Summary 313 netically useful recombinant DNA molecules. For the isolation of even larger nu- cleotide sequences, such as those of genes encoding large polypeptides (or those of eukaryotic genes that are disrupted by large introns), partial or limited digestion of DNA by restriction enzymes can be employed. However, restriction endonucle- ases that cut only at specific nucleotide sequences 8 or even 13 nucleotides in length are also available, such as NotI and SfiI. Restriction Endonucleases Can Be Used to Map the Structure of a DNA Fragment The application of these sequence-specific nucleases to problems in molecular bi- ology is considered in detail in Chapter 12, but one prominent application is de- scribed here. Because restriction endonucleases cut dsDNA at unique sites to gen- erate large fragments, they provide a means for mapping DNA molecules that are many kilobase pairs in length. Restriction digestion of a DNA molecule is in many ways analogous to proteolytic digestion of a protein by an enzyme such as trypsin (see Chapter 5): The restriction endonuclease acts only at its specific sites so that a discrete set of nucleic acid fragments is generated. This action is analogous to trypsin cleavage only at Arg and Lys residues to yield a particular set of tryptic pep- tides from a given protein. The restriction fragments represent a unique collection of different-sized DNA pieces. Fortunately, this complex mixture can be resolved by electrophoresis (see the Appendix to Chapter 5). Electrophoresis of DNA mole- cules on gels of restricted pore size (as formed in agarose or polyacrylamide me- dia) separates them according to size, the largest being retarded in their migration through the gel pores while the smallest move relatively unhindered. Figure 10.29 shows a hypothetical electrophoretogram obtained for a DNA molecule treated with two different restriction nucleases, alone and in combination. Just as cleavage of a protein with different proteases to generate overlapping fragments allows an ordering of the peptides, restriction fragments can be ordered or “mapped” ac- cording to their sizes, as deduced from the patterns depicted in Figure 10.29. SUMMARY Nucleotides and nucleic acids possess heterocyclic nitrogenous bases as principal components of their structure. Nucleotides participate as essential intermediates in virtually all aspects of cellular metabolism. Nucleic acids are the substances of heredity (DNA) and the agents of genetic information transfer (RNA). 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? The bases of nucleotides and nucleic acids are derivatives of either pyrimidine (cytosine, uracil, and thymine) or purine (adenine and gua- nine). The aromaticity of the pyrimidine and purine ring systems and the electron-rich nature of their OOH and ring nitrogen substituents allow them to undergo keto–enol tautomeric shifts and endow them with the capacity to absorb UV light. 10.2 What Are Nucleosides? Nucleosides are formed when a base is linked to a sugar. The usual sugars of nucleosides are pentoses; ribo- nucleosides contain the pentose D-ribose, whereas 2-deoxy-D-ribose is found in deoxyribonucleosides. Nucleosides are more water soluble than free bases. 10.3 What Are the Structure and Chemistry of Nucleotides? A nu- cleotide results when phosphoric acid is esterified to a sugar OOH group of a nucleoside. Successive phosphate groups can be linked to the phosphoryl group of a nucleotide through phosphoric anhydride linkages. Nucleoside 5Ј-triphosphates, as carriers of chemical energy, are indispensable agents in metabolism because phosphoric anhydride bonds are a prime source of chemical energy to do biological work. Vir- tually all of the biochemical reactions of nucleotides involve either phos- phate or pyrophosphate group transfer. The bases of nucleotides serve as “in- formation symbols.” 10.4 What Are Nucleic Acids? Nucleic acids are polynucleotides: linear polymers of nucleotides linked 3Ј to 5Ј by phosphodiester bridges. The only significant variation in the chemical structure of nucleic acids is the particular base at each nucleotide position. These bases are not part of the sugar–phosphate backbone but instead serve as distinctive side chains. 10.5 What Are the Different Classes of Nucleic Acids? The two major classes of nucleic acids are DNA and RNA. Two fundamental chemical differences distinguish DNA from RNA: The nucleotides in DNA con- tain 2-deoxyribose instead of ribose as their sugar component, and DNA contains the base thymine instead of uracil. These differences confer important biological properties on DNA. DNA consists of two antiparallel polynucleotide strands wound to- gether to form a long, slender, double helix. The strands are held together through specific base pairing of A with T and C with G. The information in DNA is encoded in digital form in terms of the se- quence of bases along each strand. Because base pairing is specific, the information in the two strands is complementary. DNA molecules may contain tens or even hundreds of millions of base pairs. In eukary- otic cells, DNA is complexed with histone proteins to form a nucleo- protein complex known as chromatin. RNA occurs in multiple forms in cells, almost all of which are single-stranded. Nevertheless, the presence of complementary nu- cleotide sequences within the strand gives rise to multiple double- stranded regions in RNA molecules. Messenger RNA (mRNA) mole- cules are direct copies of the base sequences of protein-coding genes. Ribosomal RNA (rRNA) molecules provide the structural and func- tional foundations for ribosomes, the agents for translating mRNAs into proteins. In protein synthesis, the amino acids are delivered to PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. Draw the principal ionic species of 5Ј-GMP occurring at pH 2. 2. Draw the chemical structure of pACG. 3. Chargaff’s results (Table 10.1) yielded a molar ratio of 1.29 for A to G in ox DNA, 1.43 for T to C, 1.04 for A to T, and 1.00 for G to C. Given these values, what are the approximate mole fractions of A, C, G, and T in ox DNA? 4. Results on the human genome published in Science (Science 291: 1304–1350 [2001]) indicate that the haploid human genome con- sists of 2.91 gigabase pairs (2.91 ϫ 10 9 base pairs) and that 27% of the bases in human DNA are A. Calculate the number of A, T, G, and C residues in a typical human cell. 5. Adhering to the convention of writing nucleotide sequences in the 5Ј→3Ј direction, what is the nucleotide sequence of the DNA strand that is complementary to d-ATCGCAACTGTCACTA? 6. Messenger RNAs are synthesized by RNA polymerases that read along a DNA template strand in the 3Ј→5Ј direction, polymerizing ribonucleotides in the 5Ј→3Ј direction (see Figure 10.20). Give the nucleotide sequence (5Ј→3Ј) of the DNA template strand from which the following mRNA segment was transcribed: 5Ј-UAGUGACAGUUGCGAU-3Ј. 7. The DNA strand that is complementary to the template strand copied by RNA polymerase during transcription has a nucleotide se- quence identical to that of the RNA being synthesized (except T residues are found in the DNA strand at sites where U residues oc- cur in the RNA). An RNA transcribed from this nontemplate DNA strand would be complementary to the mRNA synthesized by RNA polymerase. Such an RNA is called antisense RNA because its base sequence is complementary to the “sense” mRNA. One strategy to thwart the deleterious effects of genes activated in disease states (such as cancer) is to generate antisense RNAs in affected cells. These antisense RNAs would form double-stranded hybrids with mRNAs transcribed from the activated genes and prevent their translation into protein. Suppose transcription of a cancer-activated gene yielded an mRNA whose sequence included the segment 5Ј-UACGGUCUAAGCUGA. What is the corresponding nucleotide sequence (5Ј→3Ј) of the template strand in a DNA duplex that might be introduced into these cells so that an antisense RNA could be transcribed from it? 8. A 10-kb DNA fragment digested with restriction endonuclease EcoRI yielded fragments 4 kb and 6 kb in size. When digested with BamHI, fragments 1, 3.5, and 5.5 kb were generated. Concomitant digestion with both EcoRI and BamHI yielded fragments 0.5, 1, 3, and 5.5 kb in size. Give a possible restriction map for the original fragment. 9. Based on the information in Table 10.2, describe two different 20-base nucleotide sequences that have restriction sites for BamH1, PstI, Sal I, and SmaI. Give the sequences of the SmaI cleavage products of each. 10. (Integrates with Chapter 3.) The synthesis of RNA can be summa- rized by the reaction: n NTP ⎯⎯→(NMP) n ϩ n PP i What is the ⌬G°Ј overall for synthesis of an RNA molecule 100 nucleo- tides in length, assuming that the ⌬G°Ј for transfer of an NMP from an NTP to the 3Ј-O of polynucleotide chain is the same as the ⌬G°Ј for transfer of an NMP from an NTP to H 2 O? (Use data given in Table 3.3.) 11. Gene expression is controlled through the interaction of proteins with specific nucleotide sequences in double-stranded DNA. a. List the kinds of noncovalent interactions that might take place between a protein and DNA. b. How do you suppose a particular protein might specifically inter- act with a particular nucleotide sequence in DNA? That is, how might proteins recognize specific base sequences within the dou- ble helix? 12. Restriction endonucleases also recognize specific base sequences and then act to cleave the double-stranded DNA at a defined site. Speculate on the mechanisms by which this sequence recognition and cleavage reaction might occur by listing a set of requirements for the process to take place. 13. A carbohydrate group is an integral part of a nucleoside. a. What advantage does the carbohydrate provide? Polynucleotides are formed through formation of a sugar– phosphate backbone. b. Why might ribose be preferable for this backbone instead of glucose? c. Why might 2-deoxyribose be preferable to ribose in some situa- tions? 14. Phosphate groups are also integral parts of nucleotides, with the second and third phosphates of a nucleotide linked through phos- phoric anhydride bonds, an important distinction in terms of the metabolic role of nucleotides. a. What property does a phosphate group have that a nucleoside lacks? b. How are phosphoric anhydride bonds useful in metabolism? c. How are phosphate anhydride bonds an advantage to the ener- getics of polynucleotide synthesis? 15. The RNAs acting in RNAi are about 21 nucleotides long. To judge whether it is possible to uniquely target a particular gene with a RNA of this size, consider the following calculation: What is the ex- pected frequency of occurrence of a specific 21-nt sequence? 16. The haploid human genome consists of 3 ϫ 10 9 base pairs. Using the logic in problem 15, one can calculate the minimum length of a unique DNA sequence expected to occur by chance just once in the human genome. That is, what is the length of an oligonu- cleotide whose expected frequency of occurrence is once every 3 ϫ 10 9 bp? 17. Snake venom phosphodiesterase is an a-specific exonuclease (Fig- ure 10.28) that acts equally well on single-stranded RNA or DNA. Design a protocol based on snake venom phosphodiesterase that would allow you to determine the base sequence of an oligonu- cleotide. Hint: Adapt the strategy for protein sequencing by Edman degradation, as described on pages 80 and 102. 314 Chapter 10 Nucleotides and Nucleic Acids the ribosomes in the form of aminoacyl-tRNA (transfer RNA) deriva- tives. Small nuclear RNAs (snRNAs) are characteristic of eukaryotic cells and are necessary for processing the RNA transcripts of protein- coding genes into mature mRNA molecules. Small RNAs are a recently discovered class of regulatory RNA molecules. A prominent role of small RNAs is gene silencing, particularly in the phenomenon of RNA interference (RNAi). 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Like all biological polymers, nucleic acids are susceptible to hydrolysis, particularly hydrol- ysis of the phosphoester bonds in the polynucleotide backbone. RNA is susceptible to hydrolysis by base: DNA is not. Nucleases are hydrolytic en- zymes that cleave the phosphoester linkages in the sugar–phosphate backbone of nucleic acids. Nucleases abound in nature, with varying specificity for RNA or DNA, single- or double-stranded nucleic acids, endo versus exo action, and 3Ј- versus 5Ј-cleavage of phosphodiesters. Re- striction endonucleases of the type II class are sequence-specific endo- nucleases useful in mapping the structure of DNA molecules. FURTHER READING Nucleic Acid Biochemistry and Molecular Biology Adams, R. L. P., Knowler, J. T., and Leader, D. P., 1992. The Biochemistry of the Nucleic Acids, 11th ed. New York: Chapman and Hall (Methuen and Co., distrib.). Watson, J. D., Baker, T. A., Bell, S. T., Gann, A., et al., 2007. The Molecular Bi- ology of the Gene, 6th ed. Menlo Park, CA: Benjamin/Cummings. The History of Discovery of the DNA Double Helix Judson, H. F., 1979. The Eighth Day of Creation. New York: Simon and Schuster. DNA as Information Hood, L., and Galas, D., 2003. The digital code of DNA. Nature 421: 444–448. The Catalytic Properties of RNA and Its Role in Early Evolution Caprara, M. G., and Nilsen, T. W., 2000. RNA: Versatility in form and func- tion. Nature Structural Biology 7:831–833. Gray, M. W., and Cedergren, R., eds., 1993. The new age of RNA. The FASEB Journal 7:4–239. A collection of articles emphasizing the new apprecia- tion for RNA in protein synthesis, in evolution, and as a catalyst. Small RNAs and Their Novel Biological Roles Cartthrew, R. W., 2006. Gene regulation by microRNAs. Current Opinion in Genetics & Development 18:203–208. Hannon, G. J., 2002. RNA interference. Nature 418:244–251. A review of RNAi, a widely conserved biological response to the intracellular pres- ence of double-stranded RNA. RNAi provides an experimental method for manipulating gene expression as well as a mechanism to investigate specific gene function at the whole genome level. Pillai, R. S., et al., 2007. Repression of protein synthesis by miRNAs: How many mechanisms? Trends in Cell Biology 17:118–126. Storz, G., Altuvia, A., and Wassarman, K. M., 2005. An abundance of RNA regulators. Annual Review of Biochemistry 74:199–217. Tuschi, T., 2003. RNA sets the standard. Nature 421:220–221. Overview of the use of RNA interference to inactivate all the genes in a model or- ganism (Caenorhabditis elegans) as a means of identifying gene function. Zmora, P. D., and Haley, B., 2005. Ribo-gnome: The big world of small RNAs. Science 309:1519–1524. This review in the September 2, 2005, is- sue of Science is accompanied by a series of articles on the various non- coding RNA types. Nucleases and DNA Manipulation Linn, S. M., Lloyd, R. S., and Roberts, R. J., 1993. Nucleases, 2nd ed. Long Island, NY: Cold Spring Harbor Laboratory Press. Mishra, N. C., 2002. Nucleases: Molecular Biology and Applications. Hoboken, NJ: Wiley-Interscience. Sambrook, J., and Russell, D., 2000. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Further Reading 315 18. From the answer to problem 4 and the molecular weights of dAMP (331 D), dCMP (307 D), dGMP (347 D), and dTMP (322 D), cal- culate the mass (in daltons) of the DNA in a typical human cell. Preparing for the MCAT Exam 19. The bases of nucleotides and polynucleotides are “information sym- bols.” Their central role in providing information content to DNA and RNA is clear. What advantages might bases as “information symbols” bring to the roles of nucleotides in metabolism? 20. Structural complementarity is the key to molecular recognition, a lesson learned in Chapter 1. The principle of structural comple- mentarity is relevant to answering problems 5, 6, 7, 11, 12, and 19. The quintessential example of structural complementarity in all of biology is the DNA double helix. What features of the DNA double helix exemplify structural complementarity? Reginald H. Garrett 11 Structure of Nucleic Acids Chapter 10 presented the structure and chemistry of nucleotides and how these units are joined via phosphodiester bonds to form nucleic acids, the biological polymers for information storage and transmission. In this chapter, we investigate biochemical methods that reveal this information by determining the sequential order of nucleo- tides in a polynucleotide, the so-called primary structure of nucleic acids. Then, we consider the higher orders of structure in the nucleic acids: the secondary and tertiary levels. Although the focus here is primarily on the structural and chemical properties of these macromolecules, it is fruitful to keep in mind the biological roles of these re- markable substances. The sequence of nucleotides in nucleic acids is the embodiment of genetic information (see Part IV). We can anticipate that the cellular mechanisms for accessing this information, as well as reproducing it with high fidelity, will be illu- minated by knowledge of the chemical and structural qualities of these polymers. 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? Determining the primary structure of nucleic acids (the nucleotide sequence) would seem to be a more formidable problem than amino acid sequencing of pro- teins, simply because nucleic acids contain only 4 unique monomeric units (A, C, G, and T) whereas proteins have 20. With only four, there are apparently fewer spe- cific sites for selective cleavage, distinctive sequences are more difficult to recog- nize, and the likelihood of ambiguity is greater. The much greater number of monomeric units in most polynucleotides as compared to polypeptides is a further difficulty. However, two simple tools make nucleic acid sequencing substantially eas- ier than polypeptide sequencing. One of these tools is the set of type II restriction en- donucleases that cleave DNA at specific oligonucleotide sites, generating unique frag- ments of manageable size (see Chapter 10). The second is gel electrophoresis, a method capable of separating nucleic acid fragments that differ from one another in length by just a single nucleotide. The Nucleotide Sequence of DNA Can Be Determined from the Electrophoretic Migration of a Defined Set of Polynucleotide Fragments The most widely used protocol for nucleic acid sequencing is the chain termination or dideoxy method of Frederick Sanger, which relies on enzymatic replication of the DNA to be sequenced. Very sensitive analytical techniques that can detect the What do you suppose those masons, who created this double helix adorning the cathedral in Orvieto, Italy, some 500 years ago, might have thought about the DNA double helix and heredity? The Structure of DNA: “A melody for the eye of the intellect, with not a note wasted.” Horace Freeland Judson The Eighth Day of Creation KEY QUESTIONS 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? 11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? 11.3 Can the Secondary Structure of DNA Be Denatured and Renatured? 11.4 Can DNA Adopt Structures of Higher Complexity? 11.5 What Is the Structure of Eukaryotic Chromosomes? 11.6 Can Nucleic Acids Be Synthesized Chemically? 11.7 What Are the Secondary and Tertiary Structures of RNA? ESSENTIAL QUESTION The nucleotide sequence—the primary structure—of DNA not only determines its higher-order structure but it is also the physical representation of genetic informa- tion in organisms. RNA sequences, as copies of specific DNA segments, direct both the higher-order structure and the function of RNA molecules in information trans- fer processes. What are the higher-order structures of DNA and RNA, and what methodolo- gies have allowed scientists to probe these structures and the functions that derive from them? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? 317 newly synthesized DNA chains following electrophoretic separation are available, so Sanger sequencing can be carried out on as little as 1 attomole (amol, 10 Ϫ18 mol) of DNA contained in less than 0.1 L volume. (10 Ϫ18 moles of DNA are roughly equiv- alent to 10 Ϫ12 grams (pg) of 1-kb sized DNA molecules.) These analytical techniques typically rely on fluorescent detection of the DNA products. Sanger’s Chain Termination or Dideoxy Method Uses DNA Replication To Generate a Defined Set of Polynucleotide Fragments To appreciate the rationale of the chain termination or dideoxy method, we first must briefly examine the biochemistry of DNA replication. DNA is a double helical molecule. In the course of its replication, the sequence of nucleotides in one strand is copied in a complementary fashion to form a new second strand by the enzyme DNA polymerase. Each original strand of the double helix serves as a template for the biosynthesis that yields two daughter DNA duplexes from the parental double helix (Figure 11.1). DNA polymerase carries out this reaction in vitro in the pres- ence of the four deoxynucleotide monomers and copies single-stranded DNA, pro- vided a double-stranded region of DNA is artificially generated by adding a primer. This primer is merely an oligonucleotide capable of forming a short stretch of dsDNA by base pairing with the ssDNA (Figure 11.2). The primer must have a free 3Ј-OH end from which the new polynucleotide chain can grow as the first residue is added in the initial step of the polymerization process. DNA polymerases synthe- size new strands by adding successive nucleotides in the 5Јn3Ј direction. The Chain Termination Protocol In the chain termination method of DNA se- quencing, a DNA fragment of unknown sequence serves as a template in a polymer- ization reaction using some type of DNA polymerase, usually a genetically engi- neered version that lacks all traces of exonuclease activity that might otherwise degrade the DNA. (DNA polymerases usually have an intrinsic exonuclease activity that allows proofreading and correction of the DNA strand being synthesized; see Chapter 28.) The primer requirement is met by an appropriate oligonucleotide (this method is also known as the primed synthesis method for this reason). The reaction is run in the presence of all four deoxynucleoside triphosphates dATP, dGTP, dCTP, and dTTP, which are the substrates for DNA polymerase (Figure 11.3). In addition, the reaction mixture contains the four corresponding 2Ј,3Ј-dideoxynucleotides (ddATP, ddGTP, ddCTP, and ddTTP); it is these dideoxynucleotides that give the method its name. Because dideoxynucleotides lack 3Ј-OH groups, they cannot serve as acceptors for 5Ј-nucleotide addition in the polymerization reaction; thus, the chain is termi- nated where they become incorporated. The concentrations of the deoxynu- cleotides in each reaction mixture are significantly greater than the concentrations of the dideoxynucleotides, so incorporation of a dideoxynucleotide is infrequent. Therefore, base-specific premature chain termination is only a random, occasional event, and a population of new strands of varying length is synthesized. Neverthe- less, termination, although random, occurs everywhere in the sequence. Thus, the population of newly synthesized DNAs forms a nested set of molecules that differ in AT GC TA A GC AT GC AT GC CG A A GC AT GC GC GCCG AT AT AT GC AT AT T GC AT AT GC AT AT GC Old New Old New New Old Old Parental DNA FIGURE 11.1 DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule. Single- stranded DNA 5' 3' 5' A T G C T A C G TAGCAACT DNA polymerase Primer 3'–OH + dATP dTTP dCTP dGTP Annealing of primer creates a short stretch of double-stranded DNA ACTIVE FIGURE 11.2 Primed synthesis of a DNA template by DNA polymerase, using the four deoxynucleoside triphosphates as the substrates. Test yourself on the concepts in this figure at www.cengage.com/login. 318 Chapter 11 Structure of Nucleic Acids length by just one nucleotide. Each newly synthesized strand has a dideoxynu- cleotide at its 3Ј-end, and each of the four dideoxynucleotides used in Sanger se- quencing is distinctive because each bears a fluorescent tag of a different color. (These fluorescent tags are attached to the 5-position of pyrimidine dideoxynu- cleotides or the 7-position of purine dideoxynucleotides, where these tags do not impair the ability of DNA polymerase to add them to a growing polynucleotide chain.) The color of a particular fluorescence (as in orange for ddA, blue for ddC, green for ddG, and red for ddT) reveals which base was specified by the template and incorporated by DNA polymerase at that spot. Reading Dideoxy Sequencing Gels The sequencing products are visualized by fluorescence spectroscopy following their separation according to size by capillary electrophoresis (Figure 11.3). Because the smallest fragments migrate fastest upon electrophoresis and because fragments differing by only a single nucleotide in length are readily resolved, the sequence of nucleotides in the set of newly synthe- sized DNA fragments is given by the order of the fluorescent colors emerging from the capillary. Thus, the gel in Figure 11.3 is read TTGTCGAAGTCAG (5Јn3Ј). Be- cause of the way DNA polymerase acts, this observed sequence is complementary to the corresponding unknown template sequence. Knowing this, the template se- quence now can be written CTGACTTCGACAA (5Јn3Ј). Sanger sequencing has been fully automated. Automation is achieved through the use of robotics for preparing the samples, running the DNA sequencing reactions, ACCC G T CTT Single-stranded DNA to be sequenced TGG C A C A T A G 5' 5' T G 3' A C C T G T T G A A G 3' A C 5' T G So the sequence of the template strand is Electrophoresis and analysis using a laser to activate the fluorescent dideoxy nucleotides and a detector to distinguish the colors Larger fragments Smaller fragments G A C A A C T T C A 5' 5' G T 5' A 5' A 5' G 5' 5' T 5' 5' dCTP dTTP plus limiting amounts of fluorescently labeled dGTP dATP ddCTP ddTTP ddGTP ddATP DNA polymerase I 5' Add: 5' 3'5' 5' 5' Primer HO-3'– ANIMATED FIGURE 11.3 The chain termination or dideoxy method of DNA sequencing. A template DNA (the single-stranded DNA to be sequenced) with a complementary primer annealed at its 3Ј-end is copied by DNA polymerase in the presence of the four deoxynucleotide substrates (dATP, dCTP, dGTP,dTTP) and small amounts of the four dideoxynu- cleotide analogs of these substrates, each of which car- ries a distinctive fluorescence tag (illustrated here as orange for ddATP, blue for ddCTP,green for ddGTP,and red for ddTTP). Occasional incorporation of a dideoxy- nucleotide terminates further synthesis of that comple- mentary strand. The nested set of terminated strands can be separated by capillary electrophoresis and iden- tified by laser fluorescence spectroscopy. Test yourself on the concepts in this figure at www.cengage.com/ login. 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? 319 loading the chain-terminated DNA fragments onto capillary electrophoresis tubes, performing the electrophoresis, and imaging the results for computer analysis. These advances have made it feasible to sequence the entire genomes of organisms (see Chapter 12). Celera Genomics, the private enterprise that reported a sequence for the 2.91 billion–bp human genome in 2001, used 300 automated DNA sequencers/ EMERGING INSIGHTS INTO BIOCHEMISTRY High-Throughput DNA Sequencing by the Light of Fireflies The enormous significance of DNA sequence information to fun- damental questions in biology, medicine, and personal health is a compelling force for the development of more rapid and efficient DNA sequencing technologies, so-called next-generation sequenc- ing, or NGS, methods. One important NGS advance is 454 Tech- nology, a methodology developed by 454 Life Sciences, a division of Roche Company. Like Sanger sequencing, 454 Technology re- lies on DNA polymerase-catalyzed copying of a primed single- stranded DNA. (However, because 454 Technology does not rely on chain termination or creation of a nested set of DNA frag- ments, dideoxynucleotide terminators are not needed.) Multiple copies of unique single-stranded template DNA molecules paired with primer strands are immobilized on microscopic beads that can be loaded into micro-microtiter wells at a scale of 1.6 million different wells on a 6 cm ϫ 6 cm platform (see accompanying fig- ure). Each well receives a unique DNA template. The reagents for primed synthesis are passed over the platform in sequential order: First, a reaction mixture with DNA polymerase plus dTTP (but no other dNTPs), a wash, then a reaction mixture with enzymes but only dATP, a wash, then the dGTP-specific mixture, a wash, and fi- nally the dCTP mixture and a wash. Such cycles are repeated up to 100 times over an 8-hour period. Up to 500 cycles are possible in one run. A fiber-optic array to monitor light emission from each well is aligned with the platform. The methodology is based on detection of DNA polymerase ac- tion through light emission. To do this, the technology exploits an overlooked product of the polymerase reaction, namely, the pyro- phosphate released each time a dNTP contributes the correct complementary dNMP in the polymerase reaction. Pyrophosphate release is coupled to light emission through two reactions. The first is catalyzed by ATP sulfurylase, which uses PP i plus adenosine- 5-phosphosulfate (APS) to form ATP. The second reaction, cat- alyzed by the ATP-dependent firefly enzyme luciferase, oxidizes luciferin to form oxyluciferin with the emission of light. Reaction 1: PP i ϩ APS n ATP ϩ SO 4 2Ϫ (This is the reverse of the ATP sulfurylase reaction shown as reac- tion 1 in Figure 25.34.) Reaction 2: ATP ϩ luciferin ϩ O 2 n AMP ϩ PP i ϩ CO 2 ϩ oxyluciferin ϩ light Light detection confirms that addition of a dNMP by primed syn- thesis has occurred. Using computer recording of light emission to keep track of when in each cycle each well emitted a pulse of light al- lows reconstruction of sequence information for each of 1.6 million templates. Using this methodology, the 580,069-nucleotide sequence of Mycoplasma genitalium was confirmed in one run on the 454 Genome Sequencer. (From Margulies, M., et al., 2005. Genome sequenc- ing in microfabricated high-density picolitre reactors. Nature 437:376–380.) Oxyluciferin S OH N S N HO OH H H Luciferin S N S N O HO Polymerase Anneal primer AGAATCGGCATGCTA AAAGTC APS SO 4 2 ؊ PP i Luciferin Oxyluciferin ATP Light DNA capture bead containing millions of copies of a single clonal fragment Luciferase Sulfurylase dNTP Signal Image 320 Chapter 11 Structure of Nucleic Acids analyzers to sequence more than 1 billion bases every month. Today, the more tedious aspect of DNA sequencing is the isolation and preparation of DNA fragments of in- terest, such as cloned genes; automated sequencing makes the rest routine. 11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? Conformational Variation in Polynucleotide Strands Polynucleotide strands are inherently flexible. Each deoxyribose–phosphate segment of the backbone has six degrees of freedom (Figure 11.4a) as a consequence of the six successive single bonds per segment along the chain. Furanose rings of pentoses are not planar but instead adopt puckered conformations, four of which are shown in Figure 11.4b. A seventh degree of freedom per nucleotide unit arises because of free rotation about the C1Ј-N glycosidic bond. This freedom allows the plane of the base to rotate relative to the path of the polynucleotide strand (Figure 11.4c). DNA Usually Occurs in the Form of Double-Stranded Molecules Double-stranded DNA molecules adopt one of three secondary structures, termed A, B, and Z. In a moment, we will address the “ABZs of DNA secondary structure”; first we must consider some general features of DNA double helices. Fundamentally, double-stranded DNA is a regular two-chain structure with hydrogen bonds formed O 6 54321 1 again 4 1 C1Ј C2Ј–endo C3Ј–exo C2Ј–exo C3Ј–endo Pyrimidine: Syn C1Ј 2 6 3 5 4 1 C1Ј Anti 6 2 5 3 6 3 Purine: Syn 2 4 1 5 7 9 8 6 3 Anti 4 2 1 5 7 9 C1Ј 8 O3Ј 5ЈOP ε ␥␣ ε N1Ј of pyrimidine ϭ 135° Absent in DNA Free rotation about C1Ј–N glycosidic bond (7th degree of freedom): (c) Four puckered conformations of furanose rings: Rotation about bonds 1, 2, 3, 4, 5, and 6 correspond to 6 degrees of freedom designated ␣, , ␥, ␦, ε, and as indicated. (b) The six degrees of freedom in the sugar–PO 4 backbone: (a) Base 5Ј O 1Ј O Base Base O 3 2 4 2Ј 3Ј P 5 O O O 6 C1Ј O 4Ј 4Ј 5Ј 1Ј 3Ј 2Ј Base O 4Ј 5Ј 1Ј 4Ј 5Ј 1Ј 2Ј 2Ј 3Ј 3Ј 4Ј 5Ј 3Ј 2Ј Base 4Ј ␦ 1Ј 2Ј3Ј FIGURE 11.4 (a) The six degrees of freedom in the deoxyribose–PO 4 units of the polynucleotide chain. (b) Four puckered conformations of the furanose rings. (c) Free rotation about the C1Ј–N glycosidic bond. 11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? 321 between opposing bases on the two chains (see Chapter 10). Such H bonding is pos- sible only when the two chains are antiparallel. The polar sugar–phosphate backbones of the two chains are on the outside. The bases are stacked on the inside of the struc- ture; these heterocyclic bases, as a consequence of their -electron clouds, are hydro- phobic on their flat sides. One purely hypothetical conformational possibility for a two- stranded arrangement would be a ladderlike structure (Figure 11.5) in which the base pairs are fixed at 0.6 nm apart because this is the distance between adjacent sugars along a polynucleotide strand. Because H 2 O molecules could fit into the spaces be- tween the hydrophobic surfaces of the bases, this conformation is energetically unfa- vorable. This ladderlike structure converts to a double helix when given a simple right- handed twist. Helical twisting brings the base-pair rungs of the ladder closer together, stacking them 0.34 nm apart, without affecting the sugar–sugar distance of 0.6 nm. Be- cause this helix repeats itself approximately every 10 bp, its pitch is 3.4 nm. This is the major conformation of DNA in solution, and it is called B-DNA. Watson–Crick Base Pairs Have Virtually Identical Dimensions As indicated in Chapter 10, the base pairing in DNA is size complementary: Large bases (purines) pair with small bases (pyrimidines). Hydrogen bond formation be- tween purines and pyrimidines dictates that the purine adenine pairs with the pyrimidine thymine; the purine guanine pairs with the pyrimidine cytosine. Size complementarity means that the AϺT pair and GϺC pair have virtually identical di- mensions (Figure 11.6). Watson and Crick realized that units of such structural equivalence could serve as spatially invariant substructures to build a polymer whose exterior dimensions would be uniform along its length, regardless of the sequence of bases. That is, the pairing of smaller pyrimidines with larger purines everywhere across the double-stranded molecule allows the two polynucleotide strands to as- sume essentially identical helical conformations. The DNA Double Helix Is a Stable Structure Several factors account for the stability of the double helical structure of DNA. H Bonds Although it has long been emphasized that the two strands of DNA are held together by H bonds formed between the complementary purines and pyrim- idines, two in an AϺT pair and three in a GϺC pair (Figure 11.6), the H bonds be- tween base pairs impart little net stability to the double-stranded structure compared to the separated strands in solution. When the two strands of the double helix are separated, the H bonds between base pairs are replaced by H bonds between indi- vidual bases and surrounding water molecules. Polar atoms in the sugar–phosphate backbone do form external H bonds with surrounding water molecules, but these form with separated strands as well. Electrostatic Interactions A prominent feature of the backbone of a DNA strand is the repeating array of negatively charged phosphate groups. These ar- rays of negative charge along the strands repel each other so that their sugar–phosphate backbones are kept apart and the two strands come together through Watson–Crick base pairing. As a consequence, the negative charges are situated on the exterior surface of the double helix, such that repulsive effects are minimized. Further these charges become electrostatically shielded from one an- other because divalent cations, particularly Mg 2ϩ , bind strongly to the anionic phosphates. Van der Waals and Hydrophobic Interactions The core of the helix consists of the base pairs, and these base pairs stack together through , -electronic interac- tions (a form of van der Waals interaction), and hydrophobic forces. These base- pair stacking interactions range from Ϫ16 to Ϫ51 kJ/mol (expressed as the energy of interaction between adjacent base pairs), contributing significantly to the overall stabilizing energy. (a) Ladder Base-pair spacing 0.6 nm TA TA TA CG TA TA CG CG TA CG CG (b) Helix Base-pair spacing 0.34 nm Pitch length 3.4 nm TA TA A G GC GC TA GC GC FIGURE 11.5 (a) Double-stranded DNA as an imaginary ladderlike structure. (b) A simple right-handed twist converts the ladder to a helix. 322 Chapter 11 Structure of Nucleic Acids A stereochemical consequence of the way AϺT and GϺC base pairs form is that the sugars of the respective nucleotides have opposite orientations. This is why the sugar–phosphate backbones of the two chains run in opposite or “antiparallel” direc- tions. Furthermore, the two glycosidic bonds holding the bases in each base pair are not directly across the helix from each other, defining a common diameter (Figure 11.7). Consequently, the sugar–phosphate backbones of the helix are not equally spaced along the helix axis and the grooves between them are not the same size. In- stead, the intertwined chains create a major groove and a minor groove (Figure 11.7). Thymine C C C N C N C O O H H H H H H C C C N C N N H H N C N H 50 o To chain To chain 1.11 nm 0.28 nm 0.30 nm Adenine 51 o Major groove Minor groove C 1 ' C C N C N C H N O H H C C C N C H N O H N C N H N H 0.29 nm 0.30 nm 0.29 nm Cytosine Guanine 1.08 nm 52 o 54 o To chain To chain Major groove Minor groove H C 1 ' C 1 ' C 1 ' FIGURE 11.6 Watson–Crick AϺT and GϺC base pairs. All H bonds in both base pairs are straight. Minor groove B-DNA Top view Major groove Major groove of DNA Minor groove of DNA Glycosidic bond Glycosidic bond Radius of sugar–phosphate backbone C C C C C C C C C C O O H H H H H H H H N N N N N N N H FIGURE 11.7 The major and minor grooves of B-DNA. . migration through the gel pores while the smallest move relatively unhindered. Figure 10.29 shows a hypothetical electrophoretogram obtained for a DNA molecule treated with two different restriction. ddT) reveals which base was specified by the template and incorporated by DNA polymerase at that spot. Reading Dideoxy Sequencing Gels The sequencing products are visualized by fluorescence spectroscopy. as a consequence of their -electron clouds, are hydro- phobic on their flat sides. One purely hypothetical conformational possibility for a two- stranded arrangement would be a ladderlike structure