11.7 What Are the Secondary and Tertiary Structures of RNA? 343 5' 3' 3' 3' 3' 5' 5' Single-nucleotide bulge Three-nucleotide bulge 5' Mismatch pair or symmetric internal loop of two nucleotides 5' 3' 3' 3' 5' 5' 5' 3' 3' 3' 5' 5' Hairpin loop 3' 5' Symmetric internal loop Asymmetric internal loop FIGURE 11.30 Bulges and loops formed in RNA when aligned sequences are not fully complementary.(Adapted from Appendix Figure 1 in Gesteland, R. F.,Cech,T.R., and Atkins, J. F., eds. The RNA World, 2nd ed. New York: Cold Spring Harbor Press.) 2 2 4 4 1 3 3 1 FIGURE 11.31 Junctions and coaxial stacking in RNA. Stem junctions (or multibranched loops) are another type of RNA secondary structure. Coaxial stacking of stems or stem-loops (as in stacking of stem 1 on stem- loop 4) is a tertiary structural feature found in many RNAs. (Adapted from Figure 1 in Tyagi, R., and Matthews, D. H., 2007. Predicting coaxial stacking in multibranch loops. RNA 13:1–13. ) strongly tilted from the plane perpendicular to the helix axis (see Figure 11.9). A-form double helices are the most prominent secondary structural elements in RNA. Both tRNA and rRNA have large amounts of A-form double helix. In addition, a number of defined structural motifs recur within the loops of stem-loop struc- tures, such as U-turns (a loop motif of consensus sequence UNRN, where N is any nucleotide and R is a purine) and tetraloops (another class of four-nucleotide loops found at the termini of stem-loop structures). Stems of stem-loop structures may also have bulges (or internal loops) where the RNA strand is forced into a short single-stranded loop because one or more bases along one strand in an RNA dou- ble helix finds no base-pairing partners (Figure 11.30). Regions where several stem- loop structures meet are termed junctions (Figure 11.31). Stems, loops, bulges, and junctions are the four basic secondary structural elements in RNA. The single-stranded loops in RNA stem-loops create base-pairing opportunities between distant, complementary, single-stranded loop regions. These interactions, 344 Chapter 11 Structure of Nucleic Acids mostly based on Watson–Crick base pairing, lead to tertiary structure in RNA. Other tertiary structural motifs arise from coaxial stacking (Figure 11.31), pseudoknot formation, and ribose zippers. In coaxial stacking, the blunt, nonloop ends of stem- loops situated next to one another in the RNA sequence stack upon each other to create an uninterrupted stack of base pairs. A good example of coaxial stacking is found in the tertiary structure of tRNAs, where the acceptor end of the L-shaped tRNA is formed by coaxial stacking of the acceptor stem on the T⌿C stem-loop and the anticodon end is formed by coaxial stacking of the dihydrouracil stem-loop on the anticodon stem-loop (Figures 11.33 and 11.35). Pseudoknots occur when bases in the loops of stem-loop structures form a short double helix by base pairing with nearby single-stranded regions in the RNA (Figure 11.32). Ribose zippers are found when two antiparallel, single-stranded regions of RNA align as an H-bonded net- work forms between the 2Ј-OH groups of the respective strands, the O at the 2Ј-OH position of one strand serving as the H-bond acceptor while the H on the 2Ј-OH of the other strand is the H-bond donor. Ribose zippers and the other RNA structures mentioned here are well represented by many examples in the SCOR (Structural Classification of RNA) database at http://scor.lbl.gov/ and NDB (Nucleic Acid Database) at http://ndbserver.rutgers.edu/. Transfer RNA Adopts Higher-Order Structure Through Intrastrand Base Pairing In tRNA molecules, which contain 73 to 94 nucleotides in a single chain, a majority of the bases are hydrogen bonded to one another. Figure 11.33 shows the structure that typifies tRNAs. Hairpin turns bring complementary stretches of bases in the chain into contact so that double helical regions form, creating stem-loop sec- ondary structures. Because of the arrangement of the complementary stretches along the chain, the overall pattern of base pairing can be represented as a cloverleaf. Each cloverleaf consists of four base-paired segments—three loops and the stem where the 3Ј- and 5Ј-ends of the