Helical twist is the rotation around the axis of the double helix of one base pair relative to the next Figure 11.8a.. Successive base pairs in B-DNA show a mean rotation of 36° with res
Trang 1The edges of the base pairs have a specific relationship to these grooves The “top”
edges of the base pairs (“top” as defined by placing the glycosidic bond at the bottom,
as in Figure 11.7) are exposed along the interior surface or “floor” of the major
groove; the base-pair edges nearest to the glycosidic bond form the interior surface of
the minor groove Some proteins that bind to DNA can actually recognize specific
nu-cleotide sequences by “reading” the pattern of H-bonding possibilities presented by
the edges of the bases in these grooves Such DNA–protein interactions provide one
step toward understanding how cells regulate the expression of genetic information
encoded in DNA (see Chapter 29)
Double Helical Structures Can Adopt a Number of Stable Conformations
In solution, DNA ordinarily assumes the familar structure we have been discussing:
B-DNA However, nucleic acids also occur naturally in other double helical forms
The base-pairing arrangement remains the same, but the inherently flexible sugar–
phosphate backbone can adopt different conformations Base-pair rotations are
an-other kind of conformational variation Helical twist is the rotation (around the axis
of the double helix) of one base pair relative to the next (Figure 11.8a) Successive
base pairs in B-DNA show a mean rotation of 36° with respect to each other
Pro-pellor twist involves rotation around a different axis, namely, an axis perpendicular
to the helix axis (Figure 11.8b) Propellor twist allows greater overlap between
suc-cessive bases along a strand of DNA and diminishes the area of contact between
bases and solvent water
A-Form DNA Is an Alternative Form of Right-Handed DNA
An alternative form of the right-handed double helix is A-DNA A-DNA molecules
dif-fer from B-DNA molecules in a number of ways The pitch, or distance required to
complete one helical turn, is different In B-DNA, it is 3.4 nm, whereas in
A-DNA it is 2.46 nm One turn in A-DNA requires 11 bp to complete Depending on
local sequence, 10 to 10.6 bp define one helical turn in B-form DNA In A-DNA, the
base pairs are no longer nearly perpendicular to the helix axis but instead are tilted
19° with respect to this axis Successive base pairs occur every 0.23 nm along the axis,
as opposed to 0.332 nm in B-DNA The B-form of DNA is thus longer and thinner than
the short, squat A-form, which has its base pairs displaced around, rather than
cen-tered on, the helix axis Figure 11.9 and Table 11.1 show the relevant structural
char-acteristics of the A- and B-forms of DNA (Z-DNA, another form of DNA to be
dis-cussed shortly, is also depicted in Figure 11.9 and Table 11.1.) A comparison of the
structural properties of A-, B-, and Z-DNA is summarized in Table 11.1
Relatively dehydrated DNA fibers can adopt the A-conformation, and DNA may
be in the A-form in dehydrated structures, such as bacterial and fungal spores The
pentose conformation in A-DNA is 3-endo, as opposed to 2-endo in B-DNA
Dou-ble helical DNA⬊RNA hybrids have an A-like conformation The 2-OH in RNA
sterically prevents double helical regions of RNA chains from adopting the
B-form helical arrangement Importantly, double-stranded regions in RNA chains
often assume an A-like conformation, with their bases strongly tilted with respect to
the helix axis
Z-DNA Is a Conformational Variation in the Form of a Left-Handed
Double Helix
Z-DNAwas first discovered when X-ray analysis of crystals of the synthetic
deoxynu-cleotide dCpGpCpGpCpG revealed an antiparallel double helix of unexpected
con-formation The alternating pyrimidine–purine (Py–Pu) sequence of this
oligonu-cleotide is the key to its unusual properties The N-glycosyl bonds of G residues in
this alternating copolymer are rotated 180° with respect to their conformation in
B-DNA, so now the purine ring is in the syn rather than the anti conformation
(Fig-ure 11.10) The C residues remain in the anti form Because the G ring is “flipped,”
T = 32 °
(a)
(b)
(1)
(c)
base
base H2O
H2O
(2)
Two base pairs with 32 ° of right-handed
helical twist: the minor-groove edges are drawn with heavy shading.
