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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 4.1Structure of Nucleic Acids

DNA and RNA have great chemical similarities. In their primary structures both are linear polymers (multiple chemical units) composed of monomers (single chemical units), called nucleotides. Cellular RNAs range in length from less than one hundred to many thousands of nucleotides. Cellular DNA molecules can be as long as several hundred million nucleotides. These large DNA units in association with proteins can be stained with dyes and visualized in the light microscope as chromosomes.

Polymerization of Nucleotides Forms Nucleic Acids

DNA and RNA each consists of only four different nucleotides. All nucleotides have a common structure: a phosphate group linked by a phosphoester bond to a pentose (a five-carbon sugar molecule) that in turn is linked to an organic base (Figure 4-1a). In RNA, the pentose is ribose; in DNA, it is deoxyribose (Figure 4-1b). The only other difference in the nucleotides of DNA and RNA is that one of the four organic bases differs between the two polymers. The bases adenine, guanine, and cytosine are found in both DNA and RNA; thymine is found only in DNA, and uracil is found only in RNA. The bases are often abbreviated A, G, C, T, and U, respectively. For convenience the single letters are also used when long sequences of nucleotides are written out.

Figure 4-1. All Nucleotides have a common structure.

Figure 4-1

All Nucleotides have a common structure. (a) Chemical structure of adenosine 5′-monophosphate (AMP), a nucleotide that is present in RNA. All nucleotides are composed of a phosphate moiety, containing up to three phosphate groups, linked to the (more...)

The base components of nucleic acids are heterocyclic compounds with the rings containing nitrogen and carbon. Adenine and guanine are purines, which contain a pair of fused rings; cytosine, thymine, and uracil are pyrimidines, which contain a single ring (Figure 4-2). The acidic character of nucleotides is due to the presence of phosphate, which dissociates at the pH found inside cells, freeing hydrogen ions and leaving the phosphate negatively charged (see Figure 2-22). Because these charges attract proteins, most nucleic acids in cells are associated with proteins. In nucleotides, the 1′ carbon atom of the sugar (ribose or deoxyribose) is attached to the nitrogen at position 9 of a purine (N9) or at position 1 of a pyrimidine (N1).

Figure 4-2. The chemical structures of the principal bases in nucleic acids.

Figure 4-2

The chemical structures of the principal bases in nucleic acids. In nucleic acids and nucleotides, nitrogen 9 of purines and nitrogen 1 of pyrimidines (red) are bonded to the 1′ carbon of ribose or deoxyribose.

Cells and extracellular fluids in organisms contain small concentrations of nucleosides, combinations of a base and a sugar without a phosphate. Nucleotides are nucleosides that have one, two, or three phosphate groups esterified at the 5′ hydroxyl. Nucleoside monophosphates have a single esterified phosphate (see Figure 4-1a), diphosphates contain a prophosphate group

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and triphosphates have a third phosphate. Table 4-1 lists the names of the nucleosides and nucleotides in nucleic acids and the various forms of nucleoside phosphates. As we will see later, the nucleoside triphosphates are used in the synthesis of nucleic acids. However, these compounds also serve many other functions in the cell: ATP, for example, is the most widely used energy carrier in the cell (see Figure 2-25), and GTP plays crucial roles in intracellular signaling and acts as an energy reservoir, particularly in protein synthesis.

Table 4-1. Naming Nucleosides and Nucleotides.

Table 4-1

Naming Nucleosides and Nucleotides.

When nucleotides polymerize to form nucleic acids, the hydroxyl group attached to the 3′ carbon of a sugar of one nucleotide forms an ester bond to the phosphate of another nucleotide, eliminating a molecule of water:

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This condensation reaction is similar to that in which a peptide bond is formed between two amino acids (Chapter 3). Thus a single nucleic acid strand is a phosphate-pentose polymer (a polyester) with purine and pyrimidine bases as side groups. The links between the nucleotides are called phosphodiester bonds. Like a polypeptide, a nucleic acid strand has an end-to-end chemical orientation: the 5end has a free hydroxyl or phosphate group on the 5′ carbon of its terminal sugar; the 3end has a free hydroxyl group on the 3′ carbon of its terminal sugar (Figure 4-3). This directionality, plus the fact that synthesis proceeds 5′ to 3′, has given rise to the convention that polynucleotide sequences are written and read in the 5′ → 3′ direction (from left to right); for example, the sequence AUG is assumed to be (5′)AUG(3′). (Although, strictly speaking, the letters A, G, C, T, and U stand for bases, they are also often used in diagrams to represent the whole nucleotides containing these bases.) The 5′ → 3′ directionality of a nucleic acid strand is an extremely important property of the molecule.

