Rosalind Franklin Photo Explained Diffraction Labelled

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FIGURE 8-10 Absorption spectra of the common nucleotides. The spectra are shown as the variation in molar extinction coefficient with wavelength. The molar extinction coefficients at 260 nm and pH 7.0 (e260) are listed in the table. The spectra of corresponding ribonucleotides and deoxyribonucleotides, as well as the nucleosides, are essentially identical. For mixtures of nucleotides, a wavelength of 260 nm (dashed vertical line) is used for absorption measurements.

Adenine

Guanine

Peptidbindung

Adenine

Guanine

The most important functional groups of pyrim-idines and purines are ring nitrogens, carbonyl groups, and exocyclic amino groups. Hydrogen bonds involving the amino and carbonyl groups are the second important mode of interaction between bases in nucleic acid molecules. Hydrogen bonds between bases permit a complementary association of two (and occasionally three or four) strands of nucleic acid. The most important hydrogen-bonding patterns are those defined by James D. Watson and Francis Crick in 1953, in which A bonds specifically to T (or U) and G bonds to C (Fig. 8-11). These two types of base pairs predominate in double-stranded DNA and RNA, and the tautomers shown in Figure 8-2 are responsible for these patterns. It is this specific pairing of bases that permits the duplication of genetic information, as we shall discuss later in this chapter.

James Watson Francis Crick

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

SUMMARY 8.1 Some Basics

■ A nucleotide consists of a nitrogenous base (purine or pyrimidine), a pentose sugar, and one or more phosphate groups. Nucleic acids are polymers of nucleotides, joined together by phosphodiester linkages between the 5'-hydroxyl group of one pentose and the 3'-hydroxyl group of the next.

■ There are two types of nucleic acid: RNA and DNA. The nucleotides in RNA contain ribose, and the common pyrimidine bases are uracil and cytosine. In DNA, the nucleotides contain 2'-deoxyribose, and the common pyrimidine bases are thymine and cytosine. The primary purines are 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 was a momentous event in science, an event that gave rise to entirely new disciplines and influenced the course of many established ones. Our present understanding of the storage and utilization of a cell's genetic information is based on work made possible by this discovery, and an outline of how genetic information is processed by the cell is now a prerequisite for the discussion of any area of biochemistry. Here, we concern ourselves with DNA structure itself, the events

that led to its discovery, and more recent refinements in our understanding. 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, 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 the nucleotides in a nucleic acid can be referred to as secondary structure. All structures considered in the remainder of this chapter fall under the heading of secondary structure. The complex folding of large chromosomes within eukaryotic chromatin and bacterial nucleoids is generally considered tertiary structure; this is discussed in Chapter 24.

DNA Stores Genetic Information

The biochemical investigation of DNA began with Friedrich Miescher, who carried out the first systematic chemical studies of cell nuclei. In 1868 Miescher isolated a phosphorus-containing substance, which he called "nuclein," from the nuclei of pus cells (leukocytes) obtained from discarded surgical bandages. He found nuclein to consist of an acidic portion, which we know today as DNA, and a basic portion, protein. Miescher later found a similar acidic substance in the heads of sperm cells from salmon. Although he partially purified nuclein and studied its properties, the covalent (primary) structure of DNA (as shown in Fig. 8-7) was not known with certainty until the late 1940s.

Miescher and many others suspected that nuclein (nucleic acid) was associated in some way with cell inheritance, but the first direct evidence that DNA is the bearer of genetic information came in 1944 through a discovery made by Oswald T. Avery, Colin MacLeod, and Maclyn McCarty. These investigators found that DNA extracted from a virulent (disease-causing) strain of the bacterium Streptococcus pneumoniae, also known as pneumococcus, genetically transformed a nonvirulent strain of this organism into a virulent form (Fig. 8-12).

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FIGURE 8-12 The Avery-MacLeod-McCarty experiment. (a) When injected into mice, the encapsulated strain of pneumococcus is lethal, (b) whereas the nonencapsulated strain, (c) like the heat-killed encapsulated strain, is harmless. (d) Earlier research by the bacteriologist Frederick Griffith had shown that adding heat-killed virulent bacteria (harmless to mice) to a live nonvirulent strain permanently transformed the latter into lethal, virulent, encapsulated bacteria. (e) Avery and his colleagues extracted the DNA from heat-killed virulent pneumococci, removing the protein as completely as possible, and added this DNA to nonvirulent bacteria. The DNA gained entrance into the nonvirulent bacteria, which were permanently transformed into a virulent strain.

