impor-
pyrimidines common in DNA and RNA are aromatic tant in stabilizing the three-dimensional structure of
molecules (Fig. 8–2), a property with important conse- nucleic acids, as described later.
quences for the structure, electron distribution, and The functional groups of pyrimidines and purines
light absorption of nucleic acids. Electron delocalization are ring nitrogens, carbonyl groups, and exocyclic amino
among atoms in the ring gives most of the bonds partial groups. Hydrogen bonds involving the amino and car-
double-bond character. One result is that pyrimidines bonyl groups are the most important mode of interaction
are planar molecules and purines are very nearly planar, between two (and occasionally three or four) comple-
with a slight pucker. Free pyrimidine and purine bases mentary strands of nucleic acid. The most common

14,000
Molar extinction coefficient, e

12,000

10,000

8,000 Molar extinction

coefficient at 260 nm,
FIGURE 8–10 Absorption spectra of the common nucleotides. The 6,000 e260 (M!1cm!1)
spectra are shown as the variation in molar extinction coefficient AMP 15,400
with wavelength. The molar extinction coefficients at 260 nm and 4,000 GMP 11,700
pH 7.0 (%260) are listed in the table. The spectra of corresponding UMP 9,900
2,000 dTMP 9,200
ribonucleotides and deoxyribonucleotides, as well as the nucleo-
sides, are essentially identical. For mixtures of nucleotides, a wave- CMP 7,500
length of 260 nm (dashed vertical line) is used for absorption 230 240 250 260 270 280
measurements. Wavelength (nm)
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8.2 Nucleic Acid Structure 287

CH3
2.8 A
O
5" 3" H H C
C H
N C
3.0 A N Thymine
C N C N
G H
C N C
H C C-1"
A T Adenine C C O
N
N H
C-1"
A T
11.1 A

T
H H
A
2.9 A
N C
H C H
C O C
G Cytosine
3.0 A N
N C N
C N H C
H C C-1"
G C
Guanine C C O
N H
N N
T A C-1" 2.9 A
H
10.8 A
C G

A T
FIGURE 8–11 Hydrogen-bonding patterns in the base pairs defined by
Watson and Crick. Here as elsewhere, hydrogen bonds are represented
3" 5" by three blue lines.

hydrogen-bonding patterns are those defined by James group of one pentose and the 39-hydroxyl group of
D. Watson and Francis Crick in 1953, in which A bonds the next.
specifically to T (or U) and G bonds to C (Fig. 8–11). ! There are two types of nucleic acid: RNA and
These two types of base pairs predominate in double- DNA. The nucleotides in RNA contain ribose, and
stranded DNA and RNA, and the tautomers shown in the common pyrimidine bases are uracil and
Figure 8–2 are responsible for these patterns. It is this cytosine. In DNA, the nucleotides contain
specific pairing of bases that permits the duplication of 29-deoxyribose, and the common pyrimidine bases
genetic information, as we shall discuss later in this are thymine and cytosine. The primary purines are
chapter. adenine and guanine in both RNA and DNA.

8.2 Nucleic Acid Structure

The discovery of the structure of DNA by Watson and
Crick in 1953 gave rise to entirely new disciplines and
influenced the course of many established ones. In this
section we focus on DNA structure, some of the events
that led to its discovery, and more recent refinements
in our understanding of DNA. RNA structure is also
introduced.
As in the case of protein structure (Chapter 4), it
is sometimes useful to describe nucleic acid structure
in terms of hierarchical levels of complexity (primary,
James D. Watson Francis Crick, 1916–2004
secondary, tertiary). The primary structure of a nucleic
acid is its covalent structure and nucleotide sequence.
Any regular, stable structure taken up by some or all of
SUMMARY 8.1 Some Basics the nucleotides in a nucleic acid can be referred to as
! A nucleotide consists of a nitrogenous base secondary structure. All structures considered in the
(purine or pyrimidine), a pentose sugar, and one remainder of this chapter fall under the heading of
or more phosphate groups. Nucleic acids are secondary structure. The complex folding of large
polymers of nucleotides, joined together by chromosomes within eukaryotic chromatin and bacte-
phosphodiester linkages between the 59-hydroxyl rial nucleoids, or the elaborate folding of large tRNA
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288 Nucleotides and Nucleic Acids

or rRNA molecules, is generally considered tertiary

structure. DNA tertiary structure is discussed in
Chapter 24, and RNA tertiary structure is considered
in Chapter 26.

DNA Is a Double Helix That Stores

Genetic Information
DNA was first isolated and characterized by Friedrich
Miescher in 1868. He called the phosphorus-containing
substance “nuclein.” Not until the 1940s, with the work
of Oswald T. Avery, Colin MacLeod, and Maclyn McCarty,
was there any compelling evidence that DNA was the
FIGURE 8–12 X-ray diffraction pattern of DNA fibers. The spots form-
genetic material. Avery and his colleagues found that
ing a cross in the center denote a helical structure. The heavy bands at
DNA extracted from a virulent (disease-causing) strain
the left and right arise from the recurring bases.
of the bacterium Streptococcus pneumoniae and
injected into a nonvirulent strain of the same bacterium
transformed the nonvirulent strain into a virulent strain. of x-ray diffraction (see Box 4–5) to analyze DNA fibers.
They concluded that the DNA from the virulent strain They showed in the early 1950s that DNA produces a
carried the genetic information for virulence. Then in characteristic x-ray diffraction pattern (Fig. 8–12).
1952, experiments by Alfred D. Hershey and Martha From this pattern it was deduced that DNA molecules
Chase, in which they studied the infection of bacterial are helical with two periodicities along their long axis, a
cells by a virus (bacteriophage) with radioactively primary one of 3.4 Å and a secondary one of 34 Å. The
labeled DNA or protein, removed any remaining doubt problem then was to formulate a three-dimensional model
that DNA, not protein, carried the genetic information. of the DNA molecule that could account not only for the
Another important clue to the structure of DNA x-ray diffraction data but also for the specific A 5 T and
came from the work of Erwin Chargaff and his col- G 5 C base equivalences discovered by Chargaff and for
leagues in the late 1940s. They found that the four the other chemical properties of DNA.
nucleotide bases of DNA occur in different ratios in the
DNAs of different organisms and that the amounts of
certain bases are closely related. These data, collected
from DNAs of a great many different species, led Chargaff
to the following conclusions:
1. The base composition of DNA generally varies
from one species to another.
2. DNA specimens isolated from different tissues of
the same species have the same base composition.
3. The base composition of DNA in a given species
does not change with an organism’s age,
nutritional state, or changing environment. Maurice Wilkins,
Rosalind Franklin,
4. In all cellular DNAs, regardless of the species, the 1920–1958 1916–2004
number of adenosine residues is equal to the
number of thymidine residues (that is, A 5 T),
James Watson and Francis Crick relied on this accu-
and the number of guanosine residues is equal to
mulated information about DNA to set about deducing its
the number of cytidine residues (G 5 C). From
structure. In 1953 they postulated a three-dimensional
these relationships it follows that the sum of the
model of DNA structure that accounted for all the avail-
purine residues equals the sum of the pyrimidine
able data. It consists of two helical DNA chains wound
residues; that is, A 1 G 5 T 1 C.
around the same axis to form a right-handed double helix
These quantitative relationships, sometimes called (see Box 4–1 for an explanation of the right- or left-
“Chargaff’s rules,” were confirmed by many subsequent handed sense of a helical structure). The hydrophilic
researchers. They were a key to establishing the three- backbones of alternating deoxyribose and phosphate
dimensional structure of DNA and yielded clues to how groups are on the outside of the double helix, facing the
genetic information is encoded in DNA and passed from surrounding water. The furanose ring of each deoxyri-
one generation to the next. bose is in the C-29 endo conformation. The purine and
To shed more light on the structure of DNA, Rosalind pyrimidine bases of both strands are stacked inside the
Franklin and Maurice Wilkins used the powerful method double helix, with their hydrophobic and nearly planar
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8.2 Nucleic Acid Structure 289

distance of about 34 Å was accounted for by the pres-

ence of 10 base pairs in each complete turn of the dou-
ble helix. In aqueous solution the structure differs
slightly from that in fibers, having 10.5 base pairs per
helical turn (Fig. 8–13).
As Figure 8–14 shows, the two antiparallel poly-
Major
groove nucleotide chains of double-helical DNA are not identi-
3.4 Å cal in either base sequence or composition. Instead they
are complementary to each other. Wherever adenine
Minor occurs in one chain, thymine is found in the other;
groove
similarly, wherever guanine occurs in one chain, cyto-
sine is found in the other.
The DNA double helix, or duplex, is held together
36 Å by two forces, as described earlier: hydrogen bonding
between complementary base pairs (Fig. 8–11) and
base-stacking interactions. The complementarity
20 Å between the DNA strands is attributable to the hydro-
gen bonding between base pairs. The base-stacking
interactions, which are largely nonspecific with respect
to the identity of the stacked bases, make the major
(a) (b) (c)
contribution to the stability of the double helix.
FIGURE 8–13 Watson-Crick model for the structure of DNA. The original The important features of the double-helical model of
model proposed by Watson and Crick had 10 base pairs, or 34 Å (3.4 nm), DNA structure are supported by much chemical and bio-
per turn of the helix; subsequent measurements revealed 10.5 base logical evidence. Moreover, the model immediately sug-
pairs, or 36 Å (3.6 nm), per turn. (a) Schematic representation, showing gested a mechanism for the transmission of genetic in-
dimensions of the helix. (b) Stick representation showing the backbone formation. The essential feature of the model is the
and stacking of the bases. (c) Space-filling model. complementarity of the two DNA strands. As Watson and
Crick were able to see, well before confirmatory data
ring structures very close together and perpendicular to became available, this structure could logically be repli-
the long axis. The offset pairing of the two strands creates cated by (1) separating the two strands and (2) synthesiz-
a major groove and minor groove on the surface of the ing a complementary strand for each. Because nucleo-
duplex (Fig. 8–13). Each nucleotide base of one strand tides in each new strand are joined in a sequence specified
is paired in the same plane with a base of the other by the base-pairing rules stated above, each preexisting
strand. Watson and Crick found that the hydrogen-
bonded base pairs illustrated in Figure 8–11, G with C and
A with T, are those that fit best within the structure, provid- 5" 3"
ing a rationale for Chargaff’s rule that in any DNA, G 5 C
C
and A 5 T. It is important to note that three hydrogen G
bonds can form between G and C, symbolized GqC, but
only two can form between A and T, symbolized APT. A T
This is one reason for the finding that separation of paired
DNA strands is more difficult the higher the ratio of GqC A T
to APT base pairs. Other pairings of bases tend (to vary-
ing degrees) to destabilize the double-helical structure. T A
When Watson and Crick constructed their model,
they had to decide at the outset whether the strands of
C
DNA should be parallel or antiparallel—whether G

