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The DNA double helix is an icon for modern biology, a form represented from art

galleries to corporate logos. In chemical terms, DNA is a polymer of building blocks called

nucleotides. Each nucleotide consists of a sugar, a phosphate, and a base—adenine (A), thymine

(T), cytosine (C), or guanine (G). The way in which the sugar, phosphate, and base are arranged

in a nucleotide and how nucleotides are joined together were known by 1953. What Watson

and Crick addressed was the way in which the two strands are arranged in space as a double

helix. DNA is often thought of as an invariant twisted ladder-like structure—a one dimensional

string of letters. This chapter introduces the important concept that DNA is a dynamic molecule

with a three-dimensional shape. Not surprisingly, the three-dimensional shape of DNA has

important consequences for its function. As a result, DNA can tip, propeller twist, buckle, roll,

tilt, stagger, stretch, shear, rise, slide, and shift! Before considering the flexibility of DNA, the

more familiar primary structure will be reviewed.

Primary structure: the components of nucleic acids:

The standard abbreviations for nucleic acids are now DNA and RNA, but before this was universally

accepted, these molecules went by many different names. For example, DNA was first called thymus

nucleic acid because it was isolated from the thymus gland of cattle, while RNA was first called yeast

nucleic acid because it was isolated from yeast. Nucleic acids are a long chain, or polymer, of repeating

subunits, called nucleotides. Each nucleotide subunit is composed of three parts: a five-carbon sugar,

a phosphate group, and a nitrogen-containing base. The chemical structures of these basic

components are shown in Fig. 2.1.

Five-carbon sugars

Nucleotide subunits of RNA contain a pentose (five-carbon) sugar called ribose, while nucleotide

subunits of DNA contain the sugar deoxyribose. These sugars differ only in the presence or absence

(“deoxy”) of oxygen on the second carbon in the ring. Yet this minor distinction between RNA and DNA

dramatically influences their function. The remarkable versatility of RNA is critically dependent on this

extra hydroxyl group. To help keep all the carbons straight, the five carbon atoms in each pentose

sugar are assigned numbers 1’ through 5’. Primes are used in the numbering of the ring positions in

the sugars to differentiate them from the ring positions of the bases. Both sugars have oxygen as a

member of the five-member ring; the 5’-carbon is outside the ring.

The Structure of DNA AND RNA Molecules

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Nitrogenous bases

As their name suggests, the nitrogenous bases are nitrogen-containing molecules having the chemical

properties of a base (a substance that accepts an H+ ion or proton in solution). Two of the bases—

adenine (A) and guanine (G)—have a double carbon–nitrogen ring structure; these are called purines.

The other three bases—thymine (T), cytosine (C), and uracil (U)—have a single-ring structure; these

are called pyrimidines. Thymine is found in DNA only, while uracil is specific for RNA.

The phosphate functional group

The phosphate functional group (PO4) gives DNA and RNA the property of an acid— a substance that

releases an H+ ion or proton in solution—at physiological pH, hence the name “nucleic acid.” The

linking bonds that are formed from phosphates are esters that have the additional property of being

stable yet easily broken by enzymatic hydrolysis. When a nucleotide is removed from a DNA or RNA

chain, the nucleotide is not destroyed in the process. Further, after the phosphodiester bond is formed

(see below), one oxygen atom of the phosphate group is still negatively ionized. The negatively charged

phosphates are extremely insoluble in lipids. Why is this important? Think for moment about the

composition of the membranes surrounding a cell or the nucleus of a eukaryotic cell. In all domains of

life, phospholipids are critically important components of cell membranes. Thus, the insolubility of

nucleic acids in lipids ensures that they are retained within the cell.

Nucleosides and nucleotides

Now that the individual parts have been introduced, let’s start assembling the building blocks. A DNA

or RNA chain is formed in a series of three steps (Fig. 2.3). In the first reaction, each nitrogenous base

is covalently linked to one molecule of sugar at the 1’-carbon of the sugar, forming a compound called

a nucleoside. When a phosphate group is also covalently attached to the 5’-carbon of the same sugar,

the nucleoside is now called a nucleotide. Finally, nucleotides are joined (polymerized) by

condensation reactions to form a chain. The hydroxyl group on the 3’-carbon of a sugar of one

nucleotide forms an ester bond to the phosphate of another nucleotide, eliminating a molecule of

water and releasing two phosphates in the form of pyrophosphate (P2O 7 4- ). This covalent bond linking

the sugar components of adjacent nucleotides is called a phosphodiester bond, or 5’ → 3’

phosphodiester bond, indicating the polarity of the strand. In this context, the term “polarity” means

that the ends of a DNA or RNA chain are distinct and have different chemical properties.

