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《生物化学原理》(英文版)chapter 8 NUCLEOTIDES AND NUCLEIC ACIDS

8.1 Some Basics 273 quence in the cells DNA. A segment of a DNA molecule 8.2 Nucleic Acid Structure 275 that contains the information required for the synthesis 8.3 Nucleic Acid Chemistry 291 8.4 Other Functions of Nucleotides 300 the only known func- Watson,
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chapte NUCLEOTIDES AND NUCLEIC ACIDS 8.1 Some basics 273 quence in the cells DNA. A segment of a DNA molecule 8.2 Nucleic Acid structure 279 that contains the information required for the synthes of a functional biological product, whether protein or 8.3 Nucleic Acid Chemistry 291 RNA, is referred to as a gene. a cell typically has many 8.4 Other Functions of Nucleotides 300 thousands of genes, and DNA molecules, not surpris- ingly, tend to be very large. The storage and transmis- sion of biological information are the only known func A structure this pretty just had to exist ons of dna. -lames Watson. The Double helix. 1968 RNAs have a broader range of functions, and sev- eral classes are found in cells. Ribosomal rnas (rRNAs) are components of ribosomes, the complexes cleotides have a variety of roles in cellular metab- that carry out the synthesis of proteins. Messenger olism. They are the energy currency in metabolic RNAs(mRNAs)are intermediaries, carrying genetic transactions, the essential chemical links in the re- information from one or a few genes to a ribosome sponse of cells to hormones and other extracellular stim- where the corresponding proteins can be synthesized uli, and the structural components of an array of en- Transfer RNAs(tRNas) are adapter molecules that zyme cofactors and metabolic intermediates. And, last faithfully translate the information in mRNA into a but certainly not least, they are the constituents of nu- specific sequence of amino acids. In addition to these cleic acids: deoxyribonucleic acid(DNA)and ribonu- major classes there is a wide variety of RNAs with spe- cleic acid(RNA), the molecular repositories of genetic cial functions, described in depth in Part Ill information. The structure of every protein, and ulti- mately of every biomolecule and cellular component, is Nucleotides and Nucleic Acids Have Characteristic a product of information programmed into the nu- Bases and Pentoses cleotide sequence of a cells nucleic acids. The ability to store and transmit genetic information from one gener Nucleotides have three characteristic components ation to the next is a fundamental condition for life (1)a nitrogenous(nitrogen-containing) base, (2)apen This chapter provides an overview of the chemical se, and(3)phosphate(Fig. 8-1). The molecule with nature of the nucleotides and nucleic acids found in out the phosphate group is called a nucleoside. The most cells: a more detailed examination of the function nitrogenous bases are derivatives of two parent com- of nucleic acids is the focus of part ill of this text. pounds, pyrimidine and purine. The bases and pentoses of the common nucleotides are heterocyclic compounds 8. 1 Some basics The carbon and nitrogen atoms in the parent structures are conventionally numbered to facilitate the naming Nucleotides,Building Blocks of Nucleic Acids The amino acid and identification of the many derivative compounds sequence of every protein in a cell, and the nucleotide The convention for the pentose ring follows rules out sequence of every rNA, is specified by a nucleotide se- lined in Chapter 7, but in the pentoses of nucleotides

chapter Nucleotides have a variety of roles in cellular metab￾olism. They are the energy currency in metabolic transactions, the essential chemical links in the re￾sponse of cells to hormones and other extracellular stim￾uli, and the structural components of an array of en￾zyme cofactors and metabolic intermediates. And, last but certainly not least, they are the constituents of nu￾cleic acids: deoxyribonucleic acid (DNA) and ribonu￾cleic acid (RNA), the molecular repositories of genetic information. The structure of every protein, and ulti￾mately of every biomolecule and cellular component, is a product of information programmed into the nu￾cleotide sequence of a cell’s nucleic acids. The ability to store and transmit genetic information from one gener￾ation to the next is a fundamental condition for life. This chapter provides an overview of the chemical nature of the nucleotides and nucleic acids found in most cells; a more detailed examination of the function of nucleic acids is the focus of Part III of this text. 8.1 Some Basics Nucleotides, Building Blocks of Nucleic Acids The amino acid sequence of every protein in a cell, and the nucleotide sequence of every RNA, is specified by a nucleotide se￾quence in the cell’s DNA. A segment of a DNA molecule that contains the information required for the synthesis of a functional biological product, whether protein or RNA, is referred to as a gene. A cell typically has many thousands of genes, and DNA molecules, not surpris￾ingly, tend to be very large. The storage and transmis￾sion of biological information are the only known func￾tions of DNA. RNAs have a broader range of functions, and sev￾eral classes are found in cells. Ribosomal RNAs (rRNAs) are components of ribosomes, the complexes that carry out the synthesis of proteins. Messenger RNAs (mRNAs) are intermediaries, carrying genetic information from one or a few genes to a ribosome, where the corresponding proteins can be synthesized. Transfer RNAs (tRNAs) are adapter molecules that faithfully translate the information in mRNA into a specific sequence of amino acids. In addition to these major classes there is a wide variety of RNAs with spe￾cial functions, described in depth in Part III. Nucleotides and Nucleic Acids Have Characteristic Bases and Pentoses Nucleotides have three characteristic components: (1) a nitrogenous (nitrogen-containing) base, (2) a pen￾tose, and (3) a phosphate (Fig. 8–1). The molecule with￾out the phosphate group is called a nucleoside. The nitrogenous bases are derivatives of two parent com￾pounds, pyrimidine and purine. The bases and pentoses of the common nucleotides are heterocyclic compounds. The carbon and nitrogen atoms in the parent structures are conventionally numbered to facilitate the naming and identification of the many derivative compounds. The convention for the pentose ring follows rules out￾lined in Chapter 7, but in the pentoses of nucleotides NUCLEOTIDES AND NUCLEIC ACIDS 8.1 Some Basics 273 8.2 Nucleic Acid Structure 279 8.3 Nucleic Acid Chemistry 291 8.4 Other Functions of Nucleotides 300 A structure this pretty just had to exist. —James Watson, The Double Helix, 1968 8 273