Figure 4-3. Alternative ways of representing nucleic acid chains, in this case a single strand of DNA containing only three bases: cytosine (C), adenine (A), and guanine (G).

Figure 4-3

Alternative ways of representing nucleic acid chains, in this case a single strand of DNA containing only three bases: cytosine (C), adenine (A), and guanine (G). (a) Chemical structure of the trinucleotide CAG. Note the free hydroxyl group at the 3′ (more...)

The linear sequence of nucleotides linked by phosphodiester bonds constitutes the primary structure of nucleic acids. As we discuss in the next section, polynucleotides can twist and fold into three-dimensional conformations stabilized by noncovalent bonds; in this respect, they are similar to polypeptides. Although the primary structures of DNA and RNA are generally similar, their conformations are quite different. Unlike RNA, which commonly exists as a single polynucleotide chain, or strand, DNA contains two intertwined polynucleotide strands. This structural difference is critical to the different functions of the two types of nucleic acids.

Native DNA Is a Double Helix of Complementary Antiparallel Chains

The modern era of molecular biology began in 1953 when James D. Watson and Francis H. C. Crick proposed correctly the double-helical structure of DNA, based on the analysis of x-ray diffraction patterns coupled with careful model building. A closer look at the “thread of life,” as the DNA molecule is sometimes called, shows why the discovery of its basic structure suggests its function.

DNA consists of two associated polynucleotide strands that wind together through space to form a structure often described as a double helix. The two sugar-phosphate backbones are on the outside of the double helix, and the bases project into the interior. The adjoining bases in each strand stack on top of one another in parallel planes (Figure 4-4a). The orientation of the two strands is antiparallel; that is, their 5′ → 3′ directions are opposite. The strands are held in precise register by a regular base-pairing between the two strands: A is paired with T through two hydrogen bonds; G is paired with C through three hydrogen bonds (Figure 4-4b). This base-pair complementarity is a consequence of the size, shape, and chemical composition of the bases. The presence of thousands of such hydrogen bonds in a DNA molecule contributes greatly to the stability of the double helix. Hydrophobic and van der Waals interactions between the stacked adjacent base pairs also contribute to the stability of the DNA structure.

Figure 4-4. Two representations of contacts within the DNA double helix.

Figure 4-4

Two representations of contacts within the DNA double helix. (a) Space-filling model of B DNA, the most common form of DNA in cells. The sugar and phosphate residues (gray) in each strand form the backbone, which is traced by a red line, showing the helical twist (more...)

To maintain the geometry of the double-helical structure shown in Figure 4-4a, a larger purine (A or G) must pair with a smaller pyrimidine (C or T). In natural DNA, A almost always hydrogen bonds with T and G with C, forming A·T and G·C base pairs often called Watson-Crick base pairs. Two polynucleotide strands, or regions thereof, in which all the nucleotides form such base pairs are said to be complementary. However, in theory and in synthetic DNAs other interactions can occur. For example, a guanine (a purine) could theoretically form hydrogen bonds with a thymine (a pyrimidine), causing only a minor distortion in the helix. The space available in the helix also would allow pairing between the two pyrimidines cytosine and thymine. Although the nonstandard G·T and C·T base pairs are normally not found in DNA, G·U base pairs are quite common in double-helical regions that form within otherwise single-stranded RNA.

Two polynucleotide strands can, in principle, form either a right-handed or a left-handed helix (Figure 4-5). Because the geometry of the sugar-phosphate backbone is more compatible with the former, natural DNA is a right-handed helix. The x-ray diffraction pattern of DNA indicates that the stacked bases are regularly spaced 0.34 nm apart along the helix axis. The helix makes a complete turn every 3.4 nm; thus there are about 10 pairs per turn. This is referred to as the B form of DNA, the normal form present in most DNA stretches in cells (Figure 4-6a). On the outside of B-form DNA, the spaces between the intertwined strands form two helical grooves of different widths described as the major groove and the minor groove (see Figure 4-4a). Consequently, part of each base is accessible from outside the helix to both small and large molecules that bind to the DNA by contacting chemical groups within the grooves. These two binding surfaces of the DNA molecule are used by different classes of DNA-binding proteins.

Figure 4-5. Two possible helical forms of DNA are mirror images of each other.

Figure 4-5

Two possible helical forms of DNA are mirror images of each other. The geometry of the sugar-phosphate backbone of DNA causes natural DNA to be right-handed. (Right-handed and left-handed are defined by convention.)

Figure 4-6. Models of various DNA structures that are known to exist.

