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The double helix: a tale of two puckers
© 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology
Alexander Rich
The era of the double helix began 50 years
ago with publication of the Watson-Crick
formulation1 and the fiber X-ray diffraction patterns from groups led by Maurice
Wilkins2 and Rosalind Franklin3. Analysis
of the diffraction pattern, especially the
fibers of the hydrated B-form, could be
immediately interpreted as consistent
with a double helix: the weakness of the
first-layer line and the virtual absence of
the fourth-layer line clearly were consistent with two chains wrapping around
each other with the phosphate groups on
the outside.
Wilkins and colleagues gradually refined
the nature of the double-helical model
that could give rise to the increasingly
detailed diffraction patterns. However, the
diffraction pattern could not ‘prove’ the
structure of the molecule as there was too
little information.
The deoxyribose sugar ring contains
five atoms; they cannot all lie in one plane,
and at least one atom must be out of plane
(Box 1). With the continued analysis of
the fiber patterns, it became clear that the
B-form contained a ring pucker in which
the C2′ atom was out of plane on the same
became clear that the normal conformation in the hydrated in vivo environment
involved the C2′ endo sugar pucker of
B-form DNA.
In the original double helix formulation, Watson and Crick1 had indicated
two hydrogen bonds between the guanine
and cytosine residues. Shortly thereafter,
an analysis by Pauling and Corey4 strongly
suggested that it was likely there would be
three hydrogen bonds. This hypothesis
was confirmed experimentally in 1963 in
a single crystal analysis of the 1:1 complex
of a guanine and a cytosine derivative5.
Does RNA form a
double helix?
Watson and Crick1 suggestDeoxyguanosine with attached phosphorous atoms is shown in the two major nucleic acid sugar puckers.
The C2′ endo pucker (left) is found in B-DNA, whereas the C3′ endo pucker (right) is found in A-DNA or in RNA.
ed that it was unlikely that
The distance between successive phosphate groups is close to 7.0 Å in C2′ endo and shortens to 5.8–6 Å in the
RNA could form their proC3′ endo pucker. Nucleic acids can convert from one pucker to the other, although it takes greater energy for
posed structure because
conversion of ribonucleotides. Carbon, large open circles; hydrogen, small open circles; nitrogen, cross hatched
the added O2′ of the ribose
circles; oxygen, black circles; and phosphorous, heavily outlined open circles.
would produce prohibitive
van der Waals crowding.
Starting in 1954, attempts
were made to study RNA
fibers to see if they would
also form a double helix.
The results were ambiguous. Slightly oriented patterns could be obtained,
but they all looked the
same, independent of the
base composition of the
material6. A breakthrough
came in 1956 when, together with David Davies,
More complex and not answered at the side as the base (C2′ endo). Because of that we discovered that mixing polyuridylic
time was the question of why there were pucker, the phosphate groups could be up acid (poly(U)) and polyadenylic acid
two forms. What was the nature of the to ∼7 Å apart. Such an extended poly- (poly(A)) would form a double helix, as
less-hydrated fibers that produced the nucleotide chain influenced the rotation indicated by a well-oriented fiber diffracbetter-oriented and crystalline A-form, around the axis of successive base pairs. tion pattern7. Unlike the DNA diffraction
which could convert to the B-form? In Study of the more complex A-form led to patterns, there was no change in the patthose days a half-century ago, fiber dif- the conclusion that the C3′ atom was out tern with changing hydration, and the
fraction was the only way such large, elon- of plane (C3′ endo). In that conformation first-layer line was stronger than the
gated molecules could be studied. the phosphate groups were ∼5.8–6 Å second-layer line. In addition, the diameGenerally, the patterns revealed that the apart. Thus, the sugar phosphate back- ter of the molecule was greater than that
material in the fibers was rotationally dis- bone was shortened, producing a double of B-DNA. Eventually, it was discovered
ordered about the fiber axis. This was helix in which the base pairs were slightly that the double-stranded RNA molecule
often at the molecular level in the hydrat- displaced from the center of the helix to adopted a conformation similar to the
ed B-form and usually as elongated produce a flatter and somewhat wider A-form DNA, exclusively using a C3′ endo
microcrystals in the drier A-form. helix. Air-dried fibers were used to pro- sugar pucker (Box 1). The reason for this
Although the diffraction patterns were duce the A-type diffraction pattern. The adherence to the C3′ endo sugar pucker
limited in resolution, they were certainly A conformation was stabilized by the becomes apparent upon looking at the
consistent with the formulation. Over the smaller number of water molecules com- position of the additional oxygen that
next several years, work by Maurice pared to the B conformation. It soon would be present in the C2′ position of
BOX 1 Two major nucleic acid sugar puckers.
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© 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology
history
ribose. In the C3′ endo conformation
there is adequate separation between the
oxygen on C2′ and the oxygen on C3′, in
contrast to a van der Waals crowding that
occurs if the sugar pucker were C2′ endo.
Because of the unfavorable energetic situation of ribose in the C2′ endo conformation, RNA molecules are usually found in
the C3′ endo conformation. There is a significant energy barrier between the two
puckers for ribose; in contrast, the
deoxyribose ring has a small barrier.
Finding that poly(A) reacted with
poly(U) to form a double helix7 was surprising at the time. This was the discovery
of the first hybridization reaction in which
long polynucleotide chains formed a double helix based on the specificity of hydrogen bonds. Within a year, together with
Gary Felsenfeld, we discovered that a
second strand of poly(U) could be taken
into the helix to make a triple helix of one
poly(A) plus two poly(U)8. Because there
was no increase in the diameter of the helix,
we suggested that the additional uracil
residue was bound by two hydrogen bonds
to the amino group and N7 of adenine. This
interpretation was considerably strengthened two years later by Hoogsteen’s singlecrystal analysis of 1-methyl thymine in
complex with 9-methyl adenine9.
The DNA–RNA hybrid helix
The availability of short polynucleotides
in the deoxy series chemically synthesized
by Khorana and colleagues10 made it possible to ask whether a hybrid helix could
be made with one RNA strand and one
DNA strand. It was known at that time
that the conformation of B-DNA in solution was quite different from that seen in
the RNA–RNA duplexes; thus, it was not
obvious that they could combine. In 1960,
I could show that a two-stranded molecule would form with an RNA chain
(poly(A)) and a DNA chain (polydeoxythymidylic acid or poly(dT))11. This
reaction was important in two respects. It
was the first DNA–RNA hybridization
(which is still used today in isolating
mRNA through their poly(A) tails), and it
also represented a model for how RNA
polymerase might make an RNA strand by
forming a hybrid duplex with a single
DNA strand. One year later a purified
preparation of DNA-dependent RNA
polymerase revealed that this was precisely how the enzyme worked12. It was
only later that it was possible to show that
in the hybrid helix the DNA molecule
conformed to the RNA pucker so that
both strands had the C3′ endo sugar
pucker (Box 1). The important considera248
tion was that changing the conformation
of the RNA strand was energetically costly,
but changing the conformation of the
DNA strand was relatively easy. This
largely explained why the melting temperature of the RNA duplex was considerably
higher than a DNA duplex with the same
sequence, and the melting temperature of
the RNA–DNA hybrid was intermediate.
The double helix at atomic
resolution
A number of single crystal diffraction
studies of purine-pyrimidine co-crystals
were carried out in the 1960s. A disturbing trend was found in that all co-crystals
of adenine derivatives with uracil or
thymine derivatives had Hoogsteen pairing, but none had Watson-Crick base
pairs13. This led to a Hoogsteen model of
the DNA double helix14 that did not fit the
diