《生物化学原理》（英文版）chapter 8 NUCLEOTIDES AND NUCLEIC ACIDS.pdf_P16-P20

288 Chapter 8 Nucleotides and Nuc eic Acids that the purines can interact. Any self-complementary sequences in the molecule produce more complex struc (a) Monocistronic tures. RNa can base-pair with complementary regions of either RNA or DNA. Base pairing matches the pat 3 tern for DNA: G pairs with C and a pairs with U (or with the occasional T residue in some RNAs). One difference (b)poly cistronic is that base pairing between G and U residues-unusual FIGURE 8-24 Prokaryotic mRNA. Schematic diagrams show (a) in DNA--is fairly common in RNA (see Fig8-27).The monocistronic and (b)polycistronic mRNAs of prokaryotes. Red seg- paired strands in RNA or RNA-DNA duplexes are an ments represent RNA coding for a gene product: gray segments rep. tiparallel, as in DNA resent noncoding RNA In the polycistronic transcript, noncoding RNA RNA has no simple, regular secondary structure separates the three genes that serves as a reference point, as does the double he- lix for DNA. The three-dimensional structures of many RNAS, like those of proteins, are complex and unique if it codes for two or more different polypeptides, the Weak interactions, especially base-stacking interactions. play a major role in stabilizing RNA structures, just as mRNA is polycistronic. In eukaryotes, most mRNAs they do in DNA. Where complementary sequences are are monocistronic. (For the purposes of this discussion, cistron refers to a gene. The term itself has historical present, the predominant double-stranded structure is an A-form right-handed double helix. Z-form helices roots in the science of genetics, and its formal genetic have been made in the laboratory(under very high-salt definition is beyond the scope of this text. The mini mum length of an mRNA is set by the length of th or high-temperature conditions). The B form of RNA has not been observed. Breaks in the regular A-form he- polypeptide chain for which it codes. For example, a lix caused by mismatched or unmatched bases in one polypeptide chain of 100 amino acid residues requires and result in bulges or in termal loops(Fig. 8-26). Hairpin loops form betw (this and other details of protein synthesis are discussed nearby sell-complementary sequences. The potential for Chapter 27). However, mRNAs transcribed from dNA base-paired helical structures in many RNAs is exten- are always somewhat longer than the length needed sim- sive( Fig. 8-27), and the resulting hairpins are the most ply to code for a polypeptide sequence (or sequences). common type of secondary structure in RNA. Speci The additional, noncoding RNa includes sequences that regulate protein synthesis. Figure 8-24 summarizes the general structure of prokaryotic mRNAS Many RNAs Have More Complex Three- Dimensional structures Messenger RNa is only one of several classes of cellu- lar RNA. Transfer RNAs serve as adapter molecules in protein synthesis; covalently linked to an amino acid at one end, they pair with the mRNA in such a way that amino acids are joined to a growing polypeptide in the correct sequence. Ribosomal RNAs are components of ibosomes. There is also a wide variety of special-func tion RNAs, including some(called ribozymes) that have enzymatic activity. All the RNAs are considered in de tail in Chapter 26. The diverse and often complex func tions of these RNAs reflect a diversity of structure much richer than that observed in dna molecules The product of transcription of dNa is always single-stranded RNA. The single strand tends to assume a right-handed helical conformation dominated by base- ng interactions( Fig. 8-25), which are stronger FIGURE 8-25 Typical right-handed stacking pattern of single- tween two purines than between a purine and primi stranded RNA. The bases are shown in gray, the phosphate atoms in dine or between two pyrimidines. The purine-purine yellow, and the riboses and phosphate oxygens in green.Green is used interaction is so strong that a pyrimidine separating two to represent RNA strands in succeeding chapters, just as blue is used purines is often displaced from the stacking pattern for dna

if it codes for two or more different polypeptides, the mRNA is polycistronic. In eukaryotes, most mRNAs are monocistronic. (For the purposes of this discussion, “cistron” refers to a gene. The term itself has historical roots in the science of genetics, and its formal genetic definition is beyond the scope of this text.) The minimum length of an mRNA is set by the length of the polypeptide chain for which it codes. For example, a polypeptide chain of 100 amino acid residues requires an RNA coding sequence of at least 300 nucleotides, because each amino acid is coded by a nucleotide triplet (this and other details of protein synthesis are discussed in Chapter 27). However, mRNAs transcribed from DNA are always somewhat longer than the length needed simply to code for a polypeptide sequence (or sequences). The additional, noncoding RNA includes sequences that regulate protein synthesis. Figure 8–24 summarizes the general structure of prokaryotic mRNAs. Many RNAs Have More Complex Three-Dimensional Structures Messenger RNA is only one of several classes of cellular RNA. Transfer RNAs serve as adapter molecules in protein synthesis; covalently linked to an amino acid at one end, they pair with the mRNA in such a way that amino acids are joined to a growing polypeptide in the correct sequence. Ribosomal RNAs are components of ribosomes. There is also a wide variety of special-function RNAs, including some (called ribozymes) that have enzymatic activity. All the RNAs are considered in detail in Chapter 26. The diverse and often complex functions of these RNAs reflect a diversity of structure much richer than that observed in DNA molecules. The product of transcription of DNA is always single-stranded RNA. The single strand tends to assume a right-handed helical conformation dominated by basestacking interactions (Fig. 8–25), which are stronger between two purines than between a purine and pyrimidine or between two pyrimidines. The purine-purine interaction is so strong that a pyrimidine separating two purines is often displaced from the stacking pattern so that the purines can interact. Any self-complementary sequences in the molecule produce more complex structures. RNA can base-pair with complementary regions of either RNA or DNA. Base pairing matches the pattern for DNA: G pairs with C and A pairs with U (or with the occasional T residue in some RNAs). One difference is that base pairing between G and U residues—unusual in DNA—is fairly common in RNA (see Fig. 8–27). The paired strands in RNA or RNA-DNA duplexes are antiparallel, as in DNA. RNA has no simple, regular secondary structure that serves as a reference point, as does the double helix for DNA. The three-dimensional structures of many RNAs, like those of proteins, are complex and unique. Weak interactions, especially base-stacking interactions, play a major role in stabilizing RNA structures, just as they do in DNA. Where complementary sequences are present, the predominant double-stranded structure is an A-form right-handed double helix. Z-form helices have been made in the laboratory (under very high-salt or high-temperature conditions). The B form of RNA has not been observed. Breaks in the regular A-form helix caused by mismatched or unmatched bases in one or both strands are common and result in bulges or internal loops (Fig. 8–26). Hairpin loops form between nearby self-complementary sequences. The potential for base-paired helical structures in many RNAs is extensive (Fig. 8–27), and the resulting hairpins are the most common type of secondary structure in RNA. Specific 288 Chapter 8 Nucleotides and Nucleic Acids FIGURE 8–24 Prokaryotic mRNA. Schematic diagrams show (a) monocistronic and (b) polycistronic mRNAs of prokaryotes. Red segments represent RNA coding for a gene product; gray segments represent noncoding RNA. In the polycistronic transcript, noncoding RNA separates the three genes. FIGURE 8–25 Typical right-handed stacking pattern of singlestranded RNA. The bases are shown in gray, the phosphate atoms in yellow, and the riboses and phosphate oxygens in green. Green is used to represent RNA strands in succeeding chapters, just as blue is used for DNA

8. 2 Nucleic Acid Structure 289 Hairpin short base sequences(such as UUCG)are often found trends Intemal at the ends of RNa hairpins and are known to form par- ticularly tight and stable loops. Such sequences may act as starting points for the folding of an RNA molecule into its precise three-dimensional structure. Important additional structural contributions are made by hydro- gen bonds that are not part of standard Watson-Crick base pairs. For example, the 2-hydroxyl group of ribose can hydrogen-bond with other groups. Some of these properties are evident in the structure of the phenyl alanine transfer RNA of yeast-the tRNA responsible for inserting Phe residues into polypeptides-and in two RNA enzymes, or ribozymes, whose functions, like those of protein enzymes, depend on their three-dimensional structures(Fig. 8-28) The analysis of Rna structure and the relationship between structure and function is an emerging field of Hairpin douhe helix inquiry that has many of the same complexities as the analysis of protein structure. The importance of under FIGURE 8-26 Secondary structure of RNAs (a)Bulge, internal loop, standing RNA structure grows as we become increas- and hairpin loop. ( b)The paired regions generally have an A-form ingly aware of the large number of functional roles for ight-handed helix, as shown for a hairpin RNA molecules A-160 FIGURE 8-27 Base-paired helical structures in an RNA. Shown here is the possible secondary structure of the M1 RNA component 120 of the enzyme RNase P of E. coli, with many hairpins. RNase P, which also contains a protein component (not shown), functions in the processing of transfer RNAs(see Fig. 26-23). The two brackets G-CCUCA indicate additional complementary sequences that may be paired in the t three-dimensional structure. The blue dots indicate non Watson. Crick G=U base pairs(boxed inset). Note that G=U base pairs are allowed only when presynthesized strands of RNA fold up or anneal with each other. There are no RNA polymerases(the enzymes that synthesize RNAs on a DNA template)that insert a U opposite a template G, or vice versa, during RNA synthesis. 0 ACCGGIGUGA G-C N-H Uracil GAAGCUGACCAG UGACU

short base sequences (such as UUCG) are often found at the ends of RNA hairpins and are known to form particularly tight and stable loops. Such sequences may act as starting points for the folding of an RNA molecule into its precise three-dimensional structure. Important additional structural contributions are made by hydrogen bonds that are not part of standard Watson-Crick base pairs. For example, the 2-hydroxyl group of ribose can hydrogen-bond with other groups. Some of these properties are evident in the structure of the phenylalanine transfer RNA of yeast—the tRNA responsible for inserting Phe residues into polypeptides—and in two RNA enzymes, or ribozymes, whose functions, like those of protein enzymes, depend on their three-dimensional structures (Fig. 8–28). The analysis of RNA structure and the relationship between structure and function is an emerging field of inquiry that has many of the same complexities as the analysis of protein structure. The importance of understanding RNA structure grows as we become increasingly aware of the large number of functional roles for RNA molecules. 8.2 Nucleic Acid Structure 289 G U C A C C A G U G C A A C A G AG A G C A A C A G U G A 180 A G G C G C 120 A C G G G C G C C C A 240 A UG G C C A G C G C C G A U C C C G C C G G G G A U C G G U G G C A 160 A G G 140 A A A G C C C G G C G G U G G A 220 A A U G G C G G C U G G U C C G A C G G U A 200 A A C C GG 100 A A G G C A G G 80 C C C U A A G A A U G G G C C C A C G A U A A A G U C C G G G C A G G C U G C U U G U A G A U GA A G G A G G A G G C U U C G G G C A A A C U A C U G A C A G A C U G U C G G G A C G G C A G G C G C U U C G U G G G G C C C C G O N OH O NH2 N N N N O N H O Guanine Uracil C C G G A A A U A G G C C C A A G G U U C A G U G U C A A C G U G C C G C 280 260 A G U G G G U A 300 C A A G C G U C G C G G G U A G U U G A C 330 U A C C A G U C G A G A G U A C G U U U C GAC C U 377 360 1 C U A U U C G GC C C A A G A C A G C A C 20 60 G C G A U U U G G G C U U 40 A C FIGURE 8–26 Secondary structure of RNAs. (a) Bulge, internal loop, and hairpin loop. (b) The paired regions generally have an A-form right-handed helix, as shown for a hairpin. FIGURE 8–27 Base-paired helical structures in an RNA. Shown here is the possible secondary structure of the M1 RNA component of the enzyme RNase P of E. coli, with many hairpins. RNase P, which also contains a protein component (not shown), functions in the processing of transfer RNAs (see Fig. 26–23). The two brackets indicate additional complementary sequences that may be paired in the three-dimensional structure. The blue dots indicate non-WatsonCrick GUU base pairs (boxed inset). Note that GUU base pairs are allowed only when presynthesized strands of RNA fold up or anneal with each other. There are no RNA polymerases (the enzymes that synthesize RNAs on a DNA template) that insert a U opposite a template G, or vice versa, during RNA synthesis

8.3 Nucleic Acid Chemistry 291 pairs are stacked perpendicular to the long axis length or part of the length(partial denaturation)of the of the double helix, 3.4 A apart, with 10.5 base molecule. No covalent bonds in the DNA are broken pairs per turn. (Fig8-29) a dNA can exist in several structural forms. Two Renaturation of a DNA molecule is a rapid one-step variations of the Watson-Crick form, or B-DNA process, as long as a double-helical segment of a dozen are A- and Z-DNA. Some sequence-dependent or more residues still unites the two strands. When the structural variations cause bends in the dna temperature or pH is returned to the range in which most organisms live, the unwound segments of the two quences can form hairpin/cruciform structures strands spontaneously rewind, or anneal, to yield the or triplex or tetraplex DNA. intact duplex(Fig. 8-29). However, if the two strands are completely separated, renaturation occurs in two a Messenger RNA transfers genetic information steps. In the first, relatively slow step, the two strands from DNa to ribosomes for protein synthesis Transfer rna and ribosomal rna are also find" each other by random collisions and form a short segment of complementary double helix. The second involved in protein synthesis. RNA can be step is much faster: the remaining unpaired bases suc structurally complex; single RNA strands can cessively come into register as base pairs, and the two be folded into hairpins, double-stranded re. gions, or complex loops strands"zipper"themselves together to form the dou ble helix The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same 8. 3 Nucleic Acid Chemistry concentration of free nucleotides, and the absorption is decreased further when two complementary nucleic To understand how nucleic acids function, we must un- acids strands are paired. This is called the hypochromic derstand their chemical properties as well as their struc effect. Denaturation of a double- stranded nucleic acid tures. The role of dNa as a repository of genetic infor roduces the opposite result: an increase in absorption mation depends in part on its inherent stability. The chemical transformations that do occur are generally very slow in the absence of an enzyme catalyst. The long-term storage of information without alteration is so important H00 to a cell, however, that even very slow reactions that Dauiclesianl alter DNA structure can be physiologically significant. Processes such as carcinogenesis and aging may be Dknamuraxn s intimately linked to slowly accumulating, irreversible al- terations of dna. other nondestructive alterations also occur and are essential to function such as the strand separation that must precede DNA replication or tran- cription. In addition to providing insights into physio logical ses, our understanding of nucleic acid chemistry has given us a powerful array of technologies evs>drrelinnud that have applications in molecular biology, medicine, and forensic science. We now examine the chemical proper ties of DNa and some of these technologies Double- Helical dna and rna can be denatured 了威rcds strends be base pairing lutions of carefully isolated, native DNa are highly viscous at pH 7.0 and room temperature(25C).When such a so olution is subjected to extremes of pHor to tem- peratures above 80C, its viscosity decreases sharply indicating that the dNa has undergone a physical change. Just as heat and extremes of pH denature glob ular proteins, they also cause denaturation, or melting of double-helical DNA. Disruption of the hyo Spiral sanda bonds between paired bases and of base stacking causes o':na 11 crant ailk unwinding of the double helix to form two single strands FIGURE 8-29 Reversible denaturation and annealing(renaturation) ompletely separate from each other along the entire of DNA

pairs are stacked perpendicular to the long axis of the double helix, 3.4 Å apart, with 10.5 base pairs per turn. ■ DNA can exist in several structural forms. Two variations of the Watson-Crick form, or B-DNA, are A- and Z-DNA. Some sequence-dependent structural variations cause bends in the DNA molecule. DNA strands with appropriate sequences can form hairpin/cruciform structures or triplex or tetraplex DNA. ■ Messenger RNA transfers genetic information from DNA to ribosomes for protein synthesis. Transfer RNA and ribosomal RNA are also involved in protein synthesis. RNA can be structurally complex; single RNA strands can be folded into hairpins, double-stranded regions, or complex loops. 