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32P experiment

35S experiment

Avery and his colleagues concluded that the DNA extracted from the virulent strain carried the inheritable genetic message for virulence. Not everyone accepted these conclusions, because protein impurities present in the DNA could have been the carrier of the genetic information. This possibility was soon eliminated by the finding that treatment of the DNA with proteolytic enzymes did not destroy the transforming activity, but treatment with deoxyribonucleases (DNA-hydrolyzing enzymes) did.

A second important experiment provided independent evidence that DNA carries genetic information. In 1952 Alfred D. Hershey and Martha Chase used radioactive phosphorus (32P) and radioactive sulfur (35S) tracers to show that when the bacterial virus (bacteriophage) T2 infects its host cell, Escherichia coli, it is the phosphorus-containing DNA of the viral particle, not the sulfur-containing protein of the viral coat, that enters the host cell and furnishes the genetic information for viral replication (Fig. 8-13). These important early experiments and many other lines of evidence have shown that DNA is the exclusive chromosomal component bearing the genetic information of living cells.

DNA Molecules Have Distinctive Base Compositions

A most important clue to the structure of DNA came from the work of Erwin Chargaff and his colleagues in the late 1940s. They found that the four 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.

4. In all cellular DNAs, regardless of the species, the number of adenosine residues is equal to the number of thymidine residues (that is, A = T), and the number of guanosine residues is equal to the number of cytidine residues (G = C). From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine residues; that is, A + G = T + C.

These quantitative relationships, sometimes called "Chargaff's rules," were confirmed by many subsequent researchers. They were a key to establishing the three-dimensional structure of DNA and yielded clues to how genetic information is encoded in DNA and passed from one generation to the next.

Nonradioactive coat

Radioactive DNA

Radioactive coat

32P experiment

35S experiment

Nonradioactive coat

Radioactive DNA

Radioactive coat

Blender treatment shears off viral heads

Separation by centrifugation

Separation by centrifugation

Radioactive

Not radioactive

FIGURE 8-13 The Hershey-Chase experiment. Two batches of iso-topically labeled bacteriophage T2 particles were prepared. One was labeled with 32P in the phosphate groups of the DNA, the other with 35S in the sulfur-containing amino acids of the protein coats (capsids). (Note that DNA contains no sulfur and viral protein contains no phosphorus.) The two batches of labeled phage were then allowed to infect separate suspensions of unlabeled bacteria. Each suspension of phage-infected cells was agitated in a blender to shear the viral capsids from the bacteria. The bacteria and empty viral coats (called "ghosts") were then separated by centrifugation. The cells infected with the 32P-labeled phage were found to contain 32P, indicating that the labeled viral DNA had entered the cells; the viral ghosts contained no radioactivity. The cells infected with 35S-labeled phage were found to have no radioactivity after blender treatment, but the viral ghosts contained 35S. Progeny virus particles (not shown) were produced in both batches of bacteria some time after the viral coats were removed, indicating that the genetic message for their replication had been introduced by viral DNA, not by viral protein.

DNA Is a Double Helix

To shed more light on the structure of DNA, Rosalind Franklin and Maurice Wilkins used the powerful method of x-ray diffraction (see Box 4-4) to analyze DNA fibers. They showed in the early 1950s that DNA produces a characteristic x-ray diffraction pattern (Fig. 8-14). From this pattern it was deduced that DNA molecules are helical with two periodicities along their long axis, a primary one of 3.4 A and a secondary one of 34 A. The problem then was to formulate a three-dimensional model of the DNA molecule that could account not only for the x-ray diffraction data but also for the specific A = T and G = C base equivalences discovered by Chargaff and for the other chemical properties of DNA.

