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
Trang 1Summary 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 Sfi I.
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 5-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 pyrophosphos-phate 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 3 to 5 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
Trang 2Preparing for an exam? Create your own study path for this
chapter at www.cengage.com/login
1. Draw the principal ionic species of 5-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 109base 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
5→3 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 3→5 direction, polymerizing
ribonucleotides in the 5→3 direction (see Figure 10.20) Give
the nucleotide sequence (5→3) of the DNA template strand
from which the following mRNA segment was transcribed:
5-UAGUGACAGUUGCGAU-3
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
5-UACGGUCUAAGCUGA What is the corresponding nucleotide
sequence (5→3) 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 PP
What is the G°overallfor synthesis of an RNA molecule 100 nucleo-tides in length, assuming that the G° for transfer of an NMP from
an NTP to the 3-O of polynucleotide chain is the same as the G°
for transfer of an NMP from an NTP to H2O? (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 109base 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 109bp?
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 3- versus 5-cleavage of phosphodiesters Re-striction endonucleases of the type II class are sequence-specific endo-nucleases useful in mapping the structure of DNA molecules
Trang 3FURTHER 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 evoluapprecia-tion, 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?
Trang 4Reginald H Garrett
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?
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Trang 511.1 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, 1018mol) of
DNA contained in less than 0.1 18moles of DNA are roughly
equiv-alent to 1012grams (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
3-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 5n3 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 2,3-dideoxynucleotides
(ddATP, ddGTP, ddCTP, and ddTTP); it is these dideoxynucleotides that give the
method its name.
Because dideoxynucleotides lack 3-OH groups, they cannot serve as acceptors
for 5-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
A T
G C
T A A
G C A T G C
A T
G C
C G A A
G C
G C
G C
G
C G C
A T
A T A T G C
A T
A T T G C
A T A T
G C A
T A T
G C
New
Parental DNA
FIGURE 11.1 DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule
Single-stranded DNA
5'
A T
G C
T A
C G
T A G C A A C T
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
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Trang 6318 Chapter 11 Structure of Nucleic Acids
length by just one nucleotide Each newly synthesized strand has a dideoxynu-cleotide at its 3-end, and each of the four dideoxynudideoxynu-cleotides 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 dideoxynudideoxynu-cleotides, 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 (5n3) 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 (5n3).
Sanger sequencing has been fully automated Automation is achieved through the use of robotics for preparing the samples, running the DNA sequencing reactions,
A
G T
C
Single-stranded DNA
to be sequenced T
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
A
5'
A
5'
G
5' 5'
T
5'
5' dCTP
dTTP plus limiting amounts
of fluorescently labeled
dGTP dATP
ddCTP ddTTP ddGTP
ddATP
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 3-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
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login.
Trang 711.1 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 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 PPiplus
adenosine-5 ⴕ-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:
PPi APS n ATP SO4 (This is the reverse of the ATP sulfurylase reaction shown as reac-tion 1 in Figure 25.34.)
Reaction 2:
ATP luciferin O2n AMP PPi CO2 oxyluciferin 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
A G A A T C G G C A T G C T A A A G T CA
APS
SO42ⴚ
PPi
Luciferin Oxyluciferin ATP
Light
DNA capture bead containing millions
of copies of a single clonal fragment
Luciferase Sulfurylase
dNTP Signal Image
Trang 8320 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 C1-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
5 4 3 2 1
1 again
4
1
C1 C2–endo
C3–endo
Pyrimidine:
Syn
C1
4
1
C1 Anti
6
3
Purine:
Syn
9 8
6
3
Anti
1 5
7
9
C1 8
O
135°
Absent in DNA
Free rotation about C1–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
(b)
The six degrees of freedom in the sugar–PO4 backbone:
(a)
Base
5
O
1
O
Base Base
O
3 2
4
2
3
P
5 O
O
O
O
4
4
5
1
3
2 Base
O
4
5
1
4
5
1
2
2
3
3
4
5
3
2
Base
2
3
FIGURE 11.4 (a) The six degrees of freedom in the
deoxyribose–PO4units of the polynucleotide chain
(b) Four puckered conformations of the furanose rings.
(c) Free rotation about the C1–N glycosidic bond
Trang 911.2 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 H2O 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 A ⬊T pair and G⬊C 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 A⬊T pair and three in a G⬊C 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 Mg2, 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 16 to 51 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
T A
T A
T A
C G
C G
(b) Helix
Base-pair spacing
0.34 nm
Pitch length 3.4 nm
A
G
G C
T A
G C
FIGURE 11.5 (a) Double-stranded DNA as an imaginary
ladderlike structure (b) A simple right-handed twist
converts the ladder to a helix
Trang 10322 Chapter 11 Structure of Nucleic Acids
A stereochemical consequence of the way A⬊T and G⬊C 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
N
C
N
C
O
O
H H H
H
H H
C
N
C
N
N
H
H
N
C
N
H
50o
To chain
To chain
1.11 nm
0.28 nm 0.30 nm
Adenine
51o Major groove
Minor groove
C 1 '
N
C
N
C
H
N
O
H H
C
N
C
H N O
H
N
C
N
H
N
H
0.29 nm 0.30 nm 0.29 nm
1.08 nm
To chain
To chain
Major groove
Minor groove
H
C 1 '
FIGURE 11.6 Watson–Crick A⬊T and G⬊C base pairs All
H bonds in both base pairs are straight
Minor groove
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
O
O
H H H
H
H H
H
H
N N
N
N N
H
FIGURE 11.7 The major and minor grooves of B-DNA