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Nomenclature of nucleotides

The rather complicated nomenclature of the nucleoside and nucleotide derivatives of the DNA and

RNA bases is summarized in Table 2.1. Nucleotides may contain one phosphate unit (monophosphate),

two such units (diphosphate), or three (triphosphate). When incorporated into a nucleic acid chain, a

nucleotide contains one each of the three components.

The length of RNA and DNA chains

Molecular biologists have come a long way from the early days of thinking that DNA was composed of

just four nucleotides per molecule. Cellular RNAs range in length from less than one hundred to many

thousands of nucleotides; the number of nucleotides (nt), or “bases,” is used as a measure of length.

Cellular DNA molecules can be as long as several hundred million nucleotides. The number of base

pairs (bp) is used as a measure of length of a double-stranded DNA. In practice, the unit of length used

for DNA is the kilobase pair (kb or kbp), corresponding to 1000 base pairs, or the megabase pair (Mb

or Mbp), corresponding to 1,000,000 base pairs. In the laboratory, researchers often make use of short

chains of single-stranded DNA—usually less than 50 bases—called oligonucleotides.

Secondary structure of DNA

The secondary conformations of RNA and DNA are quite different. This structural difference is critical

to the different functions of the two types of nucleic acids. RNA commonly exists as a single chain of

nucleotides, or strand. Its secondary structure includes short regions of double helices and folded

structures called hairpins. RNA molecules can carry information as well as catalyze chemical reactions.

The remainder of this chapter will focus on DNA. As Watson and Crick deduced, DNA generally exists

in cells as two interwound strands. The DNA double helix is also referred to as double-stranded DNA

(dsDNA) or duplex DNA to distinguish it from the single stranded DNA (ssDNA) found in some viruses.

DNA’s structure allows organisms to store and replicate the hereditary information for growth and

reproduction. Various weak chemical forces drive the formation of the DNA double helix. These include

hydrogen bonds between the bases and base stacking by hydrophobic interactions.

Hydrogen bonds form between the bases

Thermodynamically stable hydrogen bonds form between the nitrogenous bases on opposite strands

of the interwound DNA chains (Fig. 2.4). Hydrogen bonds are very weak bonds that involve sharing a

hydrogen atom between two electronegative atoms, such as oxygen and nitrogen. The hydrogen

bonds provide one type of force holding the strands together. Although individually very weak,

hydrogen bonds give structural stability to a molecule with large numbers of them. Double-stranded

DNA contains a very large number of these weak bonds. Even though thermal motion is constantly

breaking apart base pairs near the ends of each strand, the two chains usually do not separate because

other hydrogen bonds are still intact. Once a bond is broken, the most likely event under physiological

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conditions is the re-forming of the same hydrogen bonds rather than the breaking of additional bonds.

The hydrogen bonding between bases is referred to as “Watson–Crick” or “complementary” base

pairing. It occurs in such a way that adenine (A) normally pairs with thymine (T) by two hydrogen bonds,

and guanine (G) pairs with cytosine (C) by three hydrogen bonds.

Base stacking provides chemical stability to the DNA double helix

The molecular processes of cellular life generally take place in a watery solution, and intracellular

components are largely molecules that are easily dissolved in water. The nitrogenous bases are an

exception because they are nonpolar and thus hydrophobic (“water hating”). On their own, they are

practically insoluble in the aqueous environment of cells. The asymmetric distribution of charge across

a polar water molecule thus has important consequences for the structure of DNA. Once the bases are

covalently attached to a sugar and a phosphate to form a nucleotide, they become soluble in water,

but even so their insolubility still places strong constraints on the overall conformation of DNA in

solution. The paired, relatively flat bases tend to stack on top of one another by means of a helical

twist (Fig. 2.6). This feature of doublestranded DNA is known as “base stacking.” This stacking

eliminates any gaps between the bases and excludes the maximum amount of water from the interior

of the double helix.