274 Chapter 8 Nucleotides and Nucleic Acids NH: pyrimidin CH Phaphate-0-P-Io-cH Adenine Oum ine Purines H OH HN aCH grimme FurT towne FIGURE 8-1 Structure of nucleotides. (a) General structure showing (RNAy Pyrimidines the numbering convention for the pentose ring. This is a ribonu- cleotide In deoxyribonucleotides the -OH group on the 2' carbon FIGURE 8-2 Major purine and pyrimidine bases of nucleic acids (in red) is replaced with-H. (b) The parent compounds of the pyrim- Some of the common names of these bases reflect the circumstances idine and purine bases of nucleotides and nucleic acids, showing the of their discovery Guanine, for example, was first isolated from guano (bird manure), and thymine was first isolated from thymus tissue and nucleosides the carbon numbers are given a prime long sequences of A, T, G, and C nucleotides in DNA are ()designation to distinguish them from the numbered the repository of genetic information. atoms of the nitrogenous bases. Although nucleotides bearing the major purines and The base of a nucleotide is joined covalently(at N-1 pyrimidines are most common, both DNA and rNa also of pyrimidines and N-9 of purines) in an N-B-glycosyl bond to the 1' carbon of the pentose, and the phosphate is esterified to the 5 carbon. The M-B-glycosyl bond is formed by removal of the elements of water (a hydroxyl group from the pentose and hydrogen from the base as in Glycosidic bond formation(see Fig. 7-31) H-C-OH Both DNA and RNa contain two major purine bases adenine(A)and guanine(G), and two major pyrim dines. In both DNA and RNa one of the pyrimidines is cytosine(C), but the second major pyrimidine is not CHOH the same in both: it is thymine(t)in DNA and uracil (a) Aldehyde 各 Furanose (U)in RNA. Only rarely does thymine occur in RNA or uracil in DNA. The structures of the five major bases corresponding nure 8-2, and the nomenclature of their shown in fi nucleotides and nucleosides is summa. C-s end rized in table 8-1 Nucleic acids have two kinds of pentoses. The urring deoxyribonucleotide units of DNA contain 2 deoxy-D-ribose, and the ribonucleotide units of RNA CA,ao ontain D-ribose. In nucleotides, both types of pentoses (b) C2 exe are in their B-furanose(closed five-membered ring FIGURE 8-3 Conformations of ribose(a)In solution, the straight form. As Figure 8-3 shows, the pentose ring is not pla- chain(aldehyde)and ring(B-furanose) forms of free ribose are in equi. nar but occurs in one of a variety of conformations gen- librium. RNA contains only the ring form, B- D-ribofuranose. Deoxy erally described as"puckered. ribose undergoes a similar interconversion in solution, but in DNA Figure 8-4 gives the structures and names of the exists solely as B-2 -deoxy-D-ribofuranose. (b)Ribofuranose rings in four major deoxyribonucleotides(deoxyribonucleo- nucleotides can exist in four different puckered conformations In all side 5'-monophosphates), the structural units of DNAS, cases, four of the five atoms are in a single plane. The fifth atom major ribonucleotides(ribonucleoside 5. (C-2' or C-3) is on either the same (endo) or the opposite(exo)side monophosphates), the structural units of RNAs. Specific of the plane relative to the C-5'atom

and nucleosides the carbon numbers are given a prime () designation to distinguish them from the numbered atoms of the nitrogenous bases. The base of a nucleotide is joined covalently (at N-1 of pyrimidines and N-9 of purines) in an N--glycosyl bond to the 1 carbon of the pentose, and the phosphate is esterified to the 5 carbon. The N--glycosyl bond is formed by removal of the elements of water (a hydroxyl group from the pentose and hydrogen from the base), as in O-glycosidic bond formation (see Fig. 7–31). Both DNA and RNA contain two major purine bases, adenine (A) and guanine (G), and two major pyrim￾idines. In both DNA and RNA one of the pyrimidines is cytosine (C), but the second major pyrimidine is not the same in both: it is thymine (T) in DNA and uracil (U) in RNA. Only rarely does thymine occur in RNA or uracil in DNA. The structures of the five major bases are shown in Figure 8–2, and the nomenclature of their corresponding nucleotides and nucleosides is summa￾rized in Table 8–1. Nucleic acids have two kinds of pentoses. The re￾curring deoxyribonucleotide units of DNA contain 2- deoxy-D-ribose, and the ribonucleotide units of RNA contain D-ribose. In nucleotides, both types of pentoses are in their -furanose (closed five-membered ring) form. As Figure 8–3 shows, the pentose ring is not pla￾nar but occurs in one of a variety of conformations gen￾erally described as “puckered.” Figure 8–4 gives the structures and names of the four major deoxyribonucleotides (deoxyribonucleo￾side 5-monophosphates), the structural units of DNAs, and the four major ribonucleotides (ribonucleoside 5- monophosphates), the structural units of RNAs. Specific long sequences of A, T, G, and C nucleotides in DNA are the repository of genetic information. Although nucleotides bearing the major purines and pyrimidines are most common, both DNA and RNA also 274 Chapter 8 Nucleotides and Nucleic Acids FIGURE 8–1 Structure of nucleotides. (a) General structure showing the numbering convention for the pentose ring. This is a ribonu￾cleotide. In deoxyribonucleotides the OOH group on the 2 carbon (in red) is replaced with OH. (b) The parent compounds of the pyrim￾idine and purine bases of nucleotides and nucleic acids, showing the numbering conventions. (b) (a) FIGURE 8–2 Major purine and pyrimidine bases of nucleic acids. Some of the common names of these bases reflect the circumstances of their discovery. Guanine, for example, was first isolated from guano (bird manure), and thymine was first isolated from thymus tissue. FIGURE 8–3 Conformations of ribose. (a) In solution, the straight￾chain (aldehyde) and ring (-furanose) forms of free ribose are in equi￾librium. RNA contains only the ring form, -D-ribofuranose. Deoxy￾ribose undergoes a similar interconversion in solution, but in DNA exists solely as -2-deoxy-D-ribofuranose. (b) Ribofuranose rings in nucleotides can exist in four different puckered conformations. In all cases, four of the five atoms are in a single plane. The fifth atom (C-2 or C-3) is on either the same (endo) or the opposite (exo) side of the plane relative to the C-5 atom