their 39,59-phosphodiester bonds should run in the same FIGURE 8–14 Complementarity of strands
or opposite directions. An antiparallel orientation pro- G C
in the DNA double helix. The comple-
duced the most convincing model, and later work with mentary antiparallel strands of DNA follow
DNA polymerases (Chapter 25) provided experimental T A the pairing rules proposed by Watson and
evidence that the strands are indeed antiparallel, a find- Crick. The base-paired antiparallel strands
ing ultimately confirmed by x-ray analysis. C differ in base composition: the left strand
G
To account for the periodicities observed in the has the composition A3T2G1C3; the right,
x-ray diffraction patterns of DNA fibers, Watson and A2T3G3C1. They also differ in sequence
A T
Crick manipulated molecular models to arrive at a struc- when each chain is read in the 59S 39
ture in which the vertically stacked bases inside the direction. Note the base equivalences:
double helix would be 3.4 Å apart; the secondary repeat 3" 5" A 5 T and G 5 C in the duplex.
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290 Nucleotides and Nucleic Acids

DNA Can Occur in Different Three-Dimensional Forms

DNA is a remarkably flexible molecule. Considerable rota-
tion is possible around several types of bonds in the
sugar–phosphate (phosphodeoxyribose) backbone, and
thermal fluctuation can produce bending, stretching, and
unpairing (melting) of the strands. Many significant devia-
tions from the Watson-Crick DNA structure are found in
5" cellular DNA, some or all of which may be important in
New 3"
New DNA metabolism. These structural variations generally do
not affect the key properties of DNA defined by Watson
and Crick: strand complementarity, antiparallel strands,
and the requirement for APT and GqC base pairs.
Structural variation in DNA reflects three things:
the different possible conformations of the deoxyri-
bose, rotation about the contiguous bonds that make
up the phosphodeoxyribose backbone (Fig. 8–16a),
and free rotation about the C-19–N-glycosyl bond (Fig.
8–16b). Because of steric constraints, purines in
Parent Parent purine nucleotides are restricted to two stable confor-
strand strand mations with respect to deoxyribose, called syn and
Daughter anti (Fig. 8–16b). Pyrimidines are generally restricted
strands to the anti conformation because of steric interference
FIGURE 8–15 Replication of DNA as suggested by Watson and Crick. between the sugar and the carbonyl oxygen at C-2 of
The preexisting or “parent” strands become separated, and each is the the pyrimidine.
template for biosynthesis of a complementary “daughter” strand (in pink).

5"
strand functions as a template to guide the synthesis of
one complementary strand (Fig. 8–15). These expecta-
1 2
tions were experimentally confirmed, inaugurating a revo-
lution in our understanding of biological inheritance.
3
Base 4
WORKED EXAMPLE 8–1 Base Pairing in DNA 5
7
In samples of DNA isolated from two unidentified spe- 6
cies of bacteria, X and Y, adenine makes up 32% and
17%, respectively, of the total bases. What relative 3"
proportions of adenine, guanine, thymine, and cytosine (a)
would you expect to find in the two DNA samples? What
NH2 NH2 NH2
assumptions have you made? One of these species was
isolated from a hot spring (64 8C). Which species is most N N N N N
likely the thermophilic bacterium, and why?
N N N N N O
Solution: For any double-helical DNA, A 5 T and G 5 C.
HOCH2 O HOCH2 O HOCH2 O
The DNA from species X has 32% A and therefore must
contain 32% T. This accounts for 64% of the bases and H H H H H H
H H H H H H
leaves 36% as GqC pairs: 18% G and 18% C. The sam-
ple from species Y, with 17% A, must contain 17% T, OH OH OH OH OH OH
accounting for 34% of the base pairs. The remaining syn-Adenosine anti-Adenosine anti-Cytidine
(b)
66% of the bases are thus equally distributed as 33% G
and 33% C. This calculation is based on the assumption FIGURE 8–16 Structural variation in DNA. (a) The conformation of a
that both DNA molecules are double-stranded. nucleotide in DNA is affected by rotation about seven different bonds.
The higher the G 1 C content of a DNA molecule, Six of the bonds rotate freely. The limited rotation about bond 4 gives
the higher the melting temperature. Species Y, having rise to ring pucker. This conformation is endo or exo, depending on
the DNA with the higher G 1 C content (66%), most whether the atom is displaced to the same side of the plane as C-59
likely is the thermophilic bacterium; its DNA has a or to the opposite side (see Fig. 8–3b). (b) For purine bases in nucle-
higher melting temperature and thus is more stable at otides, only two conformations with respect to the attached ribose
units are sterically permitted, anti or syn. Pyrimidines occur in the anti
the temperature of the hot spring.
conformation.
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8.2 Nucleic Acid Structure 291

The Watson-Crick structure is also referred to as purines, especially alternating C and G or 5-methyl-C
B-form DNA, or B-DNA. The B form is the most stable and G residues. To form the left-handed helix in Z-DNA,
structure for a random-sequence DNA molecule under the purine residues flip to the syn conformation, alter-
physiological conditions and is therefore the standard nating with pyrimidines in the anti conformation. The
point of reference in any study of the properties of major groove is barely apparent in Z-DNA, and the
DNA. Two structural variants that have been well minor groove is narrow and deep.
characterized in crystal structures are the A and Z Whether A-DNA occurs in cells is uncertain, but
forms. These three DNA conformations are shown in there is evidence for some short stretches (tracts) of
Figure 8–17, with a summary of their properties. The Z-DNA in both bacteria and eukaryotes. These Z-DNA
A form is favored in many solutions that are relatively tracts may play a role (as yet undefined) in regulating the
devoid of water. The DNA is still arranged in a right- expression of some genes or in genetic recombination.
handed double helix, but the helix is wider and the
number of base pairs per helical turn is 11, rather than
10.5 as in B-DNA. The plane of the base pairs in A-DNA Certain DNA Sequences Adopt Unusual Structures
is tilted about 20º relative to B-DNA base pairs, thus the Other sequence-dependent structural variations found
base pairs in A-DNA are not perfectly perpendicular to in larger chromosomes may affect the function and
the helix axis. These structural changes deepen the metabolism of the DNA segments in their immediate
major groove while making the minor groove shallower. vicinity. For example, bends occur in the DNA helix
The reagents used to promote crystallization of DNA wherever four or more adenosine residues appear
tend to dehydrate it, and thus most short DNA mol- sequentially in one strand. Six adenosines in a row pro-
ecules tend to crystallize in the A form. duce a bend of about 188. The bending observed with
Z-form DNA is a more radical departure from the B this and other sequences may be important in the bind-
structure; the most obvious distinction is the left-handed ing of some proteins to DNA.
helical rotation. There are 12 base pairs per helical turn, A rather common type of DNA sequence is a palin-
and the structure appears more slender and elongated. drome. A palindrome is a word, phrase, or sentence
The DNA backbone takes on a zigzag appearance. Certain that is spelled identically read either forward or back-
nucleotide sequences fold into left-handed Z helices ward; two examples are ROTATOR and NURSES RUN.
much more readily than others. Prominent examples The term is applied to regions of DNA with inverted
are sequences in which pyrimidines alternate with repeats of base sequence having twofold symmetry

FIGURE 8–17 Comparison of A, B, and Z forms of DNA. Each structure

shown here has 36 base pairs. The riboses and bases are shown in
yellow. The phosphodiester backbone is represented as a blue rope. Blue
is the color used to represent DNA strands in later chapters. The table
summarizes some properties of the three forms of DNA.

28 Å

A form B form Z form

Helical sense Right handed Right handed Left handed

Diameter !26 Å !20 Å !18 Å
Base pairs per
helical turn 11 10.5 12
Helix rise per base
pair 2.6 Å 3.4 Å 3.7 Å
Base tilt normal to
the helix axis 20° 6° 7°
Sugar pucker C-3" endo C-2" endo C-2" endo for
conformation pyrimidines;
C-3" endo for
purines
Glycosyl bond Anti Anti Anti for
conformation pyrimidines;
syn for purines
A form B form Z form
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292 Nucleotides and Nucleic Acids

Palindrome TGCGATACTCATCGCA
5" 3"
5’ T T A G C A C G T G C T A A

3’
A A T C G T G C A C G A T T
C T
A C
T A
A T
Mirror repeat G C
C G
5’ T T A G C A C C A C G A T T G C
T A
3’ 5" 3"
A A T C G T G G T G C T A A
(a) Hairpin
FIGURE 8–18 Palindromes and mirror repeats. Palindromes are sequences
of double-stranded nucleic acids with twofold symmetry. In order to
superimpose one repeat (shaded sequence) on the other, it must be rotated TGCGATACTCATCGCA
5" 3"
1808 about the horizontal axis then 1808 about the vertical axis, as shown
by the colored arrows. A mirror repeat, on the other hand, has a symmetric 3" 5"
ACGCTATGAGTAGCGT
sequence within each strand. Superimposing one repeat on the other
requires only a single 1808 rotation about the vertical axis.