8.3 Nucleic Acid Chemistry To understand how nucleic acids function, we must understand their chemical properties as well as their structures. The role of DNA as a repository of genetic information depends in part on its inherent stability. The chemical transformations that do occur are generally very slow in the absence of an enzyme catalyst. The long-term storage of information without alteration is so important to a cell, however, that even very slow reactions that alter DNA structure can be physiologically significant. Processes such as carcinogenesis and aging may be intimately linked to slowly accumulating, irreversible alterations of DNA. Other, nondestructive alterations also occur and are essential to function, such as the strand separation that must precede DNA replication or transcription. In addition to providing insights into physiological processes, our understanding of nucleic acid chemistry has given us a powerful array of technologies that have applications in molecular biology, medicine, and forensic science. We now examine the chemical properties of DNA and some of these technologies. Double-Helical DNA and RNA Can Be Denatured Solutions of carefully isolated, native DNA are highly viscous at pH 7.0 and room temperature (25 C). When such a solution is subjected to extremes of pH or to temperatures above 80 C, its viscosity decreases sharply, indicating that the DNA has undergone a physical change. Just as heat and extremes of pH denature globular proteins, they also cause denaturation, or melting, of double-helical DNA. Disruption of the hydrogen bonds between paired bases and of base stacking causes unwinding of the double helix to form two single strands, completely separate from each other along the entire length or part of the length (partial denaturation) of the molecule. No covalent bonds in the DNA are broken (Fig. 8–29). Renaturation of a DNA molecule is a rapid one-step process, as long as a double-helical segment of a dozen or more residues still unites the two strands. When the temperature or pH is returned to the range in which most organisms live, the unwound segments of the two strands spontaneously rewind, or anneal, to yield the intact duplex (Fig. 8–29). However, if the two strands are completely separated, renaturation occurs in two steps. In the first, relatively slow step, the two strands “find” each other by random collisions and form a short segment of complementary double helix. The second step is much faster: the remaining unpaired bases successively come into register as base pairs, and the two strands “zipper” themselves together to form the double helix. The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides, and the absorption is decreased further when two complementary nucleic acids strands are paired. This is called the hypochromic effect. Denaturation of a double-stranded nucleic acid produces the opposite result: an increase in absorption 8.3 Nucleic Acid Chemistry 291 FIGURE 8–29 Reversible denaturation and annealing (renaturation) of DNA

292 Chapter 8 Nucleotides and Nucleic Acids alled the hyperchromic effect. The transition from double-stranded DNa to the single-stranded, denatured form can thus be detected by monitoring the absorption of UV light. Viral or bacterial DNA molecules in solution do ture when they are heated slowly(Fig. 8-30) species of dNA has a characteristic denaturation tem perature, or melting point('m): the higher its content of G=C base pairs, the higher the melting point of the DNA. This is because G=C base pairs, with three hy- drogen bonds, require more heat energy to dissociate than a=Tbase pairs. Careful determination of the melt ing point of a DNA specimen, under fixed conditions of pH and ionic strength, can yield an estimate of its base 3 gm composition. If denaturation conditions are carefully controlled, regions that are rich in A=T base pairs will FIGURE 8-31 Partially denatured DNA. This DNA was partially de- specifically denature while most of the DNA remains natured, then fixed to prevent renaturation during sample preparation The shadowing method used to visualize the DNA in this electron mi crograph increases its diameter approximately fivefold and obliterates most details of the helix. However, length measurements can be ob- tained, and single-stranded regions are readily distinguishable from double-stranded regions. The arrows point to some single-stranded bubbles where denaturation has occurred. The regions that denature are highly reproducible and are rich in A=T base pai double-stranded. Such denatured regions(called bub bles) can be visualized with electron microscopy(Fig 8-31). Strand separation of DNA mustoccur in vivo dur- ing processes such as DNA replication and transcrip- tion. as we shall see. the dna sites where these processes are initiated are often rich in A=T base pairs Duplexes and one DNA strand(RNA-DNA hybrids) can also be Temperature (C) denatured. Notably, RNa duplexes are more stable than DNA duplexes. At neutral pH, denaturation of a double- helical RNA often requires temperatures 20%C or more higher than those required for denaturation of a dNA molecule with a comparable sequence. The stability of