NH C -0--P-0--cH O 0-P-0-CH OH H OH H A dA dAMP G dG, dGMP T d. dTMP C, dC, dCMP (a) Deoxyribonucleotides HaN 0-P-0-CH 0→P-0—CH 0-P-0 0-P-0 OHOH OH OH Nucleotide: Cytidylate(cytidine monophosphate) 5′· monophosphate) 5b包 A, AMP G GMP U UMP C, CMP Uridine (b) Ribonucleotides FIGURE 8-4 Deoxyribonucleotides and ribonucleotides of nucleic GMP, UMP, and CMP. For each nucleotide, the more common name cids. All nucleotides are shown in their free form at pH 70. The nu- is followed by the complete name in parentheses. All abbreviations cleotide units of DNA (a)are usually symbolized as A, G, T, and C, assume that the phosphate group is at the 5 position. The nucleoside sometimes as dA, dG, dT, and dC: those of RNA (b)as A, G, U, and portion of each molecule is shaded in light red. In this and the fol- C In their free form the deoxyribonucleotides are commonly abbre. lowing illustrations, the ring carbons are not shown viated dAMP dgMP dTMP and dCMP. the ribonucleotides AMP lE 8-1 Nucleotide and Nucleic Acid Nomenclature Nucleoside Nucleotide Nucleic acid Adenine Adenosine Note: 'Nucleoside and '" are Deoxyadenosine genenic terms that include both ribo- and Guanine Guanosine ribonucleotides are here desimated simpl as nucleosides and nucleotides(eg, nibo- adenosine as adenosine), and deox- Cytidylate nucleosides and daarynibonucleotides as Deoxycytidir Deoxycytidylate Thymidine or deoxythymidine Thymidylate or deoxythymidylate DNA Uridine RNA able, but the shortened names are more mmonly used Thymine is an exception

8.1 Some Basics 275 O CH2 O OH H P CH3 O HN N H H H H O T, dT, dTMP Deoxythymidine Nucleotide: Deoxyadenylate (deoxyadenosine 5-monophosphate) Deoxyguanylate (deoxyguanosine 5-monophosphate) Deoxythymidylate (deoxythymidine 5-monophosphate) Deoxycytidylate (deoxycytidine 5-monophosphate) Symbols: A, dA, dAMP Nucleoside: Deoxyadenosine O G, dG, dGMP Deoxyguanosine O C, dC, dCMP Deoxycytidine (a) Deoxyribonucleotides O O CH2 N O O OH H P NH2 O N N N H H H H O O O CH2 O OH H P HN H2N O N N N H H H H O O O CH2 O OH H P NH2 O N N H H H H O O O O CH2 N O O OH H P NH2 O N N N H H H O O O CH2 O OH H P HN H2N O N N N H H H O O O CH2 O OH H P O N N H H H O O (b) Ribonucleotides U, UMP C, CMP Uridine Nucleotide: Adenylate (adenosine 5-monophosphate) Guanylate (guanosine 5-monophosphate) Uridylate (uridine 5-monophosphate) Cytidylate (cytidine 5-monophosphate) Symbols: A, AMP Nucleoside: Adenosine G, GMP Guanosine Cytidine O CH2 O OH H P NH2 O N N H H H O O O OH OH OH OH H O O FIGURE 8–4 Deoxyribonucleotides and ribonucleotides of nucleic acids. All nucleotides are shown in their free form at pH 7.0. The nu￾cleotide units of DNA (a) are usually symbolized as A, G, T, and C, sometimes as dA, dG, dT, and dC; those of RNA (b) as A, G, U, and C. In their free form the deoxyribonucleotides are commonly abbre￾viated dAMP, dGMP, dTMP, and dCMP; the ribonucleotides, AMP, GMP, UMP, and CMP. For each nucleotide, the more common name is followed by the complete name in parentheses. All abbreviations assume that the phosphate group is at the 5 position. The nucleoside portion of each molecule is shaded in light red. In this and the fol￾lowing illustrations, the ring carbons are not shown. 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., ribo￾adenosine as adenosine), and deoxyribo￾nucleosides and deoxyribonucleotides as deoxynucleosides and deoxynucleotides (e.g., deoxyriboadenosine as deoxyadeno￾sine). Both forms of naming are accept￾able, but the shortened names are more commonly used. Thymine is an exception; “ribothymidine” is used to describe its unusual occurrence in RNA.

76 Chapter 8 Nucleotides and Nucleic Acids here)is simply to indicate the ring position of the sub- stituent by its number-for example, 5-methylcytosine 7-methylguanine, and 5-hydroxymethylcytosine(shown as the nucleosides in Fig. 8-5). The element to which the substituent is attached (N, C, O)is not identified. The convention changes when the substituted atom is exocyclic(not within the ring structure), in which case s-Alchyleytidno N Methy ladanoame the type of atom is identified and the ring position to which it is attached is denoted with a superscript. The amino nitrogen attached to C-6 of adenine is N, simi- CH OH arly, the carbonyl oxygen and amino nitrogen at C-6 and C-2 of guanine are O' and N, respectively. Examples of this nomenclature are N-methyladenosine and methylguanosine(Fig. 8-5) Riase Cells also contain nucleotides with phosphate (a) N2Mcthylgunasine groups in positions other than on the 5 carbon(Fig 8-6). Ribonucleoside 2, 3 -cyclic monophosphates are isolatable intermediates. and ribonucleoside 3 monophosphates are end products of the hydrolysis of rna by certain ribonucleases. Other variations are adenosine 3, 5'-cyclic monophosphate(CAMP)and guanosine 3, 5'-cyclic monophosphate(cGMP), consid ed at the end of this chapte Inosine Pseudouridine Phosphodiester Bonds Link Successive Nucleotides Nucleic acids The successive nucleotides of both dna and rna are HN N covalently linked through phosphate-group"bridges, "in Ribose which the 5-phosphate group of one nucleotide unit is Thicurdine Adenine FIGURE 8-5 Some minor purine and pyrimidine bases, shown as the HO→CH nucleosides. (a)Minor bases of DNA. 5-Methylcytidine occurs in the DNA of animals and higher plants, N"-methyladenosine in bacterial Adne DNA, and 5-hydroxymethylcytidine in the DNA of bacteria infected"o-p-o with certain bacteriophages. (b)Some minor bases of tRNAS Inosine H contains the base hypoxanthine. Note that pseudouridine, like uridine contains uracil; they are distinct in the point of attachment to the ribose-in uridine, uracil is attached through N-1. the usual attach- ent point for pyrimidines: in pseudouridine, through C-5 Adnasna 6monophosphate idanasne 2-mm ophasphako contain some minor bases( Fig. 8-5). In DNa the most Aden ng common of these are methylated forms of the major bases: in some viral DNAs, certain bases may be hy- droxymethylated or glucosylated. Altered or unusual bases in DNA molecules often have roles in regulating or protecting the genetic information. Minor bases of -p0 many types are also found in RNAs, especially in tRNAs (see Fig. 26-24) The nomenclature for the minor bases can be con- Adenosine s'-mmophosphat Adenoxine 2 3'syelie fusing, Like the major bases, many have common names- hypoxanthine, for example, shown as its nucleoside ino- FIGURE 8-6 Some adenosine monophosphates. Adenosine 2" sine in Figure 8-5. When an atom in the purine or monophosphate, 3-monophosphate, and 2 3 -cyclic monophosphate pyrimidine ring is substituted, the usual convention(used are formed by enzymatic and alkaline hydrolysis of RNA

contain some minor bases (Fig. 8–5). In DNA the most common of these are methylated forms of the major bases; in some viral DNAs, certain bases may be hy￾droxymethylated or glucosylated. Altered or unusual bases in DNA molecules often have roles in regulating or protecting the genetic information. Minor bases of many types are also found in RNAs, especially in tRNAs (see Fig. 26–24). The nomenclature for the minor bases can be con￾fusing. Like the major bases, many have common names— hypoxanthine, for example, shown as its nucleoside ino￾sine in Figure 8–5. When an atom in the purine or pyrimidine ring is substituted, the usual convention (used here) is simply to indicate the ring position of the sub￾stituent by its number—for example, 5-methylcytosine, 7-methylguanine, and 5-hydroxymethylcytosine (shown as the nucleosides in Fig. 8–5). The element to which the substituent is attached (N, C, O) is not identified. The convention changes when the substituted atom is exocyclic (not within the ring structure), in which case the type of atom is identified and the ring position to which it is attached is denoted with a superscript. The amino nitrogen attached to C-6 of adenine is N6 ; simi￾larly, the carbonyl oxygen and amino nitrogen at C-6 and C-2 of guanine are O6 and N2 , respectively. Examples of this nomenclature are N6 -methyladenosine and N2 - methylguanosine (Fig. 8–5). Cells also contain nucleotides with phosphate groups in positions other than on the 5 carbon (Fig. 8–6). Ribonucleoside 2,3-cyclic monophosphates are isolatable intermediates, and ribonucleoside 3- monophosphates are end products of the hydrolysis of RNA by certain ribonucleases. Other variations are adenosine 3,5-cyclic monophosphate (cAMP) and guanosine 3,5-cyclic monophosphate (cGMP), consid￾ered at the end of this chapter. Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids The successive nucleotides of both DNA and RNA are covalently linked through phosphate-group “bridges,” in which the 5-phosphate group of one nucleotide unit is 276 Chapter 8 Nucleotides and Nucleic Acids (a) (b) FIGURE 8–5 Some minor purine and pyrimidine bases, shown as the nucleosides. (a) Minor bases of DNA. 5-Methylcytidine occurs in the DNA of animals and higher plants, N6 -methyladenosine in bacterial DNA, and 5-hydroxymethylcytidine in the DNA of bacteria infected with certain bacteriophages. (b) Some minor bases of tRNAs. Inosine contains the base hypoxanthine. Note that pseudouridine, like uridine, contains uracil; they are distinct in the point of attachment to the ribose—in uridine, uracil is attached through N-1, the usual attach￾ment point for pyrimidines; in pseudouridine, through C-5. FIGURE 8–6 Some adenosine monophosphates. Adenosine 2- monophosphate, 3-monophosphate, and 2,3-cyclic monophosphate are formed by enzymatic and alkaline hydrolysis of RNA