C T
A C
over two strands of DNA (Fig. 8–18). Such sequences T A
are self-complementary within each strand and therefore A T
G C
have the potential to form hairpin or cruciform C G
G C
(cross-shaped) structures (Fig. 8–19). When the T A
inverted repeat occurs within each individual strand of 5" 3"
the DNA, the sequence is called a mirror repeat. 3" 5"
Mirror repeats do not have complementary sequences A T
C G
within the same strand and cannot form hairpin or cru- G C
ciform structures. Sequences of these types are found C
T
G
A
in virtually every large DNA molecule and can encom- A T
T G
pass a few base pairs or thousands. The extent to which
G A
palindromes occur as cruciforms in cells is not known,
(b) Cruciform
although some cruciform structures have been demon-
strated in vivo in Escherichia coli. Self-complementary FIGURE 8–19 Hairpins and cruciforms. Palindromic DNA (or RNA)
sequences cause isolated single strands of DNA (or sequences can form alternative structures with intrastrand base pairing.
RNA) in solution to fold into complex structures con- (a) When only a single DNA (or RNA) strand is involved, the structure
taining multiple hairpins. is called a hairpin. (b) When both strands of a duplex DNA are involved,
Several unusual DNA structures involve three or it is called a cruciform. Blue shading highlights asymmetric sequences
even four DNA strands. Nucleotides participating in a that can pair with the complementary sequence either in the same
Watson-Crick base pair (Fig. 8–11) can form additional strand or in the complementary strand.
hydrogen bonds, particularly with functional groups
arrayed in the major groove. For example, a cytidine strands and one purine strand; others contain two
residue (if protonated) can pair with the guanosine resi- purine strands and one pyrimidine strand.
due of a GqC nucleotide pair (Fig. 8–20); a thymidine Four DNA strands can also pair to form a tetraplex
can pair with the adenosine of an APT pair. The N-7, O6, (quadruplex), but this occurs readily only for DNA
and N6 of purines, the atoms that participate in the sequences with a very high proportion of guanosine
hydrogen bonding of triplex DNA, are often referred to residues (Fig. 8–20c, d). The guanosine tetraplex, or G
as Hoogsteen positions, and the non-Watson-Crick tetraplex, is quite stable over a wide range of condi-
pairing is called Hoogsteen pairing, after Karst Hoogs- tions. The orientation of strands in the tetraplex can
teen, who in 1963 first recognized the potential for these vary as shown in Figure 8–20e.
unusual pairings. Hoogsteen pairing allows the formation In the DNA of living cells, sites recognized by many
of triplex DNAs. The triplexes shown in Figure 8–20 sequence-specific DNA-binding proteins (Chapter 28)
(a, b) are most stable at low pH because the CqG ? C1 are arranged as palindromes, and polypyrimidine or
triplet requires a protonated cytosine. In the triplex, the polypurine sequences that can form triple helices are
pKa of this cytosine is &7.5, altered from its normal value found within regions involved in the regulation of
of 4.2. The triplexes also form most readily within long expression of some eukaryotic genes. In principle,
sequences containing only pyrimidines or only purines in synthetic DNA strands designed to pair with these
a given strand. Some triplex DNAs contain two pyrimidine sequences to form triplex DNA could disrupt gene
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8.2 Nucleic Acid Structure 293

CH3
CH3 H
H
1"-C N O O 1"-C N N N
N H H N$ H H
O N N N O O N N
H H C-1" H C-1"
N N H
N O N O
H
N N N N N
C-1" C-1" H
T A T C G C$
(a)

H C-1"
1"-C H N N N
N N
N
N
N O H
O H
H N
N H
H N
N H
H O
O N
H
N N
N N
N N N H C-1"
C-1" H
Guanosine tetraplex
(b) (c) (d)

FIGURE 8–20 DNA structures containing three or four DNA strands.

(a) Base-pairing patterns in one well-characterized form of triplex DNA.
The Hoogsteen pair in each case is shown in red. (b) Triple-helical DNA
containing two pyrimidine strands (red and white; sequence TTCCT)
and one purine strand (blue; sequence AAGGAA) (derived from PDB ID
1BCE). The blue and white strands are antiparallel and paired by normal
Watson-Crick base-pairing patterns. The third (all-pyrimidine) strand
(red) is parallel to the purine strand and paired through non-Watson-
Crick hydrogen bonds. The triplex is viewed from the side, with six trip-
lets shown. (c) Base-pairing pattern in the guanosine tetraplex structure.
(d) Four successive tetraplets from a G tetraplex structure (PDB ID Parallel Antiparallel
244D). (e) Possible variants in the orientation of strands in a G tetraplex.
(e)

expression. This approach to controlling cellular metab- on ribosomes in the cytoplasm, some molecule other
olism is of commercial interest for its potential applica- than DNA must carry the genetic message from the
tion in medicine and agriculture. nucleus to the cytoplasm. As early as the 1950s, RNA
was considered the logical candidate: RNA is found in
Messenger RNAs Code for Polypeptide Chains both the nucleus and the cytoplasm, and an increase in
We now turn our attention to the expression of the protein synthesis is accompanied by an increase in the
genetic information that DNA contains. RNA, the second amount of cytoplasmic RNA and an increase in its rate
major form of nucleic acid in cells, has many functions. of turnover. These and other observations led several
In gene expression, RNA acts as an intermediary by researchers to suggest that RNA carries genetic infor-
using the information encoded in DNA to specify the mation from DNA to the protein biosynthetic machinery
amino acid sequence of a functional protein. of the ribosome. In 1961 François Jacob and Jacques
Given that the DNA of eukaryotes is largely con- Monod presented a unified (and essentially correct)
fined to the nucleus whereas protein synthesis occurs picture of many aspects of this process. They proposed
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294 Nucleotides and Nucleic Acids

5" 3" in Chapter 26. The diverse and often complex functions
Gene
(a) Monocistronic
of these RNAs reflect a diversity of structure much
richer than that observed in DNA molecules.
The product of transcription of DNA is always
5" 3" single-stranded RNA. The single strand tends to assume
Gene 1 Gene 2 Gene 3
a right-handed helical conformation dominated by base-
(b) Polycistronic
stacking interactions (Fig. 8–22), which are stronger
FIGURE 8–21 Bacterial mRNA. Schematic diagrams show (a) monocis- between two purines than between a purine and pyrim-
tronic and (b) polycistronic mRNAs of bacteria. Red segments represent idine or between two pyrimidines. The purine-purine
RNA coding for a gene product; gray segments represent noncoding interaction is so strong that a pyrimidine separating
RNA. In the polycistronic transcript, noncoding RNA separates the three two purines is often displaced from the stacking
genes. pattern so that the purines can interact. Any self-com-
plementary sequences in the molecule produce more
complex structures. RNA can base-pair with comple-
the name “messenger RNA” (mRNA) for that portion of mentary regions of either RNA or DNA. Base pairing
the total cellular RNA carrying the genetic information matches the pattern for DNA: G pairs with C and A
from DNA to the ribosomes, where the messengers pro- pairs with U (or with the occasional T residue in some
vide the templates that specify amino acid sequences in RNAs). One difference is that base pairing between G
polypeptide chains. Although mRNAs from different and U residues—unusual in DNA—is allowed in RNA
genes can vary greatly in length, the mRNAs from a (see Fig. 8–24) when complementary sequences in two
particular gene generally have a defined size. The pro- single strands of RNA pair with each other. The paired
cess of forming mRNA on a DNA template is known as strands in RNA or RNA-DNA duplexes are antiparallel,
transcription. as in DNA.
In bacteria and archaea, a single mRNA molecule When two strands of RNA with perfectly comple-
may code for one or several polypeptide chains. If it car- mentary sequences are paired, the predominant
ries the code for only one polypeptide, the mRNA is double-stranded structure is an A-form right-handed
monocistronic; if it codes for two or more different double helix. However, strands of RNA that are per-
polypeptides, the mRNA is polycistronic. In eukary- fectly paired over long regions of sequence are uncom-
otes, most mRNAs are monocistronic. (For the purposes mon. The three-dimensional structures of many RNAs,
of this discussion, “cistron” refers to a gene. The term like those of proteins, are complex and unique. Weak
itself has historical roots in the science of genetics, and
its formal genetic definition is beyond the scope of this
text.) The minimum length of an mRNA is set by the
length of the polypeptide chain for which it codes. For
example, a polypeptide chain of 100 amino acid resi-
dues requires an RNA coding sequence of at least 300
nucleotides, because each amino acid is coded by a
nucleotide triplet (this and other details of protein syn-
thesis are discussed in Chapter 27). However, mRNAs
transcribed from DNA are always somewhat longer than
the length needed simply to code for a polypeptide
sequence (or sequences). The additional, noncoding
RNA includes sequences that regulate protein synthe-
sis. Figure 8–21 summarizes the general structure of
bacterial mRNAs.

