Coaxial stacking of stems or 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.. Stems
Trang 13'
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
1 3
3
1
FIGURE 11.31Junctions and coaxial stacking in RNA Stem junctions (or multibranched loops) are another type of RNA secondary structure Coaxial stacking of stems or 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
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, stem-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,
Trang 2mostly 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 2-OH groups of the respective strands, the O at the 2-OH position of one strand serving as the H-bond acceptor while the H on the 2-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 3- and 5-ends of the molecule meet These four segments are
desig-nated the acceptor stem, the D loop, the anticodon loop, and the T C loop (the latter
two are U-turn motifs).
A
hTR
3
C C C
G A U U
U U U
U U
L1
A A A
A A A
G 5
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 1YMO).(Adapted from Figure 2 in
Staple, D W., and Butcher, S E., 2005 Pseudoknots: RNA structures
with diverse functions PLoS Biology 3:e213.)
Trang 3tRNA 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 3-OH of the 3-terminal A nucleotide, thus
forming an aminoacyl ester (Figure 11.33) The 3-end of tRNA is invariantly
CCA-3-OH The D loop is so named because this tRNA loop often contains
dihy-drouridine, or D, residues In addition to dihydihy-drouridine, 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 5→3 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 5→3, is within the TC stem-loop It contains seven unpaired bases,
in-cluding the sequence T C, 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 T C 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 inin-teractions fold the D
and T C 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 A⬊T or G⬊C 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 T C 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
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
R U Y
A
G
H3N 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 purine;Y 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 aminoaaminoa-cyl- aminoacyl-tRNA
Trang 4Ribosomal 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 loop
D loop
10 5
C
P
D
U
U G
G
Y U Cm 30 A
A C G C U U A A
55
C
G U G 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
U U A G G C G
Constant nucleotide Constant purine or pyrimidine
Gm 7
Gm 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)
Trang 5T54
ψ55
G4
U69
A9 U12
A23
G45
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
54
56
20
44
32
38 26
12
7 69
72
76 1
4 64
60 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 3-OH acceptor end is at the top
right (b) A space-filling model of the molecule (pdb id 6TNA)
Trang 6are 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) (b)H volcanii (an archaebacterium) (c)S cerevisiae (yeast, a lower eukaryote)
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).
Trang 7Dom III
Dom I Dom II
Dom IV
Dom V
Dom VI
42
41
40
39
38
37
35.1
45 46 47
33
30
29 28 27
25 25.1 26
24 23 15
21 22
32
31
49 60 59.1 51 50
56
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
89
8887 86
8385
84 81 80 82 79 78
CC U C A G
A C
A C G G
A U G C C G A U
C U C G G G G G C G A C A C
C GG
G
A
U
G
G C CC
C
C
A
C
U G
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
C C C A C C G C
U U C G G
U U G C A A G G C U C G A
A G A C U G A G G G
G C C
A A A A
A G
U
5S rRNA
(a)
FIGURE 11.37 The secondary and tertiary structures of rRNAs in the 50S ribosomal subunit from the archaeon
Haloarcula marismortui (pdb id 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
Trang 8Riboswitches , 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 5- or 3-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 11.38Structure of the thiamine pyrophosphate
(TPP) riboswitch, a conserved region within the mRNA
that encodes enzymes for synthesis of this coenzyme
(pdb id 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 2,3-dideoxynucleotides (ddATP,
ddGTP, ddCTP, and ddTTP) As synthesis proceeds, a deoxynucleotide is
usually added to the 3-OH end of the growing chain as the newly
formed strand is extended in the 5→3 direction Occasionally, however,
a dideoxynucleotide is added and, because it lacks a 3-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 3-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, sugar–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 G⬊C-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 A⬊T and G⬊C 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 form a duplex strucrena-ture 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
Trang 9the 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 T 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 DNA⬊histone 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 3→5 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
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1.The oligonucleotide d-AGATGCCTGACT was subjected to
sequenc-ing by Sanger’s dideoxy method, ussequenc-ing 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) 0.32 nm and P (the pitch) 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 L0for 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 increasfollow-ing Tm: human, salmon, wheat,
yeast, E coli.
11. At 0.2 M Na, the melting temperature of double-stranded DNA is
given by the formula, T m 69.3 0.41 (% G C) The DNAs from mice and rats have (G C) contents of 44% and 40%, respectively
Calculate the Tms for these DNAs in 0.2 M NaCl If samples of these
DNAs were inadvertently mixed, how might they be separated from one another?
Trang 1012.The buoyant density of DNA is proportional to its (G C)
con-tent (G⬊C base pairs have more atoms per volume than A⬊T base
pairs.) Calculate the density () of avian tubercle bacillus
DNA from the data presented in Table 10.1 and the equation
1.660 0.098(GC), where (GC) is the mole fraction of (G 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 5-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 tRNAGlnis as follows:
UGGGGUAUCG10CCAAGC−GGU20AAGGCACCGG30
AUUCUGAC40CGGCAUUCCG50AGGTCGAAU60
CCUCGUACCC70CAGCCA76
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 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 G⬊C 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
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the Nucleic Acids, 11th ed London: Chapman and Hall.
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Kornberg, A., and Baker, T A., 1991 DNA Replication, 2nd ed New York:
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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
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Nunnally, B K., 2005 Analytical Techniques in DNA Sequencing Boca
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Ziebolz, B., and Droege, M 2007 Toward a new era in sequencing
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Nucleosomes
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