DNA joined to the 3-hydroxyl group of the next nucleotide, 5′End 5 End linkage(Fig. 8-7) the covalent backbones of nucleic acids consist of al o-P=0 ternating phosphate and pentose residues, and the trogenous bases may be regarded as side groups joined to the backbone at regular intervals. The backbones of 5 CH both DNA and RNa are hydrophilic. The hydroxy groups of the sugar residues form hydrogen bonds with water. The phosphate groups, with a pKa near 0. are completely ionized and negatively charged at pH 7, and Phospho- the negative charges are generally neutralized by ionic 0-P=0 0-P=0 interactions with positive charges on proteins, metal ions, and polyamines All the phosphodiester linkages have the same ori- 5 CHz 5° entation along the chain(Fig. 8-7), giving each linear nucleic acid strand a specific polarity and distinct 5'and 3ends. By definition, the 5 end lacks a nucleotide at the 5 position and the 3 end lacks a nucleotide at the 3 position. Other groups(most often one or more phos- 0P=0 phates) may be present on one or both ends The covalent backbone of DNA and RNa is subject 5 CH O 5 CH to slow, nonenzymatic hydrolysis of the phosphodiester bonds. In the test tube, RNA is hydrolyzed rapidly un der alkaline conditions, but DNA is not; the 2-hydroxyl groups in RNA (absent in DNA) are directly involved in 9 OH the process. Cyclic 2, 3-monophosphate nucleotides are the first products of the action of alkali on rNa and 3 End 3 End are rapidly hydrolyzed further to yield a mixture of 2 and 3'-nucleoside monophosphates(Fig. 8-8) FIGURE 8-7 Phosphodiester linkages in the covalent backbone of The nucleotide sequences of nucleic acids can be DNA and RNA. The phosphodiester bonds (one of which is shaded in represented schematically, as illustrated on the follow the DNA) link successive nucleotide units. The backbone of alternat. ing page by a segment of DNA with five nucleotide units ing pentose and phosphate groups in both types of nucleic acid is The phosphate groups are symbolized by and each highly polar. The 5 end of the macromolecule lacks a nucleotide at deoxyribose is symbolized by a vertical line, from C-l at the top to C-5 at the bottom(but keep in mind that deriva 00 0-H OH P- CH2oBasez FIGURE 8-8 Hydrolysis of RNA under alkaline in an intramolecular displacement. The 2, 3'-cyclic monophosphate derivative is further hydrolyzed to OH Shortened 0 OH a mixture of2·and3’, monophosphates.DNA RNA0-P=0 o-P=0 which lacks 2 hydroxyls, is stable under similar conditions

joined to the 3-hydroxyl group of the next nucleotide, creating a phosphodiester linkage (Fig. 8–7). Thus the covalent backbones of nucleic acids consist of al￾ternating phosphate and pentose residues, and the ni￾trogenous bases may be regarded as side groups joined to the backbone at regular intervals. The backbones of both DNA and RNA are hydrophilic. The hydroxyl groups of the sugar residues form hydrogen bonds with water. The phosphate groups, with a pKa near 0, are completely ionized and negatively charged at pH 7, and the negative charges are generally neutralized by ionic interactions with positive charges on proteins, metal ions, and polyamines. All the phosphodiester linkages have the same ori￾entation along the chain (Fig. 8–7), giving each linear nucleic acid strand a specific polarity and distinct 5 and 3 ends. By definition, the 5 end lacks a nucleotide at the 5 position and the 3 end lacks a nucleotide at the 3 position. Other groups (most often one or more phos￾phates) may be present on one or both ends. The covalent backbone of DNA and RNA is subject to slow, nonenzymatic hydrolysis of the phosphodiester bonds. In the test tube, RNA is hydrolyzed rapidly un￾der alkaline conditions, but DNA is not; the 2-hydroxyl groups in RNA (absent in DNA) are directly involved in the process. Cyclic 2,3-monophosphate nucleotides are the first products of the action of alkali on RNA and are rapidly hydrolyzed further to yield a mixture of 2- and 3-nucleoside monophosphates (Fig. 8–8). The nucleotide sequences of nucleic acids can be represented schematically, as illustrated on the follow￾ing page by a segment of DNA with five nucleotide units. The phosphate groups are symbolized by P, and each deoxyribose is symbolized by a vertical line, from C-1 at the top to C-5 at the bottom (but keep in mind that 8.1 Some Basics 277 O RNA CH2 O O H P H OH H O 3 5 U H O CH2 O O H P H H O O 3 5 G H O CH2 O O H P H H O O 3 5 H O H O 5 End O CH2 O O H P H H H O 3 5 A H O CH2 O O H P H H H O O 3 5 T H O CH2 O O H P H H H O O 3 5 G H O H O 5 End 3 End 3 End C 5 3 DNA Phospho￾diester linkage OH OH FIGURE 8–7 Phosphodiester linkages in the covalent backbone of DNA and RNA. The phosphodiester bonds (one of which is shaded in the DNA) link successive nucleotide units. The backbone of alternat￾ing pentose and phosphate groups in both types of nucleic acid is highly polar. The 5 end of the macromolecule lacks a nucleotide at the 5 position, and the 3 end lacks a nucleotide at the 3 position. H P H H H O OH 2,3-Cyclic monophosphate derivative O O CH2 O H P H H H O O Base1 O O O H CH2 O H P H H H O O Base2 O O H P O O CH2 H H H H O O Base2 O H P O O OH Base1 P O O O Mixture of 2- and 3-monophosphate derivatives CH2 O O RNA Shortened RNA H2O O RNA Shortened RNA FIGURE 8–8 Hydrolysis of RNA under alkaline conditions. The 2 hydroxyl acts as a nucleophile in an intramolecular displacement. The 2,3-cyclic monophosphate derivative is further hydrolyzed to a mixture of 2- and 3-monophosphates. DNA, which lacks 2 hydroxyls, is stable under similar conditions.