Figure 4-6

Models of various DNA structures that are known to exist. The sugar-phosphate backbone of each chain is on the outside in all structures (one red and one blue) with the bases (silver) oriented inward. Side views are shown at the top, and views along the (more...)

In addition to the major B form of DNA, three additional structures have been described. In very low humidity, the crystallographic structure of B DNA changes to the A form; RNA-DNA and RNA-RNA helices also exist in this form. The A form is more compact than the B form, having 11 bases per turn, and the stacked bases are tilted (Figure 4-6b). Short DNA molecules composed of alternating purine-pyrimidine nucleotides (especially Gs and Cs) adopt an alternative left-handed configuration instead of the normal right-handed helix. This structure is called Z DNA because the bases seem to zigzag when viewed from the side (Figure 4-6c). It is entirely possible that both A-form and Z-form stretches of DNA exist in cells.

Finally, a triple-stranded DNA structure can also exist at least in the test tube, and possibly during recombination and DNA repair. For example, when synthetic polymers of poly(A) and polydeoxy(U) are mixed, a three-stranded structure is formed (Figure 4-6d). Further, long homopolymeric stretches of DNA composed of C and T residues in one strand and A and G residues in the other can be targeted by short matching lengths of poly(C+T). The synthetic oligonucleotide can insert as a third strand, binding in a sequence-specific manner by so-called Hoogsteen base pairs. Specific cleavage of the DNA at the site where the triple helix ends can be achieved by attaching a chemical cleaving agent (e.g., Fe2+-EDTA) to the short oligodeoxynucleotide that makes up the third strand. Such reactions may be useful in studying site-specific DNA damage in cells.

By far the most important modifications in standard B-form DNA come about as a result of protein binding to specific DNA sequences. Although the multitude of hydrogen and hydrophobic bonds between the polynucleotide strands provide stability to DNA, the double helix is somewhat flexible about its long axis. Unlike the α helix in proteins (see Figure 3-6), there are no hydrogen bonds between successive residues in a DNA strand. This prop- erty allows DNA to bend when complexed with a DNA-binding protein. Crystallographic analyses of proteins bound to particular regions of DNA have conclusively demonstrated departures from the standard B-DNA structure in protein-DNA complexes. Two examples of DNA deformed by contact with proteins are shown in Figure 4-7. The specific DNA-protein contacts that occur in these tightly bound complexes have the ability both to untwist the DNA and to bend the axis of the helix. Although DNA in cells likely exists in the B form most of the time, particular regions bound to protein clearly depart from the standard conformation.

Figure 4-7. Bending of OF DNA resulting from protein binding.

Figure 4-7

Bending of OF DNA resulting from protein binding. (a) A linear DNA (left) is shown binding a repressor protein encoded by bacteriophage 434 (center); the resulting bend in the DNA (right) is easily seen by comparison with the linear molecule. Binding (more...)

DNA Can Undergo Reversible Strand Separation

In DNA replication and in the copying of RNA from DNA, the strands of the helix must separate at least temporarily. As we discuss later, during DNA synthesis two new strands are made (one copied from each of the original strands), resulting in two double helices identical with the original one. In the case of copying the DNA template to make RNA, the RNA is released and the two DNA strands reassociate with each other.

The unwinding and separation of DNA strands, referred to as denaturation, or “melting,” can be induced experimentally. For example, if a solution of DNA is heated, the thermal energy increases molecular motion, eventually breaking the hydrogen bonds and other forces that stabilize the double helix, and the strands separate (Figure 4-8). This melting of DNA changes its absorption of ultraviolet (UV) light (in the 260-nm range), which is routinely used to measure DNA concentration because of the high absorbance of UV light by nucleic acid bases. Native double-stranded DNA absorbs about one-half as much light at 260 nm as does the equivalent amount of single-stranded DNA (Figure 4-9a). Thus, as DNA denatures, its absorption of UV light increases. Near the denaturation temperature, a small increase in temperature causes an abrupt, near simultaneous, loss of the multiple, weak, cooperative interactions holding the two strands together, so that denaturation rapidly occurs throughout the entire length of the DNA.

Figure 4-8. The denaturation and renaturation of double-stranded DNA molecules.

Figure 4-8

The denaturation and renaturation of double-stranded DNA molecules.

Figure 4-9. Light absorption and temperature in DNA denaturation.

Figure 4-9

Light absorption and temperature in DNA denaturation. (a) Melting of doubled-stranded DNA can be monitored by the absorption of ultraviolet light at 260 nm. As regions of double-stranded DNA unpair, the absorption of light by those regions increases almost (more...)