Propellor twist, as in (2), allows greater overlap
of successive bases along the same strand and reduces the area of contact between the bases and water.
Propellor-twisted base pairs Note how the hydrogen bonds between bases are distorted by this motion, yet remain intact The minor-groove edges
of the bases are shaded gray
base base
A
T G
C
FIGURE 11.8 Helical twist and propellor twist in DNA.
(a) Successive base pairs in B-DNA show a rotation with respect to each other (b) Rotation in a different dimension—propellor twist—allows the hydrophobic
surfaces of bases to overlap better Dots represent axes perpendicular to the helix axis The view is from the
sugar–P backbone (c) Each of the bases in a base pair
shows positive propellor twist (a clockwise rotation from
the horizontal, as viewed along the N-glycosidic bond,
from the pentose C1 to the base) (Adapted from Figure
3.4 in Callandine, C R., and Drew, H R., 1992 Understanding DNA: The Molecule and How It Works London: Academic Press.)
Trang 2the C ring must also flip to maintain normal Watson–Crick base pairing However, pyrimidine nucleosides do not readily adopt the syn conformation because it cre-ates steric interference between the pyrimidine C-2 oxy substituent and atoms of the pentose Because the cytosine ring does not rotate relative to the pentose, the whole
C nucleoside (base and sugar) must flip 180° (Figure 11.11) It is topologically pos-sible for the G to go syn and the C nucleoside to undergo rotation by 180° without breaking and re-forming the G⬊C hydrogen bonds In other words, the B-to-Z struc-tural transition can take place without disrupting the bonding relationships among the atoms involved
Z-DNA
FIGURE 11.9 Comparison of the A-, B-, and Z-forms of the DNA double helix The A- and B-structures show 12 bp
of DNA; the Z-structures, 6 bp The middle Z-structure shows just one strand of a Z-DNA double helix to illustrate better the left-handed zigzag path of the polynucleotide backbones in Z-DNA (The light blue line was added to show the imaginary zigzag path.) A-DNA: pdb id 2D47, B-DNA: pdb id 355D, Z-DNA: pdb id 1DCG.
Trang 3Double Helix Type
Major groove proportions Extremely narrow but very Wide and with Flattened out on helix
Minor groove proportions Very broad but shallow Narrow and with Extremely narrow but very
intermediate depth deep
Adapted from Dickerson, R L., et al., 1983 Helix geometry and hydration in A-DNA, B-DNA, and Z-DNA Cold Spring Harbor Symposium on Quantitative Biology 47:13–24.
Deoxyguanosine in B-DNA (anti position) Deoxyguanosine in Z-DNA (syn position)
FIGURE 11.10 Comparison of the deoxyguanosine con-formation in B- and Z-DNA.
B-DNA
B-DNA
Z-DNA
B-DNA
1
2
FIGURE 11.11 The change in topological relationships
of base pairs from B- to Z-DNA A six-base-pair
GCGCGC segment of B-DNA (1) is converted to Z-DNA (2) through rotation of the base pairs, as
indi-cated by the curved arrows The purine rings (green)
of the deoxyguanosine nucleosides rotate via an anti
to syn change in the conformation of the guanine– deoxyribose glycosidic bond; the pyrimidine rings (blue) are rotated by flipping the entire
deoxycyto-sine nucleoside (base and deoxyribose).
Trang 4Because alternate nucleotides assume different conformations, the repeating unit
on a given strand in the Z-helix is the dinucleotide That is, for any number of bases,
n, along one strand, n 1 dinucleotides must be considered For example, a
GpCpGpC subset of sequence along one strand is composed of three successive
dinu-cleotide units: GpC, CpG, and GpC (In A- and B-DNA, the nudinu-cleotide conformations are essentially uniform and the repeating unit is the mononucleotide.) It follows that the CpG sequence is distinct conformationally from the GpC sequence along the al-ternating copolymer chains in the Z-double helix The conformational alterations go-ing from B to Z realign the sugar–phosphate backbone along a zigzag course that has
a left-handed orientation (Figure 11.9), thus the designation Z-DNA Note that in any GpCpGp subset, the sugar–phosphates of GpC form the horizontal “zig” while the CpG backbone segment forms the vertical “zag.” The mean rotation angle circum-scribed around the helix axis is 15° for a CpG step and 45° for a GpC step (giving
60° for the dinucleotide repeat) The minus sign denotes a left-handed or counter-clockwise rotation about the helix axis Z-DNA is more elongated and slimmer than B-DNA
Cytosine Methylation and Z-DNA The Z-form can arise in sequences that are not strictly alternating Py–Pu For example, the hexanucleotide m5CGATm5CG, a Py-Pu-Pu-Py-Py-Pu sequence containing two 5-methylcytosines (m5C), crystallizes as Z-DNA Indeed, the in vivo methylation of C at the 5-position is believed to favor a B-to-Z switch because, in B-DNA, these hydrophobic methyl groups would protrude into the aqueous environment of the major groove, a destabilizing influence In Z-DNA, the same methyl groups can form a stabilizing hydrophobic patch It is likely that the Z-conformation naturally occurs in specific regions of cellular DNA, which oth-erwise is predominantly in the B-form Furthermore, because methylation is impli-cated in gene regulation, the occurrence of Z-DNA may affect the expression of ge-netic information (see Part 4)
The Double Helix Is a Very Dynamic Structure
The long-range structure of B-DNA in solution is not a rigid, linear rod Instead, DNA behaves as a dynamic, flexible molecule Localized thermal fluctuations temporarily distort and deform DNA structure over short regions Base and backbone ensembles
of atoms undergo elastic motions on a time scale of nanoseconds To some extent, these effects represent changes in rotational angles of the bonds comprising the polynucleotide backbone These changes are also influenced by sequence-dependent variations in base-pair stacking The consequence is that the helix bends gently When these variations are summed over the great length of a DNA molecule, these bending influences give the double helix a roughly spherical shape, as might be expected for
a long, semirigid rod undergoing apparently random coiling It is also worth noting
that, on close scrutiny, the surface of the double helix is not that of a totally
feature-less, smooth, regular “barber pole” structure Different base sequences impart their own special signatures to the molecule by subtle influences on such factors as the groove width, the angle between the helix axis and base planes, and the mechanical rigidity Certain regulatory proteins bind to specific DNA sequences and participate
in activating or suppressing expression of the information encoded therein These proteins bind at unique sites by virtue of their ability to recognize novel structural characteristics imposed on the DNA by the local nucleotide sequence
Intercalating Agents Distort the Double Helix Aromatic macrocycles, flat
hydro-phobic molecules composed of fused, heterocyclic rings, such as ethidium bromide,
base pairs of DNA The bases are forced apart to accommodate these so-called
intercalating agents,causing an unwinding of the helix to a more ladderlike struc-ture The deoxyribose–phosphate backbone is almost fully extended as successive base pairs are displaced 0.7 nm from one another, and the rotational angle about the helix axis between adjacent base pairs is reduced from 36° to 10°
Trang 5Dynamic Nature of the DNA Double Helix in Solution Intercalating substances
sert with ease into the double helix, indicating that the van der Waals stacking
in-teractions that they share with the bases sandwiching them are more favorable than
similar interactions between the bases themselves Furthermore, the fact that these
agents slip in suggests that the double helix must momentarily unwind and present
gaps for these agents to occupy That is, the DNA double helix in solution must be
represented by a set of metastable alternatives to the standard B-conformation
These alternatives constitute a flickering repertoire of dynamic structures
Alternative Hydrogen-Bonding Interactions Give Rise to Novel
DNA Structures: Cruciforms, Triplexes and Quadruplexes
Cruciform Structures Arise from Inverted Repeats Inverted repeats (Figure
11.13) are duplex DNA sequences showing twofold symmetry (the 5n3 sequence
is identical in both strands) Palindromes are words, phrases, or sentences that read
the same backward or forward, such as “radar,” “sex at noon taxes,” “Madam, I’m
Adam,” and “a man, a plan, a canal, Panama.” Inverted repeats are sometimes
ferred to as palindromes (despite the inaccuracy of this description) Inverted
re-peats have the potential to adopt cruciform (meaning “cross-shaped”) structures if
the normal interstrand base pairing is replaced by intrastrand pairing In effect,
each strand forms a hairpin structure through alignment and pairing of the
self-complementary sequences along the strand Cruciforms are never as stable as
nor-mal DNA duplexes because an unpaired segment must exist in the loop region
Cru-ciforms potentially create novel structures that can serve as distinctive recognition
sites for specific DNA-binding proteins
+
N+
CH2CH3
Ethidium bromide
N(CH3)2 (CH3)2N
+ N H
Acridine orange
C O
CH3 CH3 O
N C O
NH2
O
Thr O
L -Meval Pro
Sarcosine
Thr O
L -Meval Pro
Sarcosine
Actinomycin D
Intercalating agents B-DNA before
intercalation
B-DNA after intercalation
Br–
or
or
Sar = Sarcosine = H3C N
H CH2 COOH (N-Methylglycine)
Meval = Mevalonic acid = HOCH2 CH2
CH3
OH
CH2 COOH
acri-dine orange, and actinomycin D, three intercalating agents, and their effects on DNA structure.
Trang 6Hoogsteen Base Pairs and DNA Multiplexes The A⬊T and G⬊C base pairs first seen
by Watson (Figure 11.6) are the canonical building blocks for DNA structures How-ever, Karst Hoogsteen found that adenine and thymine do not pair in this way when crystallized from aqueous solution Instead, they form two H bonds in a different arrangement (Figure 11.14) Further, Hoogsteen observed that, in mildly acidic solu-tions, guanine and cytosine form base pairs different from Watson–Crick G⬊C base pairs These Hoogsteen base pairs depend upon protonation of cytosine N-3 (Figure 11.14) and have only two H bonds, not three In both A⬊T and G⬊C Hoogsteen base pairs, the purine N-7 atom is an H-bond acceptor The functional groups of adenine and guanine that participate in Watson–Crick H bonds remain accessible in Hoog-steen base pairs Thus, base triplets can form, as shown in Figure 11.15, giving rise
G
C T
A A
T C
G T
A T
A G
C C
G A
T G
C G
C A
T T
A A
T A
T C
G A
T G
C C
G C
G T
A G
C C
G A
T A
T G
C A
T C
G T
A
G C T A A T
.
C G C G T A G C C G A T A T G C C G T A T A G C C G A T G C G C A T C G T A
.
T AT T GT C A T A A CA G
.
FIGURE 11.13 Self-complementary inverted repeats can rearrange to form hydrogen-bonded cruciform stem-loop structures. FIGURE 11.14 Hoogsteen base pairs: A⬊T (left) and C ⬊G (right).
H H N N+ N H H N N N N O O N N N N T:A C+:G N N N O O H H H CH3 N H H
A T
H
N
N
N N
N N
N N
N
O
O
O
O
H
H
H
CH3
CH3
T
N+
N
H
H
N
N
O
O
G C
C
N
N H
H
H
N O
H
N H
FIGURE 11.15 Base triplets formed when a purine interacts with one pyrimidine by Hoogsteen base pairing and another by Watson–Crick base pairing.