an RNA-DNa hybrid is generally intermediate between that of RNa and that of DNA. The physical basis for these differences in thermal stability is not known. Nucleic Acids from Different Species Can Form hybrids The ability of two complementary DNa strands to pair with one another can be used to detect similar dna quences in two different species or within the genome of a single species. If duplex DNAs isolated from human cells and from mouse cells are completely denatured by 100110 heating, then mixed and kept at 65C for many hours, m (oC) much of the dna will anneal. most of the DNA FIGURE 8-30 Heat denaturation of DNA. (a)The denaturation, or strands anneal with complementary mouse DNA strands melting, curves of two DNA specimens. The temperature at the mid- to form mouse duplex DNA; similarly, most human Dint of the transition( m) is the melting point: it depends on pH and DNA strands anneal with complementary human DNA ionic strength and on the size and base composition of the DNA. strands. However, some strands of the mouse DNa will (b)Relationship between m and the G=C content of a DNA. associate with human DNA strands to yield hybrid

called the hyperchromic effect. The transition from double-stranded DNA to the single-stranded, denatured form can thus be detected by monitoring the absorption of UV light. Viral or bacterial DNA molecules in solution denature when they are heated slowly (Fig. 8–30). Each species of DNA has a characteristic denaturation temperature, or melting point (tm): the higher its content of GqC base pairs, the higher the melting point of the DNA. This is because GqC base pairs, with three hydrogen bonds, require more heat energy to dissociate than AUT base pairs. Careful determination of the melting point of a DNA specimen, under fixed conditions of pH and ionic strength, can yield an estimate of its base composition. If denaturation conditions are carefully controlled, regions that are rich in AUT base pairs will specifically denature while most of the DNA remains double-stranded. Such denatured regions (called bubbles) can be visualized with electron microscopy (Fig. 8–31). Strand separation of DNA must occur in vivo during processes such as DNA replication and transcription. As we shall see, the DNA sites where these processes are initiated are often rich in AUT base pairs. Duplexes of two RNA strands or of one RNA strand and one DNA strand (RNA-DNA hybrids) can also be denatured. Notably, RNA duplexes are more stable than DNA duplexes. At neutral pH, denaturation of a doublehelical RNA often requires temperatures 20 C or more higher than those required for denaturation of a DNA molecule with a comparable sequence. The stability of an RNA-DNA hybrid is generally intermediate between that of RNA and that of DNA. The physical basis for these differences in thermal stability is not known. Nucleic Acids from Different Species Can Form Hybrids The ability of two complementary DNA strands to pair with one another can be used to detect similar DNA sequences in two different species or within the genome of a single species. If duplex DNAs isolated from human cells and from mouse cells are completely denatured by heating, then mixed and kept at 65 C for many hours, much of the DNA will anneal. Most of the mouse DNA strands anneal with complementary mouse DNA strands to form mouse duplex DNA; similarly, most human DNA strands anneal with complementary human DNA strands. However, some strands of the mouse DNA will associate with human DNA strands to yield hybrid 292 Chapter 8 Nucleotides and Nucleic Acids 100 G C (% of total nucleotides) 80 0 70 80 90 tm (°C) 60 100 110 60 40 20 100 Denaturation (%) 50 0 75 80 85 Temperature (°C) tm tm FIGURE 8–30 Heat denaturation of DNA. (a) The denaturation, or melting, curves of two DNA specimens. The temperature at the midpoint of the transition (tm) is the melting point; it depends on pH and ionic strength and on the size and base composition of the DNA. (b) Relationship between tm and the GqC content of a DNA. (a) (b) FIGURE 8–31 Partially denatured DNA. This DNA was partially denatured, then fixed to prevent renaturation during sample preparation. The shadowing method used to visualize the DNA in this electron micrograph increases its diameter approximately fivefold and obliterates most details of the helix. However, length measurements can be obtained, and single-stranded regions are readily distinguishable from double-stranded regions. The arrows point to some single-stranded bubbles where denaturation has occurred. The regions that denature are highly reproducible and are rich in AUT base pairs.