The melting temperature, Tm, at which the strands of DNA will separate depends on several factors. Molecules that contain a greater proportion of G·C pairs require higher temperatures to denature because the three hydrogen bonds in G·C pairs make them more stable than A·T pairs with two hydrogen bonds (see Figure 4-4b). Indeed, the percentage of G·C base pairs in a DNA sample can be estimated from its Tm (Figure 4-9b). In addition to heat, solutions of low ion concentration destabilize the double helix, causing it to melt at lower temperatures. DNA is also denatured by exposure to other agents that destabilize hydrogen bonds, such as alkaline solutions and concentrated solutions of formamide or urea:

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The single-stranded DNA molecules that result from denaturation form random coils without a regular structure. Lowering the temperature or increasing the ion concentration causes the two complementary strands to reassociate into a perfect double helix (see Figure 4-8). The extent of such renaturation is dependent on time, the DNA concentration, and the ionic content of the solution. Two DNA strands not related in sequence will remain as random coils and will not renature and, most important, will not greatly inhibit complementary DNA partner strands from finding each other. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization, a powerful technique used to study the relatedness of two DNA samples and to detect and isolate specific DNA molecules in a mixture containing numerous different DNA sequences (Chapter 7).

Many DNA Molecules Are Circular

All prokaryotic genomic DNAs and many viral DNAs are circular molecules. Circular DNA molecules also occur in mitochondria, which are present in almost all eukaryotic cells, and in chloroplasts, which are present in plants and some unicellular eukaryotes.

Each of the two strands in a circular DNA molecule forms a closed structure without free ends. Just as is the case for linear DNA, elevated temperatures or alkaline pH destroy the hydrogen bonds and other interactions that stabilize double-helical circular DNA molecules. Unlike linear DNA, however, the two strands of circular DNA cannot unwind and separate; attempts to melt such DNA result in an interlocked, tangled mass of single-stranded DNA (Figure 4-10a).

Figure 4-10. Denaturation of circular DNA.

Figure 4-10

Denaturation of circular DNA. (a) If both strands are closed circles, denaturation disrupts the double helix, but the two single strands become tangled about each other and cannot separate. (b) If one or both strands are nicked, however, the two strands (more...)

Only if a native circular DNA is nicked (i.e., one of the strands is cut), will the two strands unwind and separate when the molecule is denatured. In this case, one of the separated strands is circular, and the other is linear (Figure 4-10b). Nicking of circular DNA occurs naturally during DNA replication and can be induced experimentally with a low concentration of deoxyribonuclease (a DNA-degrading enzyme), so that only a single phosphodiester bond in the molecule is cleaved. The study of circular DNA molecules lacking free ends first uncovered the complicated geometric shape changes that the double-stranded DNA molecule must undergo when the strands are not free to separate.

Local Unwinding of DNA Induces Supercoiling

So far we have described DNA as a long regular helical structure that can have local perturbations, especially due to protein binding. In addition, when the two ends of a DNA molecule are fixed, the molecule exhibits a superstructure under certain conditions. This occurs when the base pairing is interrupted and a local region unwinds. The stress induced by unwinding is relieved by twisting of the double helix on itself, forming supercoils (Figure 4-11). Unwinding and subsequent supercoiling occurs during replication, transcription, and binding of many proteins to circular DNAs or to long DNA loops whose ends are fixed within eukaryotic chromosomes. Supercoiling is recognized and regulated by enzymes called topoisomerases. As discussed in later chapters, these enzymes have an important role in both DNA replication and the transcription of DNA into RNA.

Figure 4-11. Supercoiling in electron micrographs of DNA isolated from the SV40 virus.

Figure 4-11

Supercoiling in electron micrographs of DNA isolated from the SV40 virus. When isolated SV40 DNA is separated from its associated protein, the DNA duplex is underwound and assumes the supercoiled configuration (form I). If one strand is nicked, the strands (more...)

RNA Molecules Exhibit Varied Conformations and Functions

As noted earlier, the primary structure of RNA is generally similar to that of DNA; however, the sugar component (ribose) of RNA has an additional hydroxyl group at the 2′ position (see Figure 4-1b), and thymine in DNA is replaced by uracil in RNA (see Figure 4-2). The hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA and provides a chemically reactive group that takes part in RNA-mediated enzymatic events. As a result of this lability, RNA is cleaved into mononucleotides by alkaline solution, whereas DNA is not. Like DNA, RNA is a long polynucleotide that can be double-stranded or single-stranded, linear or circular. It can also participate in a hybrid helix composed of one RNA strand and one DNA strand; this hybrid has a slightly different conformation than the common B form of DNA.