In 1953 Watson and Crick postulated a three-dimensional model of DNA structure that accounted for all the available data. It consists of two helical DNA chains wound around the same axis to form a right-handed double helix (see Box 4-1 for an explanation of the right- or left-handed sense of a helical structure). The hydrophilic backbones of alternating deoxyribose and phosphate groups are on the outside of the double helix, facing the surrounding water. The furanose ring of each deoxyribose is in the C-2' endo conformation. The purine and pyrimidine bases of both strands are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis. The offset pairing of the two strands creates a major groove and minor groove on the surface of the duplex (Fig. 8-15). Each nucleotide base of one strand is paired in the same plane with a base of the other 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, providing a rationale for Chargaff's rule that in any DNA, G = C and A = T. It is important to note that three hydrogen bonds can form between G and C, symbolized GqC, but only two can form between A and T, symbolized A=T. This is one reason for the

FIGURE 8-14 X-ray diffraction pattern of DNA. The spots forming a cross in the center denote a helical structure. The heavy bands at the left and right arise from the recurring bases.

Rosalind Franklin, 1920-1958

Maurice Wilkins

Rosalind Franklin, 1920-1958

Maurice Wilkins finding that separation of paired DNA strands is more difficult the higher the ratio of GqC to A=T base pairs. Other pairings of bases tend (to varying degrees) to destabilize the double-helical structure.

When Watson and Crick constructed their model, they had to decide at the outset whether the strands of DNA should be parallel or antiparallel—whether their 5',3'-phosphodiester bonds should run in the same or opposite directions. An antiparallel orientation produced the most convincing model, and later work with DNA polymerases (Chapter 25) provided experimental evidence that the strands are indeed antiparallel, a finding ultimately confirmed by x-ray analysis.

To account for the periodicities observed in the x-ray diffraction patterns of DNA fibers, Watson and Crick manipulated molecular models to arrive at a structure

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FIGURE 8-15 Watson-Crick model for the structure of DNA. The original model proposed by Watson and Crick had 10 base pairs, or 34 A (3.4 nm), per turn of the helix; subsequent measurements revealed 10.5 base pairs, or 36 A (3.6 nm), per turn. (a) Schematic representation, showing dimensions of the helix. (b) Stick representation showing the backbone and stacking of the bases. (c) Space-filling model.

FIGURE 8-14 X-ray diffraction pattern of DNA. The spots forming a cross in the center denote a helical structure. The heavy bands at the left and right arise from the recurring bases.

FIGURE 8-15 Watson-Crick model for the structure of DNA. The original model proposed by Watson and Crick had 10 base pairs, or 34 A (3.4 nm), per turn of the helix; subsequent measurements revealed 10.5 base pairs, or 36 A (3.6 nm), per turn. (a) Schematic representation, showing dimensions of the helix. (b) Stick representation showing the backbone and stacking of the bases. (c) Space-filling model.

in which the vertically stacked bases inside the double helix would be 3.4 A apart; the secondary repeat distance of about 34 A was accounted for by the presence of 10 base pairs in each complete turn of the double helix. In aqueous solution the structure differs slightly from that in fibers, having 10.5 base pairs per helical turn (Fig. 8-15).

As Figure 8-16 shows, the two antiparallel polynu-cleotide chains of double-helical DNA are not identical in either base sequence or composition. Instead they are complementary to each other. Wherever adenine occurs in one chain, thymine is found in the other; similarly, wherever guanine occurs in one chain, cytosine is found in the other.

The DNA double helix, or duplex, is held together by two forces, as described earlier: hydrogen bonding between complementary base pairs (Fig. 8-11) and base-stacking interactions. The complementarity between the DNA strands is attributable to the hydrogen bonding between base pairs. The base-stacking interactions, which are largely nonspecific with respect to the identity of the stacked bases, make the major contribution to the stability of the double helix.

The important features of the double-helical model of DNA structure are supported by much chemical and

FIGURE 8-16 Complementarity of strands in the DNA double helix.

The complementary antiparallel strands of DNA follow the pairing rules proposed by Watson and Crick. The base-paired antiparallel strands differ in base composition: the left strand has the composition A3 T2 G-i C3; the right, A2 T3 G3 C. They also differ in sequence when each chain is read in the 5' —> 3' direction. Note the base equivalences: A = T and G = C in the duplex.

FIGURE 8-16 Complementarity of strands in the DNA double helix.