molecule meet. These four segments are desig- nated the acceptor stem, the D loop, the anticodon loop, and the TC loop (the latter two are U-turn motifs). A hTR 3Ј G C C C C G A U U U U U U U C G U A S2L1 S1 L2 G C A U A U AU G C U A A A A A A A C G G C G C G 5Ј C C C FIGURE 11.32 RNA pseudoknots are formed when a single-stranded region of RNA folds to base-pair with a hairpin loop. Loops L1 and L2, as shown on the sequence representation of human telomerase RNA (hTR) on the left, form a pseudoknot.The three- dimensional structure of an hTR pseudoknot is shown on the right (pdb id ϭ 1YMO). (Adapted from Figure 2 in Staple, D.W.,and Butcher, S. E., 2005. Pseudoknots: RNA structures with diverse functions. PLoS Biology 3:e213.) 11.7 What Are the Secondary and Tertiary Structures of RNA? 345 tRNA Secondary Structure The acceptor stem is where the amino acid is linked to form the aminoacyl-tRNA derivative, which serves as the amino acid–donating species in protein synthesis; this is the physiological role of tRNA. The carboxyl group of an amino acid is linked to the 3Ј-OH of the 3Ј-terminal A nucleotide, thus forming an aminoacyl ester (Figure 11.33). The 3Ј-end of tRNA is invariantly CCA-3Ј-OH. The D loop is so named because this tRNA loop often contains dihy- drouridine, or D, residues. In addition to dihydrouridine, tRNAs characteristically contain a number of unusual bases, including inosine, thiouridine, pseudouridine, and hypermethylated purines (see Figure 10.23). The anticodon stem-loop consists of a double helical segment and seven unpaired bases, three of which are the anticodon—a three-nucleotide unit that recognizes and base pairs with a particu- lar mRNA codon, a complementary three-base unit in mRNA providing the genetic information that specifies an amino acid. In the 5Ј→3Ј direction beyond the anti- codon stem-loop lies a loop that varies from tRNA to tRNA in the number of residues that it has, the so-called extra or variable loop. The last loop in the tRNA, reading 5Ј→3Ј, is within the TC stem-loop. It contains seven unpaired bases, in- cluding the sequence TC, where is the symbol for pseudouridine. Most of the invariant residues common to tRNAs lie within the non–hydrogen-bonded regions of the cloverleaf structure. tRNA Tertiary Structure Tertiary structure in tRNA arises from base-pairing inter- actions between bases in the D loop with bases in the variable and TC loops, as shown for yeast phenylalanine tRNA in Figure 11.34. Note that these base-pairing in- teractions involve the invariant nucleotides of tRNAs. These interactions fold the D and TC arms together and bend the cloverleaf into the stable L-shaped tertiary form (Figure 11.35). Many of these base-pairing interactions involve base pairs that are not canonical AϺT or GϺC pairings, as illustrated around the central ribbon dia- gram of the tRNA in Figure 11.35. Note that three of the interactions involve three bases. The amino acid acceptor stem (highlighted in green) is at one end of the in- verted, backward L shape, separated by 7 nm or so from the anticodon at the oppo- site end of the L. The D and TC loops form the corner of the L. Hydrophobic stack- ing interactions between the flat faces of the bases contributes significantly to L-form stabilization. A C 3' 5' OH Acceptor stem C Y A R C ψ T Variable loop Y Anticodon loop Anticodon G G TψC loop D loop Invariant G Invariant pyrimidine, Y Invariant TψC Invariant purine, R Anticodon CCA 3' end C P R Y U A R R U Y A G CO CH H 3 N R + FIGURE 11.33 A general diagram for the structure of tRNA.The positions of invariant bases as well as bases that seldom vary are shown in color. R ϭ purine; Y ϭ pyrimidine. Dotted lines denote sites in the D loop and variable loop regions where varying numbers of nu- cleotides are found in different tRNAs. Inset: An aminoa- cyl group can add to the 3’-OH to create an aminoacyl- tRNA. 346 Chapter 11 Structure of Nucleic Acids Ribosomal RNA also Adopts Higher-Order Structure Through Intrastrand Base Pairing rRNA Secondary Structure A large degree of intrastrand sequence complementarity is found in all ribosomal RNA strands, and all assume a highly folded pattern that allows base pairing between these complementary