Unlike DNA, which exists primarily in a single, very long three-dimensional structure, the double helix, the various types of RNA exhibit different conformations. Differences in the sizes and conformations of the various types of RNA permit them to carry out specific functions in a cell. The simplest secondary structures in single-stranded RNAs are formed by pairing of complementary bases. “Hairpins” are formed by pairing of bases within ≈5 – 10 nucleotides of each other, and “stem-loops” by pairing of bases that are separated by ≈50 to several hundred nucleotides (Figure 4-12a). These simple folds can cooperate to form more complicated tertiary structures, one of which is termed a “pseudoknot” (Figure 4-12b).

Figure 4-12. RNA secondary and tertiary structures.

Figure 4-12

RNA secondary and tertiary structures. (a) Stem-loops, hairpins, and other secondary structures can form by base pairing between distant complementary segments of an RNA molecule. In stem-loops, the single-stranded loop (dark red) between the base-paired (more...)

As discussed in detail later, tRNA molecules adopt a well-defined three-dimensional architecture in solution that is crucial in protein synthesis. Larger rRNA molecules also have locally well defined three-dimensional structures, with more flexible links in between. Secondary and tertiary structures also have been recognized in mRNA, particularly near the ends of molecules. These recently discovered structures are under active study. Clearly, then, RNA molecules are like proteins in that they have structured domains connected by less structured, flexible stretches.

The folded domains of RNA molecules not only are structurally analogous to the α helices and β strands found in proteins, but in some cases also have catalytic capacities. Such catalytic RNAs, called ribozymes, can cut RNA chains. Some RNA domains also can catalyze RNA splicing, a remarkable process in which an internal RNA sequence, an intron, is cut and removed and the two resulting chains, the exons, are sealed together. This process occurs during formation of the majority of functional mRNA molecules in eukaryotic cells, and also occurs in bacteria and archaea. Remarkably, some RNAs carry out self-splicing, with the catalytic activity residing in the intron sequence. The mechanisms of splicing and self-splicing are discussed in detail in Chapter 11. As noted later in this chapter, rRNA is thought to play a catalytic role in the formation of peptide bonds during protein synthesis.

In this chapter, we focus on the functions of mRNA, tRNA, and rRNA in gene expression — the process of getting the information in DNA converted into proteins. In later chapters we will encounter other RNAs, often associated with proteins, that participate in other cell functions.


  •  Deoxyribonucleic acid (DNA), the genetic material, carries information to specify the amino acid sequences of proteins. It is transcribed into several types of ribonucleic acid (RNA) including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), which function in protein synthesis.
  •  Both DNA and RNA are long, unbranched polymers of nucleotides. Each nucleotide consists of a heterocyclic base linked via a five-carbon sugar (deoxyribose or ribose) to a phosphate group (see Figure 4-1).
  • DNA and RNA each contain four different bases (see Figure 4-2). The purines adenine (A) and guanine (G) and the pyrimidine cytosine (C) are present in both DNA and RNA. The pyrimidine thymine (T) present in DNA is replaced by the pyrimidine uracil (U) in RNA.
  •  The bases in nucleic acids can interact via hydrogen bonds. The standard Watson-Crick base pairs are G·C, A·T (in DNA), and A·U (in RNA). Base pairing stabilizes the native three-dimensional structures of DNA and RNA.
  •  Adjacent nucleotides in a polynucleotide are linked by phosphodiester bonds. The entire strand has a chemical directionality: the 5′ end with a free hydroxyl or phosphate group on the 5′ carbon of the sugar, and the 3′ end with a free hydroxyl group on the 3′ carbon of the sugar (see Figure 4-3). Polynucleotide sequences are always written in the 5′ → 3′ direction (left to right).
  •  Natural DNA (B DNA) contains two complementary polynucleotide strands wound together into a regular right-handed double helix with the bases on the inside and the two sugar-phosphate backbones on the outside (see Figure 4-6a). Base pairing (A·T and G·C) and hydrophobic interactions between adjacent bases in the same strand stabilize this native structure.
  •  Binding of protein to DNA can deform its helical structure, causing local bending or unwinding of the DNA molecule.
  •  Heat causes the DNA strands to separate (denature). The melting temperature of DNA increases with the percentage of G·C base pairs. Under suitable conditions, separated complementary nucleic acid strands will renature.
  •  Local unwinding of the DNA helix induces stress, which is relieved by twisting of the molecule on itself, forming supercoils. This process is regulated by topoisomerases, which can add or remove supercoils.
  •  Natural RNAs are single-stranded polynucleotides that form well-defined secondary and tertiary structures (see Figure 4-12). Some RNAs, called ribozymes, have catalytic activity.
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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21514


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