The complementary antiparallel strands of DNA follow the pairing rules proposed by Watson and Crick. The base-paired antiparallel strands differ in base composition: the left strand has the composition A3 T2 G-i C3; the right, A2 T3 G3 C. They also differ in sequence when each chain is read in the 5' —> 3' direction. Note the base equivalences: A = T and G = C in the duplex.

Dna Replication

FIGURE 8-17 Replication of DNA as suggested by Watson and Crick.

The preexisting or "parent" strands become separated, and each is the template for biosynthesis of a complementary "daughter" strand (in red).

FIGURE 8-17 Replication of DNA as suggested by Watson and Crick.

The preexisting or "parent" strands become separated, and each is the template for biosynthesis of a complementary "daughter" strand (in red).

biological evidence. Moreover, the model immediately suggested a mechanism for the transmission of genetic information. The essential feature of the model is the complementarity of the two DNA strands. As Watson and Crick were able to see, well before confirmatory data became available, this structure could logically be replicated by (1) separating the two strands and (2) synthesizing a complementary strand for each. Because nucleotides in each new strand are joined in a sequence specified by the base-pairing rules stated above, each preexisting strand functions as a template to guide the synthesis of one complementary strand (Fig. 8-17). These expectations were experimentally confirmed, inaugurating a revolution in our understanding of biological inheritance.

DNA Can Occur in Different Three-Dimensional Forms

DNA is a remarkably flexible molecule. Considerable rotation is possible around a number of bonds in the sugar-phosphate (phosphodeoxyribose) backbone, and thermal fluctuation can produce bending, stretching, and unpairing (melting) of the strands. Many significant deviations from the Watson-Crick DNA structure are found in cellular DNA, some or all of which may play important roles in DNA metabolism. These structural variations generally do not affect the key properties of DNA defined by Watson and Crick: strand complementarity,

FIGURE 8-18 Structural variation in DNA. (a) The conformation of a nucleotide in DNA is affected by rotation about seven different bonds. Six of the bonds rotate freely. The limited rotation about bond 4 gives rise to ring pucker, in which one of the atoms in the five-membered furanose ring is out of the plane described by the other four. This conformation is endo or exo, depending on whether the atom is displaced to the same side of the plane as C-5' or to the opposite side (see Fig. 8-3b). (b) For purine bases in nucleotides, only two conformations with respect to the attached ribose units are sterically permitted, anti or syn. Pyrimidines generally occur in the anti conformation.

FIGURE 8-18 Structural variation in DNA. (a) The conformation of a nucleotide in DNA is affected by rotation about seven different bonds. Six of the bonds rotate freely. The limited rotation about bond 4 gives rise to ring pucker, in which one of the atoms in the five-membered furanose ring is out of the plane described by the other four. This conformation is endo or exo, depending on whether the atom is displaced to the same side of the plane as C-5' or to the opposite side (see Fig. 8-3b). (b) For purine bases in nucleotides, only two conformations with respect to the attached ribose units are sterically permitted, anti or syn. Pyrimidines generally occur in the anti conformation.

antiparallel strands, and the requirement for A=T and GqC base pairs.

Structural variation in DNA reflects three things: the different possible conformations of the deoxyribose, rotation about the contiguous bonds that make up the phosphodeoxyribose backbone (Fig. 8-18a), and free rotation about the C-1'-A^-glycosyl bond (Fig. 8-18b). Because of steric constraints, purines in purine nu-cleotides are restricted to two stable conformations with respect to deoxyribose, called syn and anti (Fig. 8-18b). Pyrimidines are generally restricted to the anti conformation because of steric interference between the sugar and the carbonyl oxygen at C-2 of the pyrimidine.

The Watson-Crick structure is also referred to as B-form DNA, or B-DNA. The B form is the most stable structure for a random-sequence DNA molecule under physiological conditions and is therefore the standard point of reference in any study of the properties of DNA. Two structural variants that have been well characterized in crystal structures are the A and Z forms. These three DNA conformations are shown in Figure 8-19, with a summary of their properties. The A form is favored in many solutions that are relatively devoid of water. The DNA is still arranged in a right-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

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  • eija-riitt
    How many bonds rotate for the confirmation of a nucleotide in DNA?
    2 years ago

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