278 Chapter 8 Nucleotide les and Nucleic Acids the sugar is always in its closed-ring B-furanose form in nucleic acids). The connecting lines between nucleotides (which pass through ( )are drawn diagonally from the middle(C-3) of the deoxyribose of one nucleotide to HON the bottom( c-5)of the next. Lactam Lactim Du bo ledi AC G T A Uracil 5 End 3 End FIGURE 8-9 Tautomeric forms of uracil. The lactam form predomi nates at pH 7.0; the other forms become more prominent as pH de- creases. The other free pyrimidines and the free purines also have tau- homeric forms, but they are more rarely encountered By convention, the structure of a single strand of nu- cleic acid is always written with the 5 end at the left and the 3 end at the right-that is, in the 5'-3' di rection. Some simpler representations of this pentade. planar, with a slight pucker. Free pyrimidine and purine oxyribonucleotide are pA-C-G-T-AoH, pApCpGp'TpA, bases may exist in two or more tautomeric forms de- and pACGTA pending on the pH Uracil, for example, occurs in a short nucleic acid is referred to as an oligonu tam, lactim, and double lactim forms(Fig.8-9).The leotide. The definition ofshort" is somewhat arbi- structures shown in Figure 8-2 are the tautomers that rary, but polymers containing 50 or fewer nucleotide predominate at pH 7.0. As a result of resonance, all nu- are generally called oligonucleotides. A longer nucleic cleotide bases absorb UV light, and nucleic acids are acid is called a polynucleotide characterized by a strong absorption at wavelengths 50m(Fg.8-10 The Properties of Nucleotide Bases Affect the three-Dimensional structure of nucleic acids ally he purine and pyrimidine bases are hydrophobic relatively insoluble in water at the near-neutral pH of the cell. At acidic or alkaline ph the bases become Free pyrimidines and purines are weakly basic com- charged and their solubility in water increases. Hy- pounds and are thus called bases. They have a variety drophobic stacking interactions in which two or more of chemical properties that affect the structure, and bases are positioned with the planes of their rings par ultimately the function, of nucleic acids. The purines (like a stack of coins) are one of two important and pyrimidines common in DNA and RNa are highly modes of interaction between bases in nucleic acids. The conjugated molecules(Fig. 8-2), a property with im- stacking also involves a combination of van der Waals portant consequences for the structure, electron distri- and dipole-dipole interactions between the bases. Base bution, and light absorption of nucleic acids. Resonance stacking helps to minimize contact of the bases with wa- among atoms in the ring gives most of the bonds par- ter, and base-stacking interactions are very important in tial double-bond character. One result is that pyrim- stabilizing the three-dimensional structure of nucleic dines are planar molecules; purines are very nearly acids, as described later. 14,000 10.000 Molar extinction FIGURE 8-10 Absorption spectra of the coefficient at 260 nm, common nucleotides. The spectra are 8.000 ∈2(Mcm-) shown as the variation in molar extinctio AMP15,400 coefficient with wavelength. The molar extinction coefficients at 260 nm and 型4000 pH 7.0 (eto) are listed in the table. The UMP 9900 spectra of corresponding ribonucleotides 2.000 dTMP 9 200 and deoxyribonucleotides, as well as the nucleosides, are essentially identical.For mixtures of nucleotides, a wavelength of 230240250260270280 260 nm(dashed vertical line) is used for

the sugar is always in its closed-ring -furanose form in nucleic acids). The connecting lines between nucleotides (which pass through P) are drawn diagonally from the middle (C-3) of the deoxyribose of one nucleotide to the bottom (C-5) of the next. By convention, the structure of a single strand of nu￾cleic acid is always written with the 5 end at the left and the 3 end at the right—that is, in the 5 n 3 di￾rection. Some simpler representations of this pentade￾oxyribonucleotide are pA-C-G-T-AOH, pApCpGpTpA, and pACGTA. A short nucleic acid is referred to as an oligonu￾cleotide. The definition of “short” is somewhat arbi￾trary, but polymers containing 50 or fewer nucleotides are generally called oligonucleotides. A longer nucleic acid is called a polynucleotide. The Properties of Nucleotide Bases Affect the Three-Dimensional Structure of Nucleic Acids Free pyrimidines and purines are weakly basic com￾pounds and are thus called bases. They have a variety of chemical properties that affect the structure, and ultimately the function, of nucleic acids. The purines and pyrimidines common in DNA and RNA are highly conjugated molecules (Fig. 8–2), a property with im￾portant consequences for the structure, electron distri￾bution, and light absorption of nucleic acids. Resonance among atoms in the ring gives most of the bonds par￾tial double-bond character. One result is that pyrim￾idines are planar molecules; purines are very nearly planar, with a slight pucker. Free pyrimidine and purine bases may exist in two or more tautomeric forms de￾pending on the pH. Uracil, for example, occurs in lac￾tam, lactim, and double lactim forms (Fig. 8–9). The structures shown in Figure 8–2 are the tautomers that predominate at pH 7.0. As a result of resonance, all nu￾cleotide bases absorb UV light, and nucleic acids are characterized by a strong absorption at wavelengths near 260 nm (Fig. 8–10). The purine and pyrimidine bases are hydrophobic and relatively insoluble in water at the near-neutral pH of the cell. At acidic or alkaline pH the bases become charged and their solubility in water increases. Hy￾drophobic stacking interactions in which two or more bases are positioned with the planes of their rings par￾allel (like a stack of coins) are one of two important modes of interaction between bases in nucleic acids. The stacking also involves a combination of van der Waals and dipole-dipole interactions between the bases. Base stacking helps to minimize contact of the bases with wa￾ter, and base-stacking interactions are very important in stabilizing the three-dimensional structure of nucleic acids, as described later. 278 Chapter 8 Nucleotides and Nucleic Acids Uracil FIGURE 8–9 Tautomeric forms of uracil. The lactam form predomi￾nates at pH 7.0; the other forms become more prominent as pH de￾creases. The other free pyrimidines and the free purines also have tau￾tomeric forms, but they are more rarely encountered. 14,000 12,000 10,000 8,000 6,000 4,000 2,000 280 Molar extinction coefficient,  Wavelength (nm) 230 240 250 260 270 Molar extinction coefficient at 260 nm, 260 (M1 cm1 ) AMP GMP UMP dTMP CMP 15,400 11,700 9,900 9,200 7,500 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 (260) 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.

8.2 Nucleic Acid Structure 27 H k-2.8A- CHe Adenine H H C HI 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 and exocyclic amino groups. Hydrogen bonds involving I A nucleotide consists of a nitrogenous base the amino and carbonyl groups are the second impor- (purine or pyrimidine), a pentose sugar, and tant mode of interaction between bases in nucleic acid one or more phosphate groups. Nucleic acids molecules. Hydrogen bonds between bases permit a are polymers of nucleotides, joined together by complementary association of two(and occasionally phosphodiester linkages between the 5 three or four) strands of nucleic acid. The most imp hydroxyl group of one pentose and the 3 tant hydrogen-bonding patterns are those defined by James D. Watson and Francis Crick in 1953. in which A hydroxyl group of the next. bonds specifically to T (or U) and G bonds to C(Fi a There are two types of nucleic acid: RNA and 8-11). These two types of base pairs predominate in DNA The nucleotides in rna contain ribose double- stranded dna and rna. and the tautomers and the common pyrimidine bases are uracil shown in Figure 8-2 are responsible for these patterns and cytosine. In dnA, the nucleotides contain It is this specific pairing of bases that permits the du 2-deoxyribose, and the common pyrimidine plication of genetic information, as we shall discuss later 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 in- fluenced the course of many established ones. Our pres ent understanding of the storage and utilization of cell's genetic information is based on work made possi ble by this discovery, and an outline of how genetic in- formation is processed by the cell is now a prerequisite the discussion of any area of biochemistry. Here, James watson Francis Crick ern ourselves with dna structure itself the events

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 impor￾tant 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 impor￾tant 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 du￾plication of genetic information, as we shall discuss later in this chapter. 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 in￾fluenced the course of many established ones. Our pres￾ent understanding of the storage and utilization of a cell’s genetic information is based on work made possi￾ble by this discovery, and an outline of how genetic in￾formation 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 8.2 Nucleic Acid Structure 279 3 C C C G C G G G A A A A A T T T T T 5 5 3 10.8 Å N C O C N C H C C H C N C N C 11.1 Å 2.8 Å 3.0 Å H N C O CH3 C O N H N C H C H C N C C N C H N C N O N H H H H 2.9 Å 3.0 Å 2.9 Å Adenine Thymine Guanine Cytosine N H C-1 C-1 C-1 H H N C N C-1 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. James Watson Francis Crick

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 Ltea capitated acid is its covalent structure and nucleotide regular, stable str cen up by some or all the nucleotides in a nucleic acid can be referred to as dary structure. All structures considered in th mainder of this chapter fall under the heading of sec- dary structure. The complex folding of large chro- 少 mosomes within eukaryotic chromatin and bacterial nucleoids is generally considered tertiary structure; this is discussed in Chapter 24 use fes 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 与 nuclein, "from the nuclei of pus cells (leukocytes)ob tained 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 (c) bateria simulant hadan nuclein and studied its properties, the covalent(pri mary)structure of DNa (as shown in Fig. 8-7)was not known with certainty until the late 1940s Hout killed virulent bacteria Miescher and many others suspected that nuclein (nucleic acid) was associated in some way with cell in- heritance. but the first direct evidence that dna is the bearer of genetic information came in 1944 through 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 L汉eDn bacterium Streptococcus pneumoniae, also known as pneumococcus, genetically transformed a nonvirulent nmvinaiat itur Mes strain of this organism into a virulent form(Fig. 8-12) nonvirulent had eria ee Lnenanvin int bateria FIGURE 8-12 The Avery-MacLeod-Mc Carty experiment. (a)When w INa isolated frmm heat killd vaunt bateria injected into mice, the encapsulated strain of pneumococcus is lethal (b)whereas the nonencapsulated strain, (c) like the heat-killed en- capsulated strain, is harmless.(d) Earlier research by the bacteriol- ist Frederick Griffith had shown that adding heat-killed virulent bac- teria (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 vir- ulent pneumococci, removing the protein as completely as possible, cneateulaled and added this DNA to nonvirulent bacteria. The DNA gained en- trance into the nonvirulent bacteria, which were permanently trans- bateria Iacapsuls ad &Ee vi kat bacteria formed into a virulent strain

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 re￾mainder of this chapter fall under the heading of sec￾ondary structure. The complex folding of large chro￾mosomes 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) ob￾tained 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 (pri￾mary) 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 in￾heritance, 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). 280 Chapter 8 Nucleotides and Nucleic Acids (a) (b) (c) (d) (e) 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 en￾capsulated strain, is harmless. (d) Earlier research by the bacteriolo￾gist Frederick Griffith had shown that adding heat-killed virulent bac￾teria (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 vir￾ulent pneumococci, removing the protein as completely as possible, and added this DNA to nonvirulent bacteria. The DNA gained en￾trance into the nonvirulent bacteria, which were permanently trans￾formed into a virulent strain