segments, giving rise to multiple stem-loop structures. Furthermore, the loop regions of stem-loops contain the characteristic structural motifs, such as U-turns, tetraloops, and bulges. Figure 11.36 shows the secondary structure of several 16S rRNAs, based on computer alignment of each nucleotide sequence into optimal H-bonding segments. The re- liability of these alignments is then tested through a comparative analysis of whether very similar secondary structures are observed. If so, then such structures are apparently conserved. The approach is based on the thesis that because ribo- somal RNA species (regardless of source) serve common roles in protein synthesis, it may be anticipated that they share structural features. These secondary struc- tures resemble one another, even though the nucleotide sequences of these 16S rRNAs exhibit little sequence similarity. Apparently, evolution is acting at the level of rRNA secondary structure, not rRNA nucleotide sequence. Similar conserved folding patterns are seen for the 5S-like rRNAs and 23S-like rRNAs that reside in the large ribosomal subunits of various species. An insightful conclusion may be drawn regarding the persistence of such strong secondary structure conservation despite the millennia that have passed since these organisms diverged: All ribosomes A C 3' 70 5' OH Acceptor stem 75 65 C Am 1 G C ψ T 60 50 Variable loop Anticodon loop 35 Anticodon D 15 TψC loopD loop 10 5 C P D U U G G Y U Cm 30 A A C G C U U A A G A C A U 55 C G UG U C U G A G G U ψ A A A 40 Gm A G A C C C 25 A G G G 20 G A C A U U A G G C G Constant nucleotide Constant purine or pyrimidine Gm 7 Gm 2 2 C Cm 7 Cm 5 Gm 2 FIGURE 11.34 Tertiary interactions in yeast phenyl- alanine tRNA.The molecule is presented in the conven- tional cloverleaf secondary structure generated by in- trastrand hydrogen bonding.Solid lines connect bases that are hydrogen bonded when this cloverleaf pattern is folded into the characteristic tRNA tertiary structure (see also Figure 11.35). 11.7 What Are the Secondary and Tertiary Structures of RNA? 347 C56 G18 1-Methyl A58 T54 ψ55 G4 U69 A9 U12 A23 G45 G10 C25 G19 G15 C48 G22 C13 7-Methyl- G46 Dimethyl G26 A44 Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose Ribose 54 56 20 44 32 38 26 12 7 69 72 76 1 4 64 60 50 15 Anticodon 3' (a) (b) FIGURE 11.35 (a) The three-dimensional structure of yeast phenylalanine tRNA.The tertiary folding is illustrated in the center of the diagram with the ribose–phosphate backbone presented as a con- tinuous ribbon; H bonds are indicated by crossbars. Unpaired bases are shown as short, uncon- nected rods.The anticodon loop is at the bottom and the -CCA 3Ј-OH acceptor end is at the top right. (b) A space-filling model of the molecule (pdb id ϭ 6TNA). 348 Chapter 11 Structure of Nucleic Acids are similar, and all function in a similar manner. As usual with RNAs, the single- stranded regions of rRNA create the possibility of base-pairing opportunities with distant, complementary, single-stranded regions. Such interactions are the driving force for tertiary structure formation in RNAs. rRNA Tertiary Structure Recently, the detailed structure of ribosomes has been revealed through X-ray crystallography and cryoelectron microscopy of ribo- somes (see Chapter 30). These detailed images not only disclose the tertiary structure of the rRNAs but also the quaternary interactions that must occur when ribosomal proteins combine with rRNAs and when the ensuing ribonucleopro- tein complexes, the small and large subunits, come together to form the com- plete ribosome. Only the rRNAs of the 50S ribosomal subunit are shown in Figure 11.37; no ribosomal proteins are shown. Note that the overall anatomy of the 50S ribosomal subunit (shown diagrammatically in Figure 10.22) is essen- tially the same as that of the rRNA molecules within this subunit, even though these rRNAs account for only 65% of the mass of this particle. An assortment of tertiary structural features are found in the rRNAs, including coaxial stacks, pseudoknots, and ribose zippers. We will consider the role of rRNA in ribosome structure and function in Chapter 30. Aptamers Are Oligonucleotides Specifically Selected for Their Ligand-Binding Ability Aptamers are synthetic oligonucleotides, usually RNA, which fold into very specific three-dimensional structures that selectively bind ligands with high affinity. Ligand binding by aptamers is based on the fundamental principle of structural comple- mentarity. The rich array of interactive possibilities presented by the four bases and the sugar–phosphate backbone, coupled with the inherent flexibility of polynu- cleotide chains, make nucleic acids very good ligand-binding candidates. The bases project polar amino and carbonyl functionalities, and their -electron density gives them nonpolar properties. The sugar–phosphate backbone presents polar OOH groups and regularly spaced, negatively charged phosphate groups. These phos- phate groups can coordinate cations and thus provide foci of positive charge. Syn- thetic aptamers designed to target a selected protein can be potent inhibitors of protein function; they are of interest in drug development. E. coli (a eubacterium) (a) H. volcanii (an archaebacterium) (b) S. cerevisiae (yeast, a lower eukaryote) (c) FIGURE 11.36 Comparison of secondary structures of 16S-like rRNAs from (a) a bacterium (E.coli), (b) an ar- chaeon (H. volcanii), and (c) a eukaryote (S. cerevisiae, a yeast). 11.7 What Are the Secondary and Tertiary Structures of RNA? 349 Dom III Dom I Dom II Dom IV Dom V Dom VI 43 44 42 41 40 39 38 37 35.1 45 46 47 33 35 34 30 29 28 27 25 25.1 26 24 23 15 21 22 32 31 36 48 49 60 59.1 51 50 56 57 59 66 65 63 62 61 64 67 68 69 71 76 77 75 74 72 73 99 100 97 96 95 94 93 92 91 90 58 53 54 52 101 20 19 11 10 8 7 6 2 1 3 4 5 13 12 14 16 18 89 88 87 86 83 85 84 81 80 82 79 78 C CU CA GC A A C C C A C G G A U G C C G A U C U C C G G G G G G C GAC A C CCGGGGAUU GGCCCCCACC U G U Loop C 40 50 60 70 80 90 120 1 105 100 30 20 Loop B Loop A Loop E Loop D Helix 3 Helix 2 Helix 5 Helix 1 Helix 4 U G C C C A C C G C U U C C G G G G U U G G C C C A A A G G G C C U C C G A A G U A C U G G A G G G GC C A A A A A G U 5S rRNA 23S rRNA 5Ј end 3Ј end (a) (b) (c) FIGURE 11.37 The secondary and tertiary structures of rRNAs in the 50S ribosomal subunit from the archaeon Haloarcula marismortui (pdb id ϭ 1FFk). (a) Secondary structure of the 23S rRNA, with various domains color- coded. (b) Secondary structure of 5S rRNA. (c) Tertiary structure of the 5S and 23S rRNAs within the 50S riboso- mal subunit.The 5S rRNA (red) lies atop the 23S rRNA. (Adapted from Figure 4 in Ban, N., et al., 2000.The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289:905–920.) 350 Chapter 11 Structure of Nucleic Acids Riboswitches, a naturally occurring class of aptamers, are conserved regions of mRNAs that reversibly bind specific metabolites and coenzymes and usually act as regulators of gene expression. Riboswitches are usually buried within the 5Ј- or 3Ј-untranslated regions of the mRNAs whose expression they regulate. Binding of the metabolite to the riboswitch typically blocks expression of the mRNA. Figure 11.38 shows the structure of the thiamine pyrophosphate riboswitch. FIGURE 11.38 Structure of the thiamine pyrophosphate (TPP) riboswitch, a conserved region within the mRNA that encodes enzymes for synthesis of this coenzyme (pdb id ϭ 2CKY).TTP, a pyrimidine-containing com- pound, is shown in orange. (From Figure 1b in Thore, S., Leibundgdut, M., and Ban, N., 2006. Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science 312:1208–1211.) SUMMARY 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? The most widely used protocol for nucleic acid sequencing is Sanger’s chain termination (also called the dideoxy or the primed syn- thesis) method. A DNA fragment of unknown sequence serves as tem- plate in a polymerization reaction using DNA polymerase. Polymeriza- tion depends on an oligonucleotide primer base-paired to the unknown sequence. All four DNA polymerase deoxynucleotide substrates—dATP, dGTP, dCTP, and dTTP—are present. In addition, the reaction mixture contains the four corresponding 2Ј,3Ј-dideoxynucleotides (ddATP, ddGTP, ddCTP, and ddTTP). As synthesis proceeds, a deoxynucleotide is usually added to the 3Ј-OH end of the growing chain as the newly formed strand is extended in the 5Ј→3Ј direction. Occasionally, however, a dideoxynucleotide is added and, because it lacks a 3Ј-OH group, it can- not serve as a deoxynucleotide acceptor in chain extension. Then syn- thesis is terminated. This base-specific premature chain termination is only a random, occasional event, and a population of new strands of vary- ing length is synthesized. The population of newly synthesized DNAs forms a nested set of molecules differing in length by just one nucleo- tide. Each has a dideoxynucleotide at its 3Ј-end. Because each of the four dideoxynucleotides bears a different fluorescent tag, the particular fluo- rescence (orange for ddA, blue for ddC, green for ddG, and red for ddT) indicates which base was specified by the template and incorpo- rated by DNA polymerase at that spot. The sequencing products are vi- sualized by fluorescence spectroscopy following capillary electrophore- sis, revealing the sequence of the newly synthesized strands. This observed sequence is complementary to the corresponding unknown template sequence. Sanger sequencing has been fully automated. 11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? DNA typically occurs as a double helical molecule, with the two DNA strands running antiparallel to one another, bases in- side, sug ar–phosphate backbone outside. The double helical arrange- ment dramatically curtails the conformational possibilities otherwise available to single-stranded DNA. DNA double helices can be in a num- ber of stable conformations, with the three predominant forms termed A-, B-, and Z-DNA. B-DNA, has about 10.5 base pairs per turn, each con- tributing about 0.332 nm to the length of the double helix. The base pairs in B-DNA are nearly perpendicular to the helix axis. In A-DNA, the pitch is 2.46 nm, with 11 bp per turn. A-DNA has its base pairs displaced around, rather than centered on, the helix axis. Z-DNA has four dis- tinctions: It is left-handed, it is GϺC-rich, the repeating unit on a given strand is the dinucleotide, and the sugar–phosphate backbone follows a zigzag course. Alternative hydrogen-bonding interactions between AϺT and GϺC gives rise to Hoogsteen base pairs. Interstrand Hoogsteen base pairing creates novel multiplex structures composed of three or four DNA strands. These multiplex structures occur naturally and have bio- logical implications. 11.3 Can the Secondary Structure of DNA Be Denatured and Rena- tured? When duplex DNA is subjected to conditions that disrupt base- pairing interactions, the double helix is denatured and the two DNA strands separate as individual random coils. Denatured DNA will rena- ture to re-form a duplex structure if the denaturing conditions are re- moved. The rate of DNA renaturation is an index of DNA sequence complexity. If DNA from two different species are mixed, denatured, and al- lowed to anneal, artificial hybrid duplexes may form, provided the DNA from one species is similar in nucleotide sequence to the DNA of the other. Nucleic acid hybridization can reveal evolutionary relationships, and it can be exploited to identify specific DNA sequences. 11.4 Can DNA Adopt Structures of Higher Complexity? Supercoils are one kind of DNA tertiary structure. In relaxed, B-form DNA, the two strands wind about each other once every 10 bp or so (once every turn of Problems 351 the helix). DNA duplexes form supercoils if the strands are underwound (negatively supercoiled) or overwound (positively supercoiled). The basic para- meter characterizing supercoiled DNA is the linking number, L. L can be equated to the twist (T) and writhe (W ), where twist is the number of he- lical turns and writhe is the number of supercoils: L ϭ T ϩ W. L can be changed only if one or both strands of the DNA are broken, the strands are wound tighter or looser, and their ends are rejoined. DNA gyrase is a topoisomerase that introduces negative supercoils into bacterial DNA. 11.5 What Is the Structure of Eukaryotic Chromosomes? The DNA in a eukaryotic cell exists as chromatin, a nucleoprotein complex mostly composed of DNA wrapped around a protein core consisting of eight histone polypeptide chains—two copies each of histones H2A, H2B, H3, and H4. This DNAϺhistone core structure is termed a nucleosome, the fundamental structural unit of chromosomes. A higher order of chromatin structure is created when the array of nu- cleosomes is wound into a solenoid, creating a 30-nm filament. This 30-nm filament then is formed into long DNA loops, and loops are arranged radially about the circumference of a single turn to form a miniband unit of a chromosome. SMC proteins mediate chomoso- mal dynamics, including chromatin condensation and chromosome formation. 