Trang 7to TAT and a CGC triplets, where each purine interacts with one of its pyrimidine
partners through Hoogsteen base pairing and the other through Watson–Crick base
pairing
H-DNA Is Triplex DNA Under certain conditions, triple-stranded DNA
struc-tures can form In H-DNA, two of the strands are pyrimidine-rich and the third
is purine-rich One pyrimidine-rich strand is hydrogen bonded to the purine-rich
strand via Watson–Crick base pairing, and the other pyrimidine-rich strand is
hydrogen bonded to the purine-rich strand by Hoogsteen base pairing Such
structures were originally referred to as H-DNA, because protonation of the
cy-tosine N-3 atom was necessary, but the name also fits because a hinge is present
between double- and triple-stranded DNA regions when H-DNA forms Consider,
for example, a long stretch of alternating C⬊T sequence in one strand of a DNA
duplex (Figure 11.16) If the C⬊T bases in half of this stretch separated from
their G⬊A partners and the unpaired C⬊T segment folded back on the C⬊T half
still paired in the C⬊T/G⬊A duplex, triplex DNA could form through Hoogsteen
base pairing Triple-stranded DNA is implicated in the regulation of some
eu-karyotic genes
DNA Quadruplex Structures Four-stranded DNA structures can form between
polynucleotide strands rich in guanine At the heart of such G-quadruplexes are
cyclic arrays of four G residues united through Hoogsteen base pairing (Figure
11.17a) The presence of metal cations (K, Na, Ca2) favors their assembly
Free-electron pairs contributed by the closely spaced O6 carbonyl oxygens of the
G-quartet coordinate the centrally located cation A variety of different G-quadruplex
structures have been reported, with different G-rich sequences leading to variations
on a common quadruplex plan Quadruplexes constructed from dGnstrands usually
form with all four strands in parallel orientation and all bases in the anti
conforma-tion (Figure 11.17b) Polynucleotides with varying sequence repeats, such as (G3N)n
or (G2N2)n, form G-quadruplexes with variations on the dGnstructural theme, such as
the (dG4T4G4)2structure in which two such strands pair in antiparallel fashion to
form the G-quadruplex (Figure 11.17c and 11.17d) G-quadruplex structures have
biological significance because they have been found in telomeres (structures that
define the ends of chromosomes), in regulatory regions of genes, in
immunoglobu-lin gene regions responsible for antibody diversity, and in sequences associated with
human diseases
C G
C
40 •
3
5
C
G
T
A
G
G
3 5
G
C
T
T
A
C
G
C
G
A
A A T
T
G
C
A
T
G
T C
C
A
T
G
C
A
T
G
C
A
T
G
C
A
T
G
C
A
T
G
C
A T
•
1 5
G
•+•+•+•+•+•+•+•+
C
C T C T C T C T C T C T C
T G
G
A T
C
• C
AGAGAGAG
3 0 AGAGAGAG A
A T
T
FIGURE 11.16 H-DNA (a) The pyrimidine-rich strands of
the duplex regions are blue, and the purine-rich strands are green The Hoogsteen base-paired pyrimidine-rich strand in the triplex (H-DNA) structure is yellow.
(b) Nucleotide sequence representation of H-DNA
formation T ⬊A Hoogsteen base pairing leading to triplex formation is shown by dots; C -G Hoogsteen base pair-ing leadpair-ing to triplex formation is shown by signs (Adapted from Htun, H., and Dahlberg, J E., 1989 Topology and
formation of triple-stranded H-DNA Science 243:1571–1576.)
Trang 811.3 Can the Secondary Structure of DNA Be Denatured
and Renatured?
Thermal Denaturation of DNA Can Be Observed by Changes
in UV Absorbance
When duplex DNA molecules are subjected to conditions of pH, temperature, or ionic strength that disrupt base-pairing interactions, the strands are no longer held
together That is, the double helix is denatured, and the strands separate as
indi-vidual random coils If temperature is the denaturing agent, the double helix is said
to melt The course of this dissociation can be followed spectrophotometrically
be-cause the relative absorbance of the DNA solution at 260 nm increases as much as
40% as the bases unstack This absorbance increase, or hyperchromic shift, is due
to the fact that the aromatic bases in DNA interact via their -electron clouds when
stacked together in the double helix Because the UV absorbance of the bases is a consequence of -electron transitions, and because the potential for these
transi-tions is diminished when the bases stack, the bases in duplex DNA absorb less 260-nm radiation than expected for their numbers Unstacking alleviates this
sup-G4 G4
G1
G3
G2
G3 G4
(a)
(c)
(b)
G2
G1
G1
G3
G2 G4
G1
G3
G2
G12 T8
T6 T5
G9
G10
G11
G2 G1 T7
G3
G4
G9
G11
G10 G4
G1
G3
G2
G12
N N
O O
H H
H H
H
H
N
N
N H H
O
N H N
H
H
H
N H
N
O H H
H N
N
N
N N
(d)
FIGURE 11.17 (a) G-quadruplex showing the
cyclic array of guanines linked by Hoogsteen
hydrogen bonding (b) Four G-rich
polynucleo-tide strands in parallel alignment with all bases
in anti conformation (c) Antiparallel dimeric
hairpin quadruplex formed from d(G 4 T 4 G 4 ) 2 (d)
Structure of d(G 4 T 4 G 4 ) 2 K solved by X-ray
crystal-lography Two d(G 4 T 4 G 4 ) strands come together
as hairpins to form a G-quadruplex The
back-bones of the two strands are traced in violet.