Avery and his colleagues concluded that the DNA ex- tracted from the virulent strain carried the inheritable ge- DNA netic message for virulence. Not everyone accepted these conclusions, because protein impurities present in the DNA could have been the carrier of the genetic informa- tion. This possibility was soon eliminated by the finding that treatment of the dNa with proteolytic enzymes did deoxyribonucleases(DNA-hydrolyzing enzymes) A second important experiment provided inde pendent evidence that DNA carries genetic information In 1952 Alfred D. Hershey and Martha Chase used ra dioactive phosphorus ('2P)and radioactive sulfur(S) tracers to show that when the bacterial virus(bacterio- phage)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 en- ters 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 compo- nent 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 radioactive Radioactive 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 organisms age, FIGURE 8-13 The Hershey-Chase experiment. Two batches of iso. nutritional state, or changing environment. topically labeled bacteriophage T2 particles were prepared. One wa labeled with"P in the phosphate groups of the DNA, the other with 4. In all cellular DNAS, regardless of the species, the 35s in the sulfur-containing amino acids of the protein coats (capsids) number of adenosine residues is equal to the (Note that DNA contains no sulfur and viral protein contains no phos- number of thymidine residues(that is, A phorus. )The two batches of labeled phage were then allowed to in. and the number of guanosine residues is equal to fect separate suspensions of unlabeled bacteria. Each suspension of the number of cytidine residues(G=C). From phage-infected cells was agitated in a blender to shear the viral these relationships it follows that the sum of the sids from the bacteria. The bacteria and empty viral coats (called purine residues equals the sum of the pyrimidine ghosts ")were then separated by centrifugation. The cells infected with residues that is a+g=T+c the3"p-labeled phage were found to containP, indicating that the labeled viral DNA had entered the cells; the viral ghosts contained These quantitative relationships, sometimes called radioactivity. The cells infected with 25s-labeled phage were found to Chargaffs rules, were confirmed by many subsequent have no radioactivity after blender treatment, but the viral ghosts con. researchers. They were a key to establishing the three- tained 35s. Progeny virus particles (not shown) were produced in both dimensional structure of DNa and yielded clues to how batches of bacteria some time after the viral coats were removed, in. genetic information is encoded in DNA and passed from dicating that the genetic message for their replication had been in. one generation to the next. troduced by viral DNA, not by viral protein

Avery and his colleagues concluded that the DNA ex￾tracted from the virulent strain carried the inheritable ge￾netic message for virulence. Not everyone accepted these conclusions, because protein impurities present in the DNA could have been the carrier of the genetic informa￾tion. 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 inde￾pendent evidence that DNA carries genetic information. In 1952 Alfred D. Hershey and Martha Chase used ra￾dioactive phosphorus (32P) and radioactive sulfur (35S) tracers to show that when the bacterial virus (bacterio￾phage) 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 en￾ters 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 compo￾nent 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. 32P experiment 35S experiment Radioactive DNA Nonradioactive coat Nonradioactive DNA Radioactive coat Injection Blender treatment shears off viral heads Separation by centrifugation Radioactive Not radioactive Phage Radioactive Not radioactive Bacterial cell 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 phos￾phorus.) The two batches of labeled phage were then allowed to in￾fect separate suspensions of unlabeled bacteria. Each suspension of phage-infected cells was agitated in a blender to shear the viral cap￾sids 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 con￾tained 35S. Progeny virus particles (not shown) were produced in both batches of bacteria some time after the viral coats were removed, in￾dicating that the genetic message for their replication had been in￾troduced by viral DNA, not by viral protein.

282 Chapter 8 Nucleotides and Nucleic Acids 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 haracteristic 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 Rosalind Franklin Maurice wilkins for the x-ray diffraction data but also for the spe- ific a=T and g=c base equivalences discovered by Chargaff and for the other chemical properties of DNa finding that separation of paired DNA strands is more In 1953 Watson and Crick postulated a three- difficult the higher the ratio of G=C to A-T base pairs dimensional model of DNA structure that accounted for Other pairings of bases tend(to varying degrees)to all the available data. It consists of two helical dna destabilize the double-helical structure. chains wound around the same axis to form a right- When Watson and Crick constructed their model handed double helix (see Box 4-1 for an explanation of they had to decide at the outset whether the strands the right- or left-handed sense of a helical structure) of DNA should be parallel or antiparallel--whether The hydrophilic backbones of alternating deoxyribose their 5 3"phosphodiester bonds should run in the same and phosphate groups are on the outside of the double or opposite directions. An antiparallel orientation pro helix, facing the surrounding water. The furanose ring duced the most convincing model, and later work with of each deoxyribose is in the C-2 endo conformation. DNA polymerases( Chapter 25)provided experimental The purine and pyrimidine bases of both strands are evidence that the strands are indeed antiparallel, a find stacked inside the double helix, with their hydrophobic ing ultimately confirmed by x-ray analysis. and nearly planar ring structures very close together To account for the periodicities observed in the x and perpendicular to the long axis. The offset pairing of ray diffraction patterns of DNA fibers, Watson and Crick the two strands creates a major groove and minor groove on the surface of the duplex(Fig. 8-15). Each manipulated molecular models to arrive at a structure 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 Fig ure 8-11.G with C and a with T are those that fit best rule that in any DNA, G=C and A=T. It is important gimel to note that three hydrogen bonds can form between G and C, symbolized G=C, but only two can form between A and T, symbolized A-T. This is one reason for the M FIGURE 8-15 Watson-Crick model for the structure of dNA. The original model proposed by Watson and Crick had 10 base pairs,or A(3.4 nm), per turn of the helix subsequent measurements revealed FIGURE 8-14 X-ray diffraction pattern of DNA. The spots forming a 10.5 base pairs, or 36 A(3.6 nm), per turn. (a)Schematic represen- cross in the center denote a helical structure. The heavy bands at the tation, showing dimensions of the helix. (b) Stick representation show left and right arise from the recurring bases ing the backbone and stacking of the bases. (c)Space-filling model

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 Å and a secondary one of 34 Å. 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 spe￾cific 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 Fig￾ure 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 AUT. This is one reason for the finding that separation of paired DNA strands is more difficult the higher the ratio of GqC to AUT 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 pro￾duced the most convincing model, and later work with DNA polymerases (Chapter 25) provided experimental evidence that the strands are indeed antiparallel, a find￾ing 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 282 Chapter 8 Nucleotides and Nucleic Acids 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 Å (3.4 nm), per turn of the helix; subsequent measurements revealed 10.5 base pairs, or 36 Å (3.6 nm), per turn. (a) Schematic represen￾tation, showing dimensions of the helix. (b) Stick representation show￾ing the backbone and stacking of the bases. (c) Space-filling model. Rosalind Franklin, 1920–1958 Maurice Wilkins

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