Structure of the Watson–Crick DNA double helix

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Alternating deoxyribose sugars and phosphate groups form the backbone of DNA. The bases are

attached to the sugars and are located between the backbones of the DNA strands, lying perpendicular

to the long axis of the strands. As the backbones of the two strands wind around each other, they form

a double helix (Fig. 2.8).

Major and minor grooves

The two bonds that attach a base pair to its deoxyribose sugar rings are not directly opposite.

Therefore, the sugar–phosphate backbone is not equally spaced. This results in what are called the

major and minor grooves of DNA that wind around the central axis of the molecule (see Fig. 2.8).

The major groove has a significant role in sequence-specific DNA–protein interactions. Proteins that

carry out their function by binding DNA “read” the string of base letters exposed in the groove. The

edges of the base-paired purines and pyrimidines are solvent accessible. In particular, the solvent-

exposed nitrogen and oxygen atoms of the bases that line the major grooves of DNA can make

hydrogen bonds with the side chains of the amino acids of a protein. The pattern of these hydrogen-

bonding groups— whether they are donors or acceptors—is different for AT, TA, GC, and CG base pairs

(Fig. 2.9). Thus, the major groove carries a message in a form that can be read by DNA-binding proteins.

The minor groove of DNA is thought to be harder for proteins to “read” than the major groove. In the

minor groove, the hydrogen-bonding patterns are the same regardless of which way the base pair is

flipped, and there is only one difference in the pattern between AT and GC base pairs. However,

recently molecular biologists have learned that the shape of the minor groove can be recognized by

some DNA-binding proteins. The width of the minor groove varies depending on which nucleotides are

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present in the surrounding DNA. For example, a short run of adenine nucleotides (called an A-tract)

tends to form a narrow minor groove that serves as a site for specific recognition by a DNA-binding

protein. Most proteins involved in regulating gene expression bind DNA in the major groove. A well-

known exception is the TATA-binding protein (TBP). TBP binds the minor groove at a specific DNA

sequence known as the TATA box and plays a role in initiating gene transcription in eukaryotes.

Distinguishing between features of alternative double-helical structures

By now you probably appreciate that DNA is much more than a simple double-stranded string of

nucleotides. DNA is a dynamic molecule, and its overall shape is critical for its biological function. With

this in mind, researchers wondered whether DNA could adopt different helical shapes. To answer this

question, they turned to X-ray diffraction analysis. As you may recall from lec.1 , 2, the X-ray diffraction

analysis performed by Rosalind Franklin provided a key clue that allowed Watson and Crick to

complete their model of the DNA double helix. Oligonucleotides of any desired sequence can be

synthesized in sufficient quantity and purity to be studied by single-crystal X-ray. However, even with

highly pure material, X-ray crystallography is still a challenging technique. It is often likened to

gardening by scientists—some researchers have a “green thumb” and others do not when it comes to

growing crystals successfully. Structures have been determined for a number of synthetic

oligonucleotides—usually 4–24 self-complementary bases that fold into a double helix—under various

conditions. These studies have revealed the basic structure of three fundamental types of double helix:

Watson–Crick or B-DNA, A-DNA, and Z-DNA (Table 2.2).

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B-DNA (Watson–Crick DNA)

B-DNA represents the type of double helix proposed by Watson and Crick. B-DNA is a right-

handed helix; it turns in a clockwise manner when viewed down its axis (see Table 2.2). The

bases are stacked almost exactly perpendicular to the main axis with 10.5 bases per turn. The

major groove is wide and of moderate depth, while the minor groove is of moderate depth

but is much narrower. Of particular note, B-DNA occurs under conditions of high humidity

(95%) and relatively low salt. Since the inside of a cell is mostly water with a relatively low salt

concentration, it follows that the predominant form of DNA in vivo is B-DNA.

A-DNA

After showing that B-DNA formed under physiological conditions, X-ray crystallographers then

began to experiment with the conditions during crystal growth. If the water content is

decreased and the salt concentration increased during crystal formation, the A form of DNA

(A-DNA) will occur (see Table 2.2). In this right-handed helix, the bases are tilted with respect

to the axis and there are 11 bases per turn. The major groove of A-DNA is deep and narrow,

while the minor groove is shallow and broad. Although it is possible to make this type of helix

in vitro, it is considered unlikely that A-DNA is present in any lengthy sections in cells.