Many RNAs Have More Complex

Three-Dimensional Structures
Messenger RNA is only one of several classes of cellular
RNA. Transfer RNAs are adapter molecules in protein
synthesis; covalently linked to an amino acid at one end,
they pair with the mRNA in such a way that amino acids
are joined to a growing polypeptide in the correct
sequence. Ribosomal RNAs are components of ribo- FIGURE 8–22 Typical right-handed stacking pattern of single-stranded
somes. There is also a wide variety of special-function RNA. The bases are shown in yellow, the phosphorus atoms in orange,
RNAs, including some (called ribozymes) that have and the riboses and phosphate oxygens in green. Green is used to repre-
enzymatic activity. All the RNAs are considered in detail sent RNA strands in succeeding chapters, just as blue is used for DNA.
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8.2 Nucleic Acid Structure 295

interactions, especially base-stacking interactions, Hairpin

help stabilize RNA structures, just as they do in DNA. Single A C
Z-form helices have been made in the laboratory strands Internal loop A
Bulge U A
G A C
(under very high-salt or high-temperature conditions). C A U A C A
A UG CUA
The B form of RNA has not been observed. Breaks in ACCU CC CGU
U AG
G
the regular A-form helix caused by mismatched or G
A
unmatched bases in one or both strands are common G C G A UGG
GGA

G
U G C A

C
C
and result in bulges or internal loops (Fig. 8–23). A

C
G G

C
U A
Hairpin loops form between nearby self-complementary

G
G
(palindromic) sequences. The potential for base- (a)
paired helical segments in many RNAs is extensive
(Fig. 8–24), and the resulting hairpins are the most
common type of secondary structure in RNA. Specific
short base sequences (such as UUCG) are often found
at the ends of RNA hairpins and are known to form
particularly tight and stable loops. Such sequences
may act as starting points for the folding of an RNA
molecule into its precise three-dimensional structure. Hairpin double helix
Other contributions are made by hydrogen bonds that (b)
are not part of standard Watson-Crick base pairs. For
FIGURE 8–23 Secondary structure of RNAs. (a) Bulge, internal loop,
example, the 29-hydroxyl group of ribose can hydro-
and hairpin loop. (b) The paired regions generally have an A-form right-
gen-bond with other groups. Some of these properties
handed helix, as shown for a hairpin (derived from PDB ID 1GID).
are evident in the structure of the phenylalanine trans-
fer RNA of yeast—the tRNA responsible for inserting
Phe residues into polypeptides—and in two RNA G CA
C
A 160
enzymes, or ribozymes, whose functions, like those of G G
C C
C G
G C G
GU G
A A
FIGURE 8–24 Base-paired helical structures in an RNA. Shown here is G
C
U
C A
the possible secondary structure of the M1 RNA component of the C G
140 G G
enzyme RNase P of E. coli, with many hairpins. RNase P, which also con- C U A
A
AGC AAC GA
tains a protein component (not shown), functions in the processing of AG G
G G
A U
C G 180
transfer RNAs (see Fig. 26–26). The two brackets indicate additional A A
A A
G U A
complementary sequences that may be paired in the three dimensional G
C A
G CG A
UG
U GG
structure. The blue dots indicate non-Watson-Crick GPU base pairs G C G
ACG G A
A CC
(boxed inset). Note that GPU base pairs are allowed only when presyn- C G 200
C CGC
thesized strands of RNA fold up or anneal with each other. There are no A 100 GG C
UG G
G C A GUG U
RNA polymerases (the enzymes that synthesize RNAs on a DNA tem- 12 0 C G A G CC
GAC
A
A C C 220 U A
U
plate) that insert a U opposite a template G, or vice versa, during RNA C
C C G
C
A
C U G G
A G
synthesis. A G
G G G
G C
A
U
G
A G CG A A
CC UCA
U A
G C
G C 240
GCA AA
G C G
G U G UACG C
G 260 G
AUA C G A U AG U CA C
G A G G U
A U G C
C G C
80 U A A GU
G C AA 280
C C C C
CAAG C G
GG G G A
G
G
C C U GG
C U
U G 300
G C
A U
A U
A G A
G GC
G CA
U
O 60 A
G
A C CG
GU
GU
GA
G
G G 20 G
A CG C
G AG C G U A A G U UU
C G C 330 A G
GG C G
HON G U UC G
C C U A
GA UG
N O N GA GC
G C
A U
GG CU
GG U C G
O C UC
C G 1
A U
U UC
N NOH Uracil
GAAGC UGA CC AG CC
A
40 C
CU U UGAC UGG G
N A C A
C U C
2NH C A A
U 377 360 U G
Guanine U
C A
A
GG CC
C
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296 Nucleotides and Nucleic Acids

D Ribose
O
D !
Cytosine OPPOO
G
H O
D G Ribose
Ribose N N H
G A Ribose
N H
HON N N
O H O
H N N "
N
N N H G
O CH 3
H N
N 7-Methylguanine
Guanine
Ribose
(a)

H
Adenine G
N NOH O N

N N HON N
Ribose N N Ribose
N 2
G N -Dimethylguanine
CH 3 CH 3
(b)

O Uracil

N
Ribose E
H Ribose 2#
O
N N
O
H i
D H
N N
G
N H N
Adenine N NH2

Adenine
N N
Ribose
(c)

FIGURE 8–25 Three-dimensional structure in RNA. (a) Three-dimensional Ribozymes, or RNA enzymes, catalyze a variety of reactions, primarily in
structure of phenylalanine tRNA of yeast (PDB ID 1TRA). Some unusual RNA metabolism and protein synthesis. The complex three-dimensional
base-pairing patterns found in this tRNA are shown. Note also the structures of these RNAs reflect the complexity inherent in catalysis,
involvement of the oxygen of a ribose phosphodiester bond in one as described for protein enzymes in Chapter 6. (c) A segment of mRNA
hydrogen-bonding arrangement, and a ribose 29-hydroxyl group in known as an intron, from the ciliated protozoan Tetrahymena thermophila
another (both in red). (b) A hammerhead ribozyme (so named because (derived from PDB ID 1GRZ). This intron (a ribozyme) catalyzes its
the secondary structure at the active site looks like the head of a hammer), own excision from between exons in an mRNA strand (discussed in
derived from certain plant viruses (derived from PDB ID 1MME). Chapter 26).

protein enzymes, depend on their three-dimensional SUMMARY 8.2 Nucleic Acid Structure
structures (Fig. 8–25).
The analysis of RNA structure and the relationship ! Many lines of evidence show that DNA bears
between its structure and its function is an emerging genetic information. Some of the earliest evidence
field of inquiry that has many of the same complexities came from the Avery-MacLeod-McCarty
as the analysis of protein structure. The importance of experiment, which showed that DNA isolated from
understanding RNA structure grows as we become one bacterial strain can enter and transform the
increasingly aware of the large number of functional cells of another strain, endowing it with some of
roles for RNA molecules. the inheritable characteristics of the donor. The
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8.3 Nucleic Acid Chemistry 297

Hershey-Chase experiment showed that the DNA bonds between paired bases and of base stacking causes
of a bacterial virus, but not its protein coat, carries unwinding of the double helix to form two single
the genetic message for replication of the virus in strands, completely separate from each other along the
a host cell. entire length or part of the length (partial denaturation)
! Putting together the available data, Watson and of the molecule. No covalent bonds in the DNA are
Crick postulated that native DNA consists of two broken (Fig. 8–26).
antiparallel chains in a right-handed double-helical Renaturation of a DNA molecule is a rapid one-step
arrangement. Complementary base pairs, APT process, as long as a double-helical segment of a dozen or
and GqC, are formed by hydrogen bonding within more residues still unites the two strands. When the tem-
the helix. The base pairs are stacked perpendicular perature or pH is returned to the range in which most
to the long axis of the double helix, 3.4 Å apart, organisms live, the unwound segments of the two strands
with 10.5 base pairs per turn. spontaneously rewind, or anneal, to yield the intact
duplex (Fig. 8–26). However, if the two strands are com-
! DNA can exist in several structural forms. Two pletely separated, renaturation occurs in two steps. In
variations of the Watson-Crick form, or B-DNA, are the first, relatively slow step, the two strands “find” each
A- and Z-DNA. Some sequence-dependent other by random collisions and form a short segment of
structural variations cause bends in the DNA complementary double helix. The second step is much
molecule. DNA strands with appropriate sequences faster: the remaining unpaired bases successively come
can form hairpin or cruciform structures or triplex into register as base pairs, and the two strands “zipper”
or tetraplex DNA. themselves together to form the double helix.
! Messenger RNA transfers genetic information from The close interaction between stacked bases in a
DNA to ribosomes for protein synthesis. Transfer nucleic acid has the effect of decreasing its absorption
RNA and ribosomal RNA are also involved in of UV light relative to that of a solution with the same
protein synthesis. RNA can be structurally concentration of free nucleotides, and the absorption is
complex; single RNA strands can fold into hairpins, decreased further when two complementary nucleic
double-stranded regions, or complex loops. acid strands are paired. This is called the hypochromic

8.3 Nucleic Acid Chemistry

The role of DNA as a repository of genetic information
depends in part on its inherent stability. The chemical
transformations that do occur are generally very slow in Double-helical DNA
the absence of an enzyme catalyst. The long-term stor-
age of information without alteration is so important to
Annealing Denaturation
a cell, however, that even very slow reactions that alter
DNA structure can be physiologically significant. Pro-
cesses such as carcinogenesis and aging may be inti-
mately linked to slowly accumulating, irreversible alter-
ations of DNA. Other, nondestructive alterations also
occur and are essential to function, such as the strand Partially denatured DNA
separation that must precede DNA replication or tran-
scription. In addition to providing insights into physio-
logical processes, our understanding of nucleic acid
Association of
chemistry has given us a powerful array of technologies strands by base
Separation
that have applications in molecular biology, medicine, of strands
pairing
and forensic science. We now examine the chemical
properties of DNA and some of these technologies.

Double-Helical DNA and RNA Can Be Denatured

Solutions of carefully isolated, native DNA are highly Separated strands
of DNA in random coils
viscous at pH 7.0 and room temperature (25 8C). When
such a solution is subjected to extremes of pH or to
temperatures above 80 8C, its viscosity decreases sharply,
indicating that the DNA has undergone a physical
change. Just as heat and extremes of pH denature
globular proteins, they also cause denaturation, or melt- FIGURE 8–26 Reversible denaturation and annealing (renaturation) of
ing, of double-helical DNA. Disruption of the hydrogen DNA.
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298 Nucleotides and Nucleic Acids

effect. Denaturation of a double-stranded nucleic acid

produces the opposite result: an increase in absorption
called the hyperchromic effect. The transition from
double-stranded DNA to the single-stranded, denatured
form can thus be detected by monitoring UV absorption
at 260 nm.
Viral or bacterial DNA molecules in solution dena-
ture when they are heated slowly (Fig. 8–27). Each
species of DNA has a characteristic denaturation tem-
perature, or melting point (tm; formally, the tempera-
ture at which half the DNA is present as separated sin-
gle strands): the higher its content of GqC base pairs,
the higher the melting point of the DNA. This is because
GqC base pairs, with three hydrogen bonds, require 3 !m
more heat energy to dissociate than APT base pairs.
Thus the melting point of a DNA molecule, determined FIGURE 8–28 Partially denatured DNA. This DNA was partially dena-
tured, then fixed to prevent renaturation during sample preparation. The
under fixed conditions of pH and ionic strength, can
shadowing method used to visualize the DNA in this electron micro-
yield an estimate of its base composition. If denatur-
graph increases its diameter approximately fivefold and obliterates most
ation conditions are carefully controlled, regions that
details of the helix. However, length measurements can be obtained, and
are rich in APT base pairs will specifically denature
single-stranded regions are readily distinguishable from double-stranded
while most of the DNA remains double-stranded. Such regions. The arrows point to some single-stranded bubbles where dena-
turation has occurred. The regions that denature are highly reproducible
and are rich in APT base pairs.

100

denatured regions (called bubbles) can be visualized

with electron microscopy (Fig. 8–28). Note that in the
Denaturation (%)

strand separation of DNA that occurs in vivo during

tm tm
processes such as DNA replication and transcription,
50
the sites where these processes are initiated are often
rich in APT base pairs, as we shall see.
Duplexes of two RNA strands or one RNA strand
and one DNA strand (RNA-DNA hybrids) can also be
denatured. Notably, RNA duplexes are more stable to
0
75 80 85 heat denaturation than DNA duplexes. At neutral pH,
(a) Temperature (°C) denaturation of a double-helical RNA often requires
temperatures 20 8C or more higher than those required
for denaturation of a DNA molecule with a comparable
100
sequence, assuming the strands in each molecule are
perfectly complementary. The stability of an RNA-DNA
G + C (% of total nucleotides)

80 hybrid is generally intermediate between that of RNA

and DNA duplexes. The physical basis for these differ-
60 ences in thermal stability is not known.

40 Nucleic Acids from Different Species Can

Form Hybrids
20 The ability of two complementary DNA strands to pair
with one another can be used to detect similar DNA
0 sequences in two different species or within the genome
60 70 80 90 100 110
of a single species. If duplex DNAs isolated from human
(b) tm (°C)
cells and from mouse cells are completely denatured by
FIGURE 8–27 Heat denaturation of DNA. (a) The denaturation, or heating, then mixed and kept at about 25 8C below their
melting, curves of two DNA specimens. The temperature at the mid- tm for many hours, much of the DNA will anneal. The
point of the transition (tm) is the melting point; it depends on pH and rate of DNA annealing is affected by temperature, the
ionic strength and on the size and base composition of the DNA. length and concentration of the DNA fragments being
(b) Relationship between tm and the G1C content of a DNA. annealed, the concentration of salts in the reaction
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8.3 Nucleic Acid Chemistry 299

mixture, and properties of the sequence itself (e.g., essential to the practice of modern molecular genetics.
complexity and GqC content). Temperature is espe- A specific DNA sequence or gene can be detected in
cially important. If the temperature is too low, short the presence of many other sequences if one already
sequences with coincidental similarity from distant, has an appropriate complementary DNA strand (usu-
heterologous parts of the DNA molecules will anneal ally labeled in some way) to hybridize with it (Chapter 9).
unproductively and interfere with the more general The complementary DNA can be from a different spe-
alignment of complementary DNA strands. Tempera- cies or from the same species, or it can be synthesized
tures that are too high will favor denaturation. Most of chemically in the laboratory using techniques described
the reannealing occurs between complementary mouse later in this chapter. Hybridization techniques can be
DNA strands to form mouse duplex DNA; similarly, varied to detect a specific RNA rather than DNA. The
most human DNA strands anneal with complementary isolation and identification of specific genes and
human DNA strands. However, some strands of the RNAs rely on these hybridization techniques. Appli-
mouse DNA will associate with human DNA strands to cations of this technology make possible the identifi-
yield hybrid duplexes, in which segments of a mouse cation of an individual on the basis of a single hair left
DNA strand form base-paired regions with segments of at the scene of a crime or the prediction of the onset
a human DNA strand (Fig. 8–29). This reflects a com- of a disease decades before symptoms appear (see
mon evolutionary heritage; different organisms gener- Box 9–1).
ally have many proteins and RNAs with similar func-
tions and, often, similar structures. In many cases, the
DNAs encoding these proteins and RNAs have similar
Nucleotides and Nucleic Acids Undergo
sequences. The closer the evolutionary relationship Nonenzymatic Transformations
between two species, the more extensively their DNAs Purines and pyrimidines, along with the nucle-
will hybridize. For example, human DNA hybridizes otides of which they are a part, undergo
much more extensively with mouse DNA than with DNA spontaneous alterations in their covalent structure.
from yeast. The rate of these reactions is generally very slow, but
The hybridization of DNA strands from different they are physiologically significant because of the
sources forms the basis for a powerful set of techniques cell’s very low tolerance for alterations in its genetic
information. Alterations in DNA structure that pro-
duce permanent changes in the genetic information
encoded therein are called mutations, and much evi-
dence suggests an intimate link between the accumu-
lation of mutations in an individual organism and the
process of aging and carcinogenesis.
Several nucleotide bases undergo spontaneous
loss of their exocyclic amino groups (deamination)
(Fig. 8–30a). For example, under typical cellular con-
ditions, deamination of cytosine (in DNA) to uracil
Hybrid occurs in about one of every 107 cytidine residues in 24
Sample 1 Mix duplex hours. This corresponds to about 100 spontaneous
and cool Duplex of
sample 1
events per day, on average, in a mammalian cell.
Duplex of
Deamination of adenine and guanine occurs at about
sample 2 1/100th this rate.
The slow cytosine deamination reaction seems
innocuous enough, but is almost certainly the reason
why DNA contains thymine rather than uracil. The
product of cytosine deamination (uracil) is readily
recognized as foreign in DNA and is removed by a
Sample 2 repair system (Chapter 25). If DNA normally con-
FIGURE 8–29 DNA hybridization. Two DNA samples to be compared
tained uracil, recognition of uracils resulting from
are completely denatured by heating. When the two solutions are
cytosine deamination would be more difficult, and
mixed and slowly cooled, DNA strands of each sample associate with unrepaired uracils would lead to permanent sequence
their normal complementary partner and anneal to form duplexes. If changes as they were paired with adenines during rep-
the two DNAs have significant sequence similarity, they also tend to lication. Cytosine deamination would gradually lead to
form partial duplexes or hybrids with each other: the greater the a decrease in GqC base pairs and an increase in APU
sequence similarity between the two DNAs, the greater the number of base pairs in the DNA of all cells. Over the millennia,
hybrids formed. Hybrid formation can be measured in several ways. cytosine deamination could eliminate GqC base pairs
One of the DNAs is usually labeled with a radioactive isotope to sim- and the genetic code that depends on them. Establish-
plify their detection and measurement. ing thymine as one of the four bases in DNA may well
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300 Nucleotides and Nucleic Acids

O
NH2 O
N
HN
N HN
H 2N N N
O N O N O"
O P O CH2 O Guanosine residue
Cytosine Uracil (in DNA)
O H H
H H

NH2 O O H
CH 3 CH 3
N HN
H2 O

O N O N O
O"
N
HN
5-Methylcytosine Thymine # O P O CH2 O OH
H2N N N
H O H H
H H
Guanine
NH 2 O O H
N N
N HN
Apurinic residue

N N N N
(b) Depurination
Adenine Hypoxanthine
FIGURE 8–30 Some well-characterized nonenzymatic reactions of
nucleotides. (a) Deamination reactions. Only the base is shown.
O O (b) Depurination, in which a purine is lost by hydrolysis of the N-!-
glycosyl bond. Loss of pyrimidines via a similar reaction occurs, but
N N
HN HN much more slowly. The resulting lesion, in which the deoxyribose is
present but the base is not, is called an abasic site or an AP site (apu-
H2 N N N O N N
rinic site or, rarely, apyrimidinic site). The deoxyribose remaining after
H
depurination is readily converted from the !-furanose to the aldehyde
Guanine Xanthine
form (see Fig. 8–3), further destabilizing the DNA at this position.
(a) Deamination More nonenzymatic reactions are illustrated in Figures 8–31 and 8–32.