11.6 Can Nucleic Acids Be Synthesized Chemically? Laboratory syn- thesis of oligonucleotide chains of defined sequence is accomplished through orthogonal solid-phase methods based on phosphoramidite chemistry. Chemical synthesis takes place in the 3Ј→5Ј direction (the reverse of the biological polymerization direction). Commercially avail- able automated instruments called DNA synthesizers can synthesize oligonucleotide chains with 150 bases or more. 11.7 What Are the Secondary and Tertiary Structures of RNA? Compared to double-stranded DNA, single-stranded RNA has many more conformational possibilities, but intramolecular interactions and other stabilizing influences limit these possibilities. RNA mole- cules have many double-stranded regions formed via intrastrand hy- drogen bonding . Such double-stranded regions give rise to hairpin stem-loop structures. A number of defined structural motifs recur within the loops of stem-loop structures, such as U-turns and tetraloops. Single-stranded loops in RNA stem-loops create base-pairing oppor- tunities between distant, complementary, single-stranded loop regions. Other tertiary structural motifs arise from coaxial stacking, pseudoknot formation, and ribose zippers. In tRNAs, the formation of stem-loops leads to a cloverleaf pattern of secondary structure formed from four base-paired segments: the acceptor stem, the D loop, the anticodon loop, and the TC loop. Base-pairing in- teractions between bases in the D and TC loops give rise to tertiary structure by bending the cloverleaf into the stable L-shaped form. Substantial intrastrand sequence complementarity also is found in ribosomal RNA molecules, leading to a highly folded pattern based on base pairing between complementary segments. The complete three-dimensional structure of rRNAs has revealed an assortment of the tertiary structural features common to RNAs, including coaxial stacks, pseudoknots, and ribose zippers. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. The oligonucleotide d-AGATGCCTGACT was subjected to sequenc- ing by Sanger’s dideoxy method, using fluorescent-tagged dideoxy- nucleotides and capillary electrophoresis, essentially as shown in Figure 11.3. Draw a diagram of the gel-banding pattern within the capillary. 2. The output of an automated DNA sequence determination by the Sanger dideoxy chain termination method performed as illustrated in Figure 11.3 is displayed at right. What is the sequence of the orig- inal oligonucleotide? 3. X-ray diffraction studies indicate the existence of a novel double- stranded DNA helical conformation in which ⌬Z (the rise per base pair) ϭ 0.32 nm and P (the pitch) ϭ 3.36 nm. What are the other pa- rameters of this novel helix: (a) the number of base pairs per turn, (b) ⌬ (the mean rotation per base pair), and (c) c (the true repeat)? 4. A 41.5-nm-long duplex DNA molecule in the B-conformation adopts the A-conformation upon dehydration. How long is it now? What is its approximate number of base pairs? 5. If 80% of the base pairs in a duplex DNA molecule (12.5 kbp) are in the B-conformation and 20% are in the Z-conformation, what is the length of the molecule? 6. A “relaxed,” circular, double-stranded DNA molecule (1600 bp) is in a solution where conditions favor 10 bp per turn. What is the value of L 0 for this DNA molecule? Suppose DNA gyrase introduces 12 negative supercoils into this molecule. What are the values of L, W, and T now? What is the superhelical density, ? 7. Suppose one double helical turn of a superhelical DNA molecule changes conformation from B- to Z-form. What are the changes in L, W, and T? Why do you suppose the transition of DNA from B- to Z-form is favored by negative supercoiling? 