(Adapted from Keniry, M A., 2001 Quadruplex structures
in nucleic acids Biopolymers 56:123–146.)
Trang 9pression of UV absorbance The rise in absorbance coincides with strand
separa-tion, and the midpoint of the absorbance increase is termed the melting
tempera-ture, Tm(Figure 11.18) DNAs differ in their Tmvalues because they differ in
rela-tive G C content The higher the G C content of a DNA, the higher its melting
temperature because G⬊C pairs have higher base stacking energies than A⬊T pairs
Also, Tm is dependent on the ionic strength of the solution; the lower the ionic
strength, the lower the melting temperature Because cations suppress the
electro-static repulsion between the negatively charged phosphate groups in the
comple-mentary strands of the double helix, the double-stranded form of DNA is more
sta-ble in dilute salt solutions DNA in pure water melts even at room temperature
pH Extremes or Strong H-Bonding Solutes also Denature DNA Duplexes
At pH values greater than 10, the bases of DNA become deprotonated, which
de-stroys their base-pairing potential, thus denaturing the DNA duplex Extensive
pro-tonation of the bases below pH 2.3 also disrupts base pairing Alkali is the preferred
denaturant because, unlike acid, it does not hydrolyze the glycosidic bonds linking
purine bases to the sugar–phosphate backbone Small solutes that readily form H
bonds can also denature duplex DNA at temperatures below Tm.If present in
suffi-ciently high concentrations, such small solutes will form H bonds with the bases,
thereby disrupting H-bonding interactions between the base pairs Examples
in-clude formamide and urea
Single-Stranded DNA Can Renature to Form DNA Duplexes
Denatured DNA will renature to re-form the duplex structure if the denaturing
con-ditions are removed (that is, if the solution is cooled, the pH is returned to
neutral-ity, or the denaturants are diluted out) Renaturation requires reassociation of the
DNA strands into a double helix, a process termed reannealing For this to occur, the
strands must realign themselves so that their complementary bases are once again in
register and the helix can be zippered up (Figure 11.19) Renaturation is dependent
on both DNA concentration and time Many of the realignments are imperfect, and
thus the strands must dissociate again to allow for proper pairings to be formed The
process occurs more quickly if the temperature is warm enough to promote diffusion
of the large DNA molecules but not so warm as to cause melting
The Rate of DNA Renaturation Is an Index of DNA Sequence Complexity
The renaturation rate of DNA is an excellent indicator of the sequence complexity of
DNA For example, the DNA of bacteriophage T4 contains 2 105base pairs; an
Escherichia coli cell contains more than ten times as much (4.64 106 base pairs)
Temperature ( ⬚C) 1.0
1.2
1.4
Pneumococcus
(38% G + C)
E coli (52%)
S marcescens (58%)
M phlei (66%)
FIGURE 11.18 Heat denaturation of DNA from various sources, so-called melting curves (From Marmur, J., 1959.
Heterogenity in deoxyribonucleic acids Nature 183:1427–1429.)