have been one of the crucial turning points in evolu- on the same DNA strand (Fig. 8–31). A second type of
tion, making the long-term storage of genetic informa- pyrimidine dimer, called a 6-4 photoproduct, is also
tion possible. formed during UV irradiation. Ionizing radiation (x rays
Another important reaction in deoxyribonucleo- and gamma rays) can cause ring opening and fragmen-
tides is the hydrolysis of the N-!-glycosyl bond tation of bases as well as breaks in the covalent back-
between the base and the pentose, to create a DNA bone of nucleic acids.
lesion called an AP (apurinic, apyrimidinic) site or Virtually all forms of life are exposed to energy-
abasic site (Fig. 8–30b). This occurs at a higher rate rich radiation capable of causing chemical changes in
for purines than for pyrimidines. As many as one in DNA. Near-UV radiation (with wavelengths of 200 to
105 purines (10,000 per mammalian cell) are lost from 400 nm), which makes up a significant portion of the
DNA every 24 hours under typical cellular conditions. solar spectrum, is known to cause pyrimidine dimer
Depurination of ribonucleotides and RNA is much formation and other chemical changes in the DNA of
slower and generally is not considered physiologically bacteria and of human skin cells. We are subject to a
significant. In the test tube, loss of purines can be constant field of ionizing radiation in the form of cos-
accelerated by dilute acid. Incubation of DNA at pH 3 mic rays, which can penetrate deep into the earth, as
causes selective removal of the purine bases, resulting well as radiation emitted from radioactive elements,
in a derivative called apurinic acid. such as radium, plutonium, uranium, radon, 14C, and
3
Other reactions are promoted by radiation. UV light H. X rays used in medical and dental examinations
induces the condensation of two ethylene groups to and in radiation therapy of cancer and other diseases
form a cyclobutane ring. In the cell, the same reaction are another form of ionizing radiation. It is estimated
between adjacent pyrimidine bases in nucleic acids that UV and ionizing radiations are responsible for
forms cyclobutane pyrimidine dimers. This happens about 10% of all DNA damage caused by environmen-
most frequently between adjacent thymidine residues tal agents.
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8.3 Nucleic Acid Chemistry 301

O H
C N
N C O
C C
P H CH3
Adjacent
thymines
O H
C N
N C O
C C
H CH3
UV light UV light

A
T
O H O H Kink
A
C N C N T
N 6 5
C O N 6
C O
C C C C
P H CH3 P H CH3
OH

O H O H
C N C N
N 6 5
C O N 4 C OH
C C C C
H CH3 H CH3

Cyclobutane thymine dimer 6-4 Photoproduct

(a) (b)

FIGURE 8–31 Formation of pyrimidine dimers induced by UV light. linkage between C-6 of one pyrimidine and C-4 of its neighbor.
(a) One type of reaction (on the left) results in the formation of a cyclo- (b) Formation of a cyclobutane pyrimidine dimer introduces a bend or
butyl ring involving C-5 and C-6 of adjacent pyrimidine residues. An kink into the DNA (PDB ID 1TTD).
alternative reaction (on the right) results in a 6-4 photoproduct, with a

DNA also may be damaged by reactive chemicals salts, is a potent accelerator of the deamination of
introduced into the environment as products of indus- bases. Bisulfite has similar effects. Both agents are
trial activity. Such products may not be injurious per se used as preservatives in processed foods to prevent
but may be metabolized by cells into forms that are. the growth of toxic bacteria. They do not seem to
There are two prominent classes of such agents (Fig. increase cancer risks significantly when used in this
8–32): (1) deaminating agents, particularly nitrous acid way, perhaps because they are used in small amounts
(HNO2) or compounds that can be metabolized to and make only a minor contribution to the overall lev-
nitrous acid or nitrites, and (2) alkylating agents. els of DNA damage. (The potential health risk from
Nitrous acid, formed from organic precursors food spoilage if these preservatives were not used is
such as nitrosamines and from nitrite and nitrate much greater.)

FIGURE 8–32 Chemical agents that cause

DNA damage. (a) Precursors of nitrous COO" CH3
G
O O O O O

#
acid, which promotes deamination reactions. H3NO C O H O O
NH2 G J
(b) Alkylating agents. Only S-adenosylmethi- CH3 S
onine acts enzymatically. CH2 N G D M
methionine N NONPO O O
CH2 D D
N CH3 CH3
NaNO2 NaNO3 N
#
S OO CH2 O Dimethylnitrosamine Dimethylsulfate
Sodium nitrite Sodium nitrate
CH3
H H
R1 H H CH2 O CH2 OCl
G D
NONPO OH OH H3C ON
D G
R 2 adenosine CH2 O CH2 O Cl
Nitrosamine S-Adenosylmethionine Nitrogen mustard

(a) Nitrous acid precursors (b) Alkylating agents

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302 Nucleotides and Nucleic Acids

Alkylating agents can alter certain bases of DNA. For has two prominent methylation systems. One serves as
example, the highly reactive chemical dimethylsulfate part of a defense mechanism that helps the cell to dis-
(Fig. 8–32b) can methylate a guanine to yield O6-methyl- tinguish its DNA from foreign DNA by marking its own
guanine, which cannot base-pair with cytosine. DNA with methyl groups and destroying (foreign) DNA
without the methyl groups (this is known as a restriction-
O
modification system; see p. 314). The other system
6 N
HN methylates adenosine residues within the sequence
N
(59)GATC(39) to N6-methyladenosine (Fig. 8–5a). This
H2N N
H is mediated by the Dam (DNA adenine methylation)
Guanine methylase, a component of a system that repairs mis-
tautomers matched base pairs formed occasionally during DNA
OH OCH3 replication (see Fig. 25–21).
N N In eukaryotic cells, about 5% of cytidine residues in
N 6 (CH3)2SO4 N 6
DNA are methylated to 5-methylcytidine (Fig. 8–5a).
H2N N N H2N N N Methylation is most common at CpG sequences, produc-
H H ing methyl-CpG symmetrically on both strands of the
O6-Methylguanine DNA. The extent of methylation of CpG sequences varies
by molecular region in large eukaryotic DNA molecules.
Many similar reactions are brought about by alkylating
agents normally present in cells, such as S-adenosyl
methionine. The Sequences of Long DNA Strands Can Be Determined
The most important source of mutagenic alterations In its capacity as a repository of information, a DNA
in DNA is oxidative damage. Excited-oxygen species molecule’s most important property is its nucleotide
such as hydrogen peroxide, hydroxyl radicals, and sequence. Until the late 1970s, determining the
superoxide radicals arise during irradiation or as a sequence of a nucleic acid containing even five or ten
byproduct of aerobic metabolism. Of these species, the nucleotides was very laborious. The development of two
hydroxyl radicals are responsible for most oxidative new techniques in 1977, one by Alan Maxam and Walter
DNA damage. Cells have an elaborate defense system to Gilbert and the other by Frederick Sanger, made possi-
destroy reactive oxygen species, including enzymes such ble the sequencing of larger DNA molecules with an
as catalase and superoxide dismutase that convert reac- ease unimagined just a few years before. The tech-
tive oxygen species to harmless products. A fraction of niques depend on an improved understanding of nucle-
these oxidants inevitably escape cellular defenses, how- otide chemistry and DNA metabolism, and on electro-
ever, and damage to DNA occurs through any of a large, phoretic methods for separating DNA strands differing
complex group of reactions ranging from oxidation of in size by only one nucleotide. Electrophoresis of DNA
deoxyribose and base moieties to strand breaks. Accu- is similar to that of proteins (see Fig. 3–18). Polyacryl-
rate estimates for the extent of this damage are not yet amide is often used as the gel matrix in work with short
available, but every day the DNA of each human cell is DNA molecules (up to a few hundred nucleotides); aga-
subjected to thousands of damaging oxidative reactions. rose is generally used for longer pieces of DNA.
This is merely a sampling of the best-understood In both Sanger and Maxam-Gilbert sequencing, the
reactions that damage DNA. Many carcinogenic com- general principle is to reduce the DNA to four sets of
pounds in food, water, or air exert their cancer-causing labeled fragments. The reaction producing each set is
effects by modifying bases in DNA. Nevertheless, the base-specific, so the lengths of the fragments corre-
integrity of DNA as a polymer is better maintained than spond to positions in the DNA sequence where a certain
that of either RNA or protein, because DNA is the only base occurs. For example, for an oligonucleotide with
macromolecule that has the benefit of extensive bio- the sequence pAATCGACT, labeled at the 59 end (the
chemical repair systems. These repair processes left end), a reaction that breaks the DNA after each C
(described in Chapter 25) greatly lessen the impact of residue will generate two labeled fragments: a four-
damage to DNA. ■ nucleotide and a seven-nucleotide fragment; a reaction
that breaks the DNA after each G will produce only one
Some Bases of DNA Are Methylated labeled, five-nucleotide fragment. Because the frag-
Certain nucleotide bases in DNA molecules are enzy- ments are radioactively labeled at their 59 ends, only the
matically methylated. Adenine and cytosine are methyl- fragment to the 59 side of the break is visualized. The
ated more often than guanine and thymine. Methylation fragment sizes correspond to the relative positions of C
is generally confined to certain sequences or regions of and G residues in the sequence. When the sets of frag-
a DNA molecule. In some cases the function of methyla- ments corresponding to each of the four bases are elec-
tion is well understood; in others the function remains trophoretically separated side by side, they produce a
unclear. All known DNA methylases use S-adenosylme- ladder of bands from which the sequence can be read
thionine as a methyl group donor (Fig. 8–32b). E. coli directly (Fig. 8–33). We illustrate only the Sanger
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8.3 Nucleic Acid Chemistry 303