8. Assume that there is one nucleosome for every 200 bp of eukaryotic DNA. How many nucleosomes are there in a diploid human cell? Nucleosomes can be approximated as disks 11 nm in diameter and 6 nm long. If all the DNA molecules in a diploid human cell are in the B-conformation, what is the sum of their lengths? If this DNA is now arrayed on nucleosomes in the beads-on-a-string motif, what would be the approximate total height of the nucleosome column if these disks were stacked atop one another? 9. The characteristic secondary structures of tRNA and rRNA mole- cules are achieved through intrastrand hydrogen bonding. Even for the small tRNAs, remote regions of the nucleotide sequence inter- act via H bonding when the molecule adopts the cloverleaf pattern. Using Figure 11.33 as a guide, draw the primary structure of a tRNA and label the positions of its various self-complementary regions. 10. Using the data in Table 10.1, arrange the DNAs from the follow- ing sources in order of increasing T m : human, salmon, wheat, yeast, E. coli. 11. At 0.2 M Na ϩ , the melting temperature of double-stranded DNA is given by the formula, T m ϭ 69.3 ϩ 0.41 (% G ϩ C). The DNAs from mice and rats have (G ϩ C) contents of 44% and 40%, respectively. Calculate the T m s for these DNAs in 0.2 M NaCl. If samples of these DNAs were inadvertently mixed, how might they be separated from one another? 352 Chapter 11 Structure of Nucleic Acids 12. The buoyant density of DNA is proportional to its (G ϩ C) con- tent. (GϺC base pairs have more atoms per volume than AϺT base pairs.) Calculate the density () of avian tubercle bacillus DNA from the data presented in Table 10.1 and the equation ϭ 1.660 ϩ 0.098(GC), where (GC) is the mole fraction of (G ϩ C) in DNA. 13. (Integrates with Chapter 10.) Pseudouridine () is an invariant base in the TC loop of tRNA; is also found in strategic places in rRNA. (Figure 10.23 shows the structure of pseudouridine.) Draw the structure of the base pair that might form with G. 14. The plasmid pBR322 is a closed circular dsDNA containing 4363 base pairs. What is the length in nm of this DNA (that is, what is its circumference if it were laid out as a perfect circle)? The E. coli K12 chromosome is a closed circular dsDNA of about 4,639,000 base pairs. What would be the circumference of a perfect circle formed from this chromosome? What is the diameter of a dsDNA molecule? Calculate the ratio of the length of the circular plasmid pBR322 to the diameter of the DNA of which it’s made. Do the same for the E. coli chromosome. 15. Listed below are four DNA sequences. Which one contains a type-II restriction endonuclease (“six-cutter”) hexanucleotide site? Which one that is likely to form a cruciform structure? Which one is likely to be found in Z-DNA? Which one represents the 5Ј-end of a tRNA gene? Which one is most likely to be found in a triplex DNA structure? a. CGCGCGCCGCGCACGCGCTCGCGCGCCGC b. GAACGTCGTATTCCCGTACGACGTTC c. CAGGTCTCTCTCTCTCTCTCTC d. TGGTGCGAATTCTGTGGAT e. ATCGGAATTCATCG 16. The nucleotide sequence of E. coli tRNA Gln is as follows: UGGGGUAUCG 10 CCAAGC−GGU 20 AAGGCACCGG 30 AUUCUGA⌿⌿C 40 CGGCAUUCCG 50 AGGT⌿CGAAU 60 CCUCGUACCC 70 CAGCCA 76 From this primary structure information, draw the secondary struc- ture (cloverleaf) of this RNA and identify its anticodon. 17. The Protein Data Bank (PDB) is also a repository for nucleic acid structures. Go to the PDB at www.rcsb.org and enter pdb id ϭ 1YI2. 1YI2 is the PDB ID for the structure of the H. marismortui 50S ribo- somal subunit with erythromycin bound. Erythromycin is an antibi- otic that acts by inhibiting bacterial protein synthesis. In the list of the display options under the image of the 50S subunit, click on the “KiNG” viewing option to view the structure. Using the tools of the KiNG viewer, zoom in and locate erythromycin within this structure. If the 50S ribosomal subunit can be compared to a mitten, where in the mitten is erythromycin? 