Trang 10E coli DNA is considerably more complex in that it encodes more information
Ex-pressed in another way, for any fixed amount of single-stranded DNA (in grams), the
nucleotide sequences represented in an E coli sample will show greater sequence
variation than those in an equal weight of phage T4 DNA Thus, it will take longer for
the E coli DNA strands to find their complementary partners and reanneal Because
the rate of DNA duplex formation depends on complementary DNA sequences en-countering one another and beginning the process of sequence alignment and rean-nealing, the time necessary for reconstituting double-stranded DNA molecules is an excellent index of the degree of sequence complementarity in a DNA sample
Nucleic Acid Hybridization: Different DNA Strands of Similar Sequence Can Form Hybrid Duplexes
If DNA from two different species are mixed, denatured, and allowed to cool slowly
so that reannealing can occur, hybrid duplexes may form, provided the DNA from
one species is similar in nucleotide sequence to the DNA of the other The degree
A DEEPER LOOK
The Buoyant Density of DNA
Density gradient ultracentrifugation is a variant of the basic
tech-nique of ultracentrifugation (discussed in the Appendix to
Chap-ter 5) The densities of DNAs are about the same as those of
con-centrated solutions of cesium chloride, CsCl (1.6 to 1.8 g/mL)
Centrifugation of CsCl solutions at very high rotational speeds,
where the centrifugal force becomes 105times stronger than the
force of gravity, causes the formation of a density gradient within
the solution This gradient is the result of a balance that is
estab-lished between the sedimentation of the salt ions toward the
bot-tom of the tube and their diffusion upward toward regions of
lower concentration If DNA is present in the centrifuged CsCl
solution, it moves to a position of equilibrium in the gradient
equivalent to its buoyant density (as shown in the figure) For this
reason, this technique is also called isopycnic centrifugation.
Cesium chloride centrifugation is an excellent means of
re-moving RNA and proteins in the purification of DNA The density
of DNA is typically slightly greater than 1.7 g/cm3, whereas the
density of RNA is more than 1.8 g/cm3 Proteins have densities less
than 1.3 g/cm3 In CsCl solutions of appropriate density, the DNA
bands near the center of the tube, RNA pellets to the bottom, and
the proteins float near the top Single-stranded DNA is denser
than double helical DNA The irregular structure of randomly
coiled ssDNA allows the atoms to pack together through van der
Waals interactions These interactions compact the molecule into
a smaller volume than that occupied by a hydrogen-bonded
dou-ble helix
The net movement of solute particles in an ultracentrifuge is the result of two processes: diffusion (from regions of higher con-centration to regions of lower concon-centration) and sedimentation due to centrifugal force (in the direction away from the axis of ro-tation) In general, diffusion rates for molecules are inversely pro-portional to their molecular weight—larger molecules diffuse more slowly than smaller ones On the other hand, sedimentation rates increase with increasing molecular weight A macromolecu-lar species that has reached its position of equilibrium in isopycnic centrifugation has formed a concentrated band of material Essentially three effects are influencing the movement of the molecules in creating this concentration zone: (1) diffusion away
to regions of lower concentration, (2) sedimentation of molecules situated at positions of slightly lower solution density in the den-sity gradient, and (3) flotation (buoyancy or “reverse sedimenta-tion”) of molecules that have reached positions of slightly greater solution density in the gradient The consequence of the physics
of these effects is that, at equilibrium, the width of the concentration
band established by the macromolecular species is inversely proportional to the square root of its molecular weight That is, a population of large
molecules will form a concentration band that is narrower than the band formed by a population of small molecules For example, the bandwidth formed by dsDNA will be less than the bandwidth formed by the same DNA when dissociated into ssDNA
CsCl solution
[6 M; density
()~1.7]
Cell extract
Mix CsCl solution and cell extract and place in centrifuge Centrifuge at
high speed for
~48 hours.
Proteins and nucleic acids absorb UV light The positions
of these molecules within the centrifuge can be determined
by ultraviolet optics.
=1.65
=1.80
CsCl density
DNA
RNA
Protein
1.80 1.65
Molecules move to positions where their density equals that
of the CsCl solution.
Density ()
in g/mL
RNA DNA Protein