However, the A-type helix turns out to be an important structural feature of RNA. RNA adopts

an A-form helix when it forms double-stranded regions. This is because the 2’-hydroxyl group

on the ribose sugar hinders formation of B-form RNA.

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Z-DNA In 1979, Alexander Rich and his colleagues at the Massachusetts Institute of

Technology (MIT) made a novel discovery. They found that oligonucleotides composed of

repeating GC sequences on one strand, with the complementary CG sequences on the other,

formed a left-handed helix. A left-handed helix turns counterclockwise away from the viewer

when viewed down its axis (see Table 2.2). Because the backbone formed a zigzag structure,

they called the structure Z-DNA. Z-DNA has 12 base pairs per turn. The minor groove is very

deep and narrow. In contrast, the major groove is shallow to the point of being virtually

nonexistent. Z-DNA was first formed under conditions of high salt or in the presence of

alcohol. Later, it was shown that this form of double helix can be stabilized in physiologically

normal conditions if methyl groups are added to the cytosines. Cytosine methylation is a

common feature of eukaryotic DNA.

DNA can undergo reversible strand separation

As Watson and Crick realized, the double helix structure holds the key to the biological

functions of DNA. Each strand of DNA in the helix has the base sequence of its complementary strand, and from one strand, the other can be made. This important characteristic of the molecule allows for the fidelity of DNA replication, transcription (making an RNA copy of the

DNA), and translation (decoding the RNA message to make a protein). During DNA replication

and transcription, the strands of the helix must separate transiently and reversibly.

Denaturation of DNA:

The same feature that allows DNA to fulfill these biological roles also makes it possible to

manipulate DNA in vitro. The unwinding and separation of DNA strands, referred to as

denaturation or “melting,” can be induced in the laboratory. The hydrogen bonds between

the base pairs can be broken and the DNA strands separated by heating the DNA molecule,

whereas the covalent phosphodiester bonds remain intact (Fig. 2.11).

A point is reached in which thermal agitation overcomes the hydrogen bonds, hydrophobic interactions, and other forces that stabilize the double helix, and the molecule “melts.” This

strand separation of DNA changes its absorption of ultraviolet (UV) light in the 260-nm range.

Native double-stranded DNA absorbs less light at 260 nm by about 40% than does the

equivalent amount of single-stranded DNA. This is because base stacking in duplex DNA quenches the capacity of the bases to absorb UV light. Thus, as DNA denatures, its absorption of UV light increases, a phenomenon known as “hyperchromicity.” The temperature at which

half the base pairs in a double-stranded DNA sample have denatured is denoted the melting

temperature (Tm) (Fig. 2.12). Near the denaturation temperature, a small increase in

temperature causes an abrupt loss of the multiple weak interactions holding the two strands together, so denaturation occurs rapidly along the entire length of the DNA.

The G+C content of a DNA molecule has a significant effect on its Tm. Since a GC base pair has

three hydrogen bonds to every two in an AT base pair, the higher the GC content in a given

molecule of DNA, the higher the temperature required to denature the DNA. More

importantly, the base-stacking hydrophobic interactions of GC base pairs with neighboring

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base pairs are more favorable energetically than interactions of AT base pairs with their

adjacent base pairs. In addition to heat, other methods can be used to denature DNA.

Lowering the salt concentration of a DNA solution promotes denaturation by removing the

cations that shield the negative charges on the two strands from each other.

Renaturation of DNA

If the DNA is rapidly cooled, it remains single-stranded. When heated solutions of denatured

DNA are slowly cooled, sequences that are complementary will find each other and eventually

base-pair again to form a new double helix. This is called “renaturation” or “annealing.” The

capacity to renature denatured DNA molecules permits hybridization—the complementary

base pairing of strands from two different sources (see Fig. 2.11). In addition, the study of DNA

renaturation has contributed to molecular biologists’ understanding of the types of DNA

sequences present in various organisms.

Unusual DNA secondary structures

Originally, DNA was thought of as a static, linear string of genetic information. Since the mid-

1960s, there has been a rapid expansion in awareness of its flexibility in form. Unusual

secondary structures adopted by short regions of DNA have gained attention recently because

of the role they play in hereditary neurological diseases. There are three well-characterized

secondary structures—slipped structures, cruciforms, and triple helix DNA—that form in

stretches of DNA with repetitive sequences (Fig. 2.16). Supercoiling provides the necessary

driving energy for their formation, due to the release of torsional strain.

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Tertiary structure of DNA

Both strands of the Watson–Crick double helix follow a right-handed helical path around a

central axis with 10.5 base pairs per turn. It is relatively easy for long DNA molecules to lose

or gain a few turns or “twists.” This can happen by the DNA unwinding locally during DNA

replication or by binding to certain proteins. If the two ends of the DNA are not free to rotate,

then a small change in the number of turns can cause the DNA to coil through space rather

than following a straight path. For example, many naturally occurring DNA molecules are

circular, with no free 5’ or 3’ end. Due to the polarity of the strands of the DNA double helix,

the 5’ end of one strand can only join its own 3’ end to covalently close a circle.

Thus, circular, double-stranded DNA is essentially two circles of single-stranded DNA twisted

around each other. Such circular DNA molecules often become overwound or underwound,

with respect to the number of complete turns of the DNA double helix. This DNA can then

become supercoiled (under torsional stress). A supercoil is a twisted, three-dimensional

structure that is more favorable energetically. Consider a short double-stranded linear DNA

molecule of 10 complete turns (or twists, T = 10) with 10.5 bp/turn. If the ends of the DNA

molecule are sealed together, the result is an energetically relaxed circle that lies flat. Since

each chain is seen to cross the other 10 times, this relaxed circle has a linking number (L) of

10. But if the double helix is underwound by one full turn to the left and then the ends are

sealed together, the result is a strained circle with 11.67 bp/turn, where L = 9 and T = 9.

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Once the two ends become covalently linked, the linking number cannot change. Generally,

changes in the average number of base pairs per turn of the double helix will be counteracted

by the formation of an appropriate number of supercoils in the opposite direction. In this

example, one negative (left-handed) supercoil is introduced spontaneously, reestablishing the

total number of original “turns” of the helix (T = 10, L = 9). Overtwisting of the double helix

usually leads to positive (right-handed) supercoiling. For example, if the double helix is

overwound by one full turn to the right and then the ends are sealed together, the result is a

strained circle with 9.5 bp/turn, where L = 11, T = 11. The introduction of one positive (right-

handed) supercoil restores the total number of original turns of the helix (T = 10, L = 11).

RNA STRUCTURE

RNA Contains Ribose and Uracil and Is Usually Single-Stranded

We now turn our attention to RNA, which differs from DNA in three

respects. First, the backbone of RNA contains ribose rather than 2_-

deoxyribose. That is, ribose has a hydroxyl group at the 2_ position.

Second, RNA contains uracil in place of thymine. Uracil has the same

single-ringed structure as thymine, except that it lacks the 5_ methyl group.

Thymine is in effect 5_methyl-uracil. Third, RNA is usually found as a

single polynucleotide chain. Except for the case of certain viruses, RNA is

not the genetic material and does not need to be capable of serving as a

template for its own replication. Rather, RNA functions as the intermediate,

the mRNA, between the gene and the protein-synthesizing machinery.

Another function of RNA is as an adaptor, the tRNA, between the codons

in the mRNA and amino acids. RNA can also play a structural role as in

the case of the RNA components of the ribosome. Yet another role for RNA

is as a regulatory molecule, which through sequence complementarity

binds to, and interferes with the translation of, certain mRNAs. Finally,

some RNAs (including one of the structural RNAs of the ribosome) are

enzymes that catalyze essential reactions in the cell. In all of these cases,

the RNA is copied as a single strand off only one of the two strands of the

DNA template, and its complementary strand does not exist. RNA is

capable of forming long double helices, but these are unusual in nature.

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ο RNA Chains Fold Back on Themselves to Form Local Regions of Double Helix Similar

to A-Form DNA.

ο Despite being single-stranded, RNA molecules often exhibit a great deal of double-

helical character.

ο RNA Can Fold Up into Complex Tertiary Structures.

ο Some RNAs Are Enzymes: The Hammerhead Ribozyme Cleaves RNA by the

Formation of a 2’, 3’ Cyclic Phosphate.