18. Online resources provide ready access to detailed information about the human genome. Go the National Center for Biotechnology Infor- mation (NCBI) genome database at http://www.ncbi.nlm.nih.gov/ Genomes/index.html and click on Homo sapiens in the Map Viewer genome annotation updates list to access the chromosome map and organization of the human genome. Next, go to http://www.ncbi .nlm.nih.gov/genome/. In the “Search For” box, type in the follow- ing diseases to discover the chromosomal location of the affected gene and, by exploring links highlighted by the search results, dis- cover the name of the protein affected by the disease: a. Sickle cell anemia b. Tay Sachs disease c. Leprechaunism d. Hartnup disorder Preparing for the MCAT Exam 19. (Integrates with Chapter 10.) Erwin Chargaff did not have any DNA samples from thermoacidophilic bacteria such as those that thrive in the geothermal springs of Yellowstone National Park. (Such bacteria had not been isolated by 1951 when Chargaff re- ported his results.) If he had obtained such a sample, what do you think its relative GϺC content might have been? Why? 20. Think about the structure of DNA in its most common B-form double helical conformation and then list its most important struc- tural features (deciding what is “important” from the biological role of DNA as the material of heredity). Arrange your answer with the most significant features first. FURTHER READING General References Adams, R. L. P., Knowler, J. T., and Leader, D. P., 1992. The Biochemistry of the Nucleic Acids, 11th ed. London: Chapman and Hall. Gesteland, R. F., et al., eds. 2006. The RNA World, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Kornberg, A., and Baker, T. A., 1991. DNA Replication, 2nd ed. New York: W. H. Freeman. Sinden, R. R., 1994. DNA Structure and Function. St. Louis: Elsevier/ Academic Press. Watson, J. D., et al., 2007. The Molecular Biology of the Gene, 6th ed. Menlo Park, CA: Pearson/Benjamin Cummings. DNA Sequencing Meldrum, D., 2000. Automation for genomics, Part One: Preparation for sequencing. Genome Research 10:1081–1092. Meldrum, D., 2000. Automation for genomics, Part Two: Sequencers, mi- croarrays, and future trends. Genome Research 10:1288–1303. Nunnally, B. K., 2005. Analytical Techniques in DNA Sequencing. Boca Raton, FL: CRC Group, Taylor and Francis. Ziebolz, B., and Droege, M. 2007. Toward a new era in sequencing. Biotechnology Annual Review 13:1–26. Higher-Order DNA Structure Bates, A. D., and Maxwell, A., 1993. DNA Topology. New York: IRL Press at Oxford University Press. Benner, S. A., 2004. Redesigning genetics. Science 306:625–626. Callandine, C. R., et al., 2004. Understanding DNA: The Molecule and How It Works, 3rd ed. London: Academic Press. Frank-Kamenetskii, M. D., and Mirkin, S. A. M., 1995. Triplex DNA struc- tures. Annual Review of Biochemistry 64:65–95. Fry, M., 2007. Tetraplex DNA and its interacting proteins. Frontiers in Bio- sciences 12:4336–4351. Htun, H., and Dahlberg, J. E., 1989. Topology and formation of triple- stranded H-DNA. Science 243:1571–1576. Keniry, M. A., 2001. Quadruplex structures in nucleic acids. Biopolymers 56:123–146. Rich, A., 2003. The double helix: A tale of two puckers. Nature Structural Biology 10:247–249. Rich, A., Nordheim, A., and Wang, A. H-J., 1984. The chemistry and bi- ology of left-handed Z-DNA. Annual Review of Biochemistry 53: 791–846. Watson, J. D., ed., 1983. Structures of DNA. Cold Spring Harbor Symposia on Quantitative Biology, Volume XLVII. New York: Cold Spring Harbor Laboratory. Wells, R. D., 1988. Unusual DNA structures. Journal of Biological Chemistry 263:1095–1098. Zain, R., and Sun, J S., 2003. So natural triple-helical structurs occur and function in vivo? Cellular and Molecular Life Sciences 60:862–870. Nucleosomes Cobbe, N., and Heck, M. M. S., 2000. Review: SMCs in the world of chro- mosome biology—from prokaryotes to higher eukaryotes. Journal of Structural Biology 129: 123–143. Hirano, T ., 2005. SMC proteins and chromosome mechanics: From bac- teria to humans. Philosophical Transactions of the Royal Society London, Series B 360:507–514. . provide foci of positive charge. Syn- thetic aptamers designed to target a selected protein can be potent inhibitors of protein function; they are of interest in drug development. E. coli (a eubacterium) (a) H indicates which base was specified by the template and incorpo- rated by DNA polymerase at that spot. The sequencing products are vi- sualized by fluorescence spectroscopy following capillary electrophore- sis,