P FIGURE 8–33 DNA sequencing by the Sanger method. This method

P makes use of the mechanism of DNA synthesis by DNA polymerases
P
dATP (Chapter 25). (a) DNA polymerases require both a primer (a short
P P
oligonucleotide strand), to which nucleotides are added, and a tem-
dGTP
P plate strand to guide selection of each new nucleotide. In cells, the
5$ OH
39-hydroxyl group of the primer reacts with an incoming deoxynucleo-
P P P P OH OH
Primer side triphosphate (dNTP) to form a new phosphodiester bond. (b) The
strand Sanger sequencing procedure uses dideoxynucleoside triphosphate
A
(ddNTP) analogs to interrupt DNA synthesis. (The Sanger method is
T G C C G
also known as the dideoxy method.) When a ddNTP is inserted in
3$ A C G G C T A C T place of a dNTP, strand elongation is halted after the analog is added,
because it lacks the 39-hydroxyl group needed for the next step. (c)
Template The DNA to be sequenced is used as the template strand, and a short
strand
P P P P P P P P P primer, radioactively or fluorescently labeled, is annealed to it. By addi-
(a) tion of small amounts of a single ddNTP, for example ddCTP, to an
otherwise normal reaction system, the synthesized strands will be pre-
maturely terminated at some locations where dC normally occurs.
Given the excess of dCTP over ddCTP, the chance that the analog will
O" O" O"
be incorporated whenever a dC is to be added is small. However,
"
O P O P O P O CH2 Base ddCTP is present in sufficient amounts to ensure that each new strand
O
has a high probability of acquiring at least one ddC at some point during
O O O
H H synthesis. The result is a solution containing a mixture of labeled frag-
H H ments, each ending with a C residue. Each C residue in the sequence
H H generates a set of fragments of a particular length, such that the different-
(b) ddNTP analog sized fragments, separated by electrophoresis, reveal the location of C
residues. This procedure is repeated separately for each of the four
ddNTPs, and the sequence can be read directly from an autoradiogram
of the gel. Because shorter DNA fragments migrate faster, the frag-
Primer ments near the bottom of the gel represent the nucleotide positions
5! OH closest to the primer (the 59 end), and the sequence is read (in the
59S39 direction) from bottom to top. Note that the sequence obtained
3! CTAAGCTCGACT
is that of the strand complementary to the strand being analyzed.
Template
"
dCTP, dGTP, dATP, dTTP

+ ddATP + ddCTP + ddGTP + ddTTP

GATTCGAGCTGddA GATTCGAGddC GATTCGAGCTddG GATTCGAGCddT
GATTCGddA GATTddC GATTCGAddG GATddT
GddA GATTCddG GAddT
ddG

A C G T
3!
12 A
11 G
10 T
9 C
8 G
7 A
6 G
5 C
4 T
3 T
2 A
1 G

Autoradiogram of Sequence of
electrophoresis gel complementary
(c) strand
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304 Nucleotides and Nucleic Acids

method, because it has proved to be technically easier Primer

and is in more widespread use. It requires the enzy- 3! Template of
unknown sequence
matic synthesis of a DNA strand complementary to the
strand under analysis, using a radioactively labeled
“primer” and dideoxynucleotides.
Since these first practical DNA-sequencing meth-
ods appeared, methodology has improved rapidly.
Much of the advance has been fueled by the Human
DNA polymerase,
Genome Project, described in Chapter 9. A variation of four dNTPs,
Sanger’s sequencing method, in which the dideoxynu- four ddNTPs
cleotides used for each reaction are labeled with a dif-
ferently colored fluorescent tag (Fig. 8–34), was used
in early efforts to automate large DNA-sequencing A T
efforts. With this technology, researchers can sequence T
DNA molecules containing thousands of nucleotides in C
a few hours. This approach was heavily used in the G
G
initial efforts to sequence entire organism genomes,
and is still used for routine sequencing of genes or DNA
segments. However, modern genomic sequencing now
Denature
makes use of vastly more efficient methods, sometimes
referred to as next-generation or next-gen sequencing.
These are described in Chapter 9. Dideoxy Sequencing
A Dye-labeled
of DNA segments of DNA,
T
C copied from
The Chemical Synthesis of DNA Has Been Automated G
template with
unknown sequence
G
An important practical advance in nucleic acid chemis- T
try was the rapid and accurate synthesis of short oligo-
nucleotides of known sequence. The methods were
pioneered by H. Gobind Khorana and his colleagues in
the 1970s. Refinements by Robert Letsinger and Marvin
Caruthers led to the chemistry now in widest use, called
the phosphoramidite method (Fig. 8–35). The synthe-
Dye-labeled segments
sis is carried out with the growing strand attached to a applied to a capillary
solid support, using principles similar to those used by DNA gel and subjected to
Merrifield for peptide synthesis (see Fig. 3–32), and is migration electrophoresis
readily automated. The efficiency of each addition step
is very high, allowing the routine synthesis of polymers
containing 70 or 80 nucleotides and, in some laborato-
ries, much longer strands. The availability of relatively
inexpensive DNA polymers with predesigned sequences
is having a powerful impact on all areas of biochemistry
(Chapter 9).
Laser beam

Detector Laser

FIGURE 8–34 Strategy for automating DNA-sequencing reactions.

Each dideoxynucleotide used in the Sanger method can be linked to a
fluorescent molecule that gives all the fragments terminating in that
nucleotide a particular color. All four labeled ddNTPs are added to a
single tube. The resulting colored DNA fragments are then separated by
size in a single electrophoretic gel contained in a capillary tube (a refine-
ment of gel electrophoresis that allows for faster separations). All frag-
ments of a given length migrate through the capillary gel in a single
peak, and the color associated with each peak is detected using a laser
beam. The DNA sequence is read by determining the sequence of colors C C T G T T T G A T G G T G GT T CC G A A A T C G G
in the peaks as they pass the detector. This information is fed directly to Computer-generated result after
a computer, which determines the sequence. bands migrate past detector
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8.3 Nucleic Acid Chemistry 305

DMT

O
Nucleoside
5" CH2 Base1 protected
O at 5" hydroxyl DMT DMT
Nucleotide
H H activated
O at 3" position O
H H
3" CH2 Base2 CH2 Base2
OH H O O

Nucleoside H H H H
1 attached to H H H H
silica support Cyanoethyl
protecting group O H O H
DMT
NC (CH2)2 O P Phosphoramidite NC (CH2) 2 O P
O OH
(CH3)2CH N! CH(CH3)2 O
CH2 Base1 CH2 Base1 H
O O CH2 Base1
Diisopropylamino activating group O
H H DMT H H
H H H H H H
H H
O H 2 O H 3
O H
R Protecting R Next nucleotide
group removed added R
Si Si (CH3)2CH N CH(CH3)2
H Si
Diisopropylamine byproduct
4
Oxidation to
DMT form triester

O
CH2 Base2
Repeat steps 2 to 4 until all residues are added O

H H
H H
5 Remove protecting groups from bases
O H
6 Remove cyanoethyl groups from phosphates
NC (CH2)2 O P O
7 Cleave chain from silica support
O
CH2 Base1
5" 3" O

H H
Oligonucleotide chain H H

O H
R

Si

FIGURE 8–35 Chemical synthesis of DNA by the phosphoramidite replaced by an amino group or substituted amine. In the common variant
method. Automated DNA synthesis is conceptually similar to the syn- shown, one of the phosphoramidite oxygens is bonded to the deoxyri-
thesis of polypeptides on a solid support. The oligonucleotide is built up bose, the other is protected by a cyanoethyl group, and the third position
on the solid support (silica), one nucleotide at a time, in a repeated series is occupied by a readily displaced diisopropylamino group. Reaction with
of chemical reactions with suitably protected nucleotide precursors. the immobilized nucleotide forms a 59,39 linkage, and the diisopropyl-
1 The first nucleoside (which will be the 39 end) is attached to the silica amino group is eliminated. In step 4, the phosphite linkage is oxidized with
support at the 39 hydroxyl (through a linking group, R) and is protected at iodine to produce a phosphotriester linkage. Reactions 2 through 4 are
the 59 hydroxyl with an acid-labile dimethoxytrityl group (DMT). The repeated until all nucleotides are added. At each step, excess nucleotide
reactive groups on all bases are also chemically protected. 2 The pro- is removed before addition of the next nucleotide. In steps 5 and 6 the
tecting DMT group is removed by washing the column with acid (the remaining protecting groups on the bases and the phosphates are
DMT group is colored, so this reaction can be followed spectrophotomet- removed, and in 7 the oligonucleotide is separated from the solid sup-
rically). 3 The next nucleotide has a reactive phosphoramidite at its 39 port and purified. The chemical synthesis of RNA is somewhat more
position: a trivalent phosphite (as opposed to the more oxidized pentava- complicated because of the need to protect the 29 hydroxyl of ribose
lent phosphate normally present in nucleic acids) with one linked oxygen without adversely affecting the reactivity of the 39 hydroxyl.
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306 Nucleotides and Nucleic Acids

SUMMARY 8.3 Nucleic Acid Chemistry nucleoside triphosphates provides the chemical energy
!
to drive many cellular reactions. Adenosine 59-triphos-
Native DNA undergoes reversible unwinding and
phate, ATP, is by far the most widely used for this
separation of strands (melting) on heating or at
purpose, but UTP, GTP, and CTP are also used in some
extremes of pH. DNAs rich in GqC pairs have
reactions. Nucleoside triphosphates also serve as the
higher melting points than DNAs rich in APT
activated precursors of DNA and RNA synthesis, as
pairs.
described in Chapters 25 and 26.
! Denatured single-stranded DNAs from two The energy released by hydrolysis of ATP and the
species can form a hybrid duplex, the degree of other nucleoside triphosphates is accounted for by
hybridization depending on the extent of the structure of the triphosphate group. The bond
sequence similarity. Hybridization is the basis for between the ribose and the " phosphate is an ester
important techniques used to study and isolate linkage. The ",! and !,# linkages are phosphoanhy-
specific genes and RNAs. drides (Fig. 8–37). Hydrolysis of the ester linkage
! DNA is a relatively stable polymer. Spontaneous yields about 14 kJ/mol under standard conditions,
reactions such as deamination of certain bases, whereas hydrolysis of each anhydride bond yields about
hydrolysis of base-sugar N-glycosyl bonds, 30 kJ/mol. ATP hydrolysis often plays an important
radiation-induced formation of pyrimidine dimers, thermodynamic role in biosynthesis. When coupled to
and oxidative damage occur at very low rates, yet a reaction with a positive free-energy change, ATP
are important because of a cell’s very low hydrolysis shifts the equilibrium of the overall process
tolerance for changes in genetic material. to favor product formation (recall the relationship
!
between equilibrium constant and free-energy change
DNA sequences can be determined with a range of
described by Eqn 6–3 on p. 194).
modern methods.
! Oligonucleotides of known sequence can be
synthesized rapidly and accurately.
Adenine Nucleotides Are Components of Many
Enzyme Cofactors
8.4 Other Functions of Nucleotides A variety of enzyme cofactors serving a wide range of
In addition to their roles as the subunits of nucleic chemical functions include adenosine as part of their
acids, nucleotides have a variety of other functions in structure (Fig. 8–38). They are unrelated structurally
every cell: as energy carriers, components of enzyme except for the presence of adenosine. In none of these
cofactors, and chemical messengers. cofactors does the adenosine portion participate directly
in the primary function, but removal of adenosine gen-
erally results in a drastic reduction of cofactor activities.
Nucleotides Carry Chemical Energy in Cells For example, removal of the adenine nucleotide (39-phos-
The phosphate group covalently linked at the 59 hydroxyl phoadenosine diphosphate) from acetoacetyl-CoA, the
of a ribonucleotide may have one or two additional coenzyme A derivative of acetoacetate, reduces its reac-
phosphates attached. The resulting molecules are tivity as a substrate for !-ketoacyl-CoA transferase (an
referred to as nucleoside mono-, di-, and triphosphates enzyme of lipid metabolism) by a factor of 106. Although
(Fig. 8–36). Starting from the ribose, the three phos- this requirement for adenosine has not been investi-
phates are generally labeled ", !, and #. Hydrolysis of gated in detail, it must involve the binding energy between

$ # "
O! O! O!
!
O P O P O P O CH2 Base
O Abbreviations of ribonucleoside Abbreviations of deoxyribonucleoside
O O O 5%-phosphates 5%-phosphates
H H
H H
Base Mono- Di- Tri- Base Mono- Di- Tri-
OH OH
Adenine AMP ADP ATP Adenine dAMP dADP dATP
NMP
Guanine GMP GDP GTP Guanine dGMP dGDP dGTP
NDP Cytosine CMP CDP CTP Cytosine dCMP dCDP dCTP
Uracil UMP UDP UTP Thymine dTMP dTDP dTTP
NTP

FIGURE 8–36 Nucleoside phosphates. General structure of the nucleo- their standard abbreviations. In the deoxyribonucleoside phosphates
side 59-mono-, di-, and triphosphates (NMPs, NDPs, and NTPs) and (dNMPs, dNDPs, and dNTPs), the pentose is 29-deoxy-D-ribose.
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8.4 Other Functions of Nucleotides 307

Ester enzyme and substrate (or cofactor) that is used both in

O! O! O! catalysis and in stabilizing the initial enzyme-substrate
!
O P O P O P O CH2 Adenine complex (Chapter 6). In the case of !-ketoacyl-CoA
O transferase, the nucleotide moiety of coenzyme A seems
O O O to be a binding “handle” that helps to pull the substrate
H H
Anhydride
Anhydride
H H (acetoacetyl-CoA) into the active site. Similar roles may
be found for the nucleoside portion of other nucleotide
OH OH
ATP cofactors.
Why is adenosine, rather than some other large
H3C C O C CH3 H3C C O CH3 molecule, used in these structures? The answer here
O O O
may involve a form of evolutionary economy. Adenosine
Acetic anhydride, Methyl acetate,
is certainly not unique in the amount of potential bind-
a carboxylic acid a carboxylic acid ing energy it can contribute. The importance of adenos-
anhydride ester ine probably lies not so much in some special chemical
characteristic as in the evolutionary advantage of using
FIGURE 8–37 The phosphate ester and phosphoanhydride bonds of
one compound for multiple roles. Once ATP became the
ATP. Hydrolysis of an anhydride bond yields more energy than hydrolysis
universal source of chemical energy, systems developed
of the ester. A carboxylic acid anhydride and carboxylic acid ester are
shown for comparison.

Coenzyme A NH2
N
H H H CH3 O! O! N
5%
HS CH2 CH2 N C CH2 CH2 N C C C CH2 O P O P O CH2 N N
O
O O OH CH3 O O 4% 1%
H H
!-Mercaptoethylamine Pantothenic acid H H
3% 2%
O OH

FIGURE 8–38 Some coenzymes containing adenosine. The adenosine O P O!

portion is shaded in light red. Coenzyme A (CoA) functions in acyl O!
group transfer reactions; the acyl group (such as the acetyl or acetoacetyl
3%-Phosphoadenosine diphosphate
group) is attached to the CoA through a thioester linkage to the (3%-P-ADP)
!-mercaptoethylamine moiety. NAD1 functions in hydride transfers, and
FAD, the active form of vitamin B2 (riboflavin), in electron transfers. Another
coenzyme incorporating adenosine is 59-deoxyadenosylcobalamin, the O
active form of vitamin B12 (see Box 17–2), which participates in intramolec- H3C N
ular group transfers between adjacent carbons.
NH

H3C N N O
O
CH2 Riboflavin
C NH2
CHOH
Nicotinamide
&
CHOH
O CH2 N
O
CHOH
H H CH2
O P O! H H
O
OH OH
!O P O
O
O
NH2 !O NH2
O P O! P O
N N
N O N

O CH2 N N CH2 N N
O O

H H H H
H H H H

OH OH OH OH
Nicotinamide adenine dinucleotide (NAD") Flavin adenine dinucleotide (FAD)
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308 Nucleotides and Nucleic Acids

to synthesize ATP in greater abundance than the other the inner face of the plasma membrane. Cyclic AMP
nucleotides; because it is abundant, it becomes the serves regulatory functions in virtually every cell out-
logical choice for incorporation into a wide variety of side the plant kingdom. Guanosine 39,59-cyclic mono-
structures. The economy extends to protein structure. phosphate (cGMP) occurs in many cells and also has
A single protein domain that binds adenosine can be regulatory functions.
used in different enzymes. Such a domain, called a Another regulatory nucleotide, ppGpp (Fig. 8–39),
nucleotide-binding fold, is found in many enzymes is produced in bacteria in response to a slowdown in
that bind ATP and nucleotide cofactors. protein synthesis during amino acid starvation. This
nucleotide inhibits the synthesis of the rRNA and tRNA
Some Nucleotides Are Regulatory Molecules molecules (see Fig. 28–22) needed for protein synthe-
sis, preventing the unnecessary production of nucleic
Cells respond to their environment by taking cues from
acids.
hormones or other external chemical signals. The inter-
action of these extracellular chemical signals (“first
messengers”) with receptors on the cell surface often SUMMARY 8.4 Other Functions of Nucleotides
leads to the production of second messengers inside ! ATP is the central carrier of chemical energy in
the cell, which in turn leads to adaptive changes in the cells. The presence of an adenosine moiety in a
cell interior (Chapter 12). Often, the second messenger variety of enzyme cofactors may be related to
is a nucleotide (Fig. 8–39). One of the most common is binding-energy requirements.
adenosine 39,59-cyclic monophosphate (cyclic ! Cyclic AMP, formed from ATP in a reaction
AMP, or cAMP), formed from ATP in a reaction cata- catalyzed by adenylyl cyclase, is a common second
lyzed by adenylyl cyclase, an enzyme associated with messenger produced in response to hormones and
other chemical signals.

5"
O CH2 Adenine
O

H H
Key Terms
H H Terms in bold are defined in the glossary.
3"
O P O OH gene 281 major groove 289
O! ribosomal RNA minor groove 289
Adenosine 3",5"-cyclic monophosphate (rRNA) 281 B-form DNA 291
(cyclic AMP; cAMP) messenger RNA A-form DNA 291
5" (mRNA) 281 Z-form DNA 291
O CH2 Guanine
O transfer RNA palindrome 291
(tRNA) 281 hairpin 292
H H cruciform 292
H H
nucleotide 281
3" nucleoside 281 triplex DNA 292
O P O OH pyrimidine 282 G tetraplex 292
O! purine 282 transcription 294
Guanosine 3",5"-cyclic monophosphate deoxyribonucleotides 283 monocistronic
(cyclic GMP; cGMP) ribonucleotide 283 mRNA 294
phosphodiester polycistronic mRNA 294
O! O! linkage 285 mutation 299
5"
!O P O P O CH2 Guanine 59 end 285 second messenger 308
O
39 end 285 adenosine 39,59-cyclic
O O
H H oligonucleotide 286 monophosphate (cyclic
H H polynucleotide 286 AMP, cAMP) 308
3"
O OH base pair 287
A
!O P O
O
!O P O
Further Reading
O! General
Guanosine 5"-diphosphate,3"-diphosphate Cox, M.M., Doudna, J.A., & O’Donnell, M. (2012) Molecular
(guanosine tetraphosphate) Biology: Principles and Practice, W. H. Freeman and Company,
(ppGpp) New York.
The best place to start to learn more about nucleic acid structure
FIGURE 8–39 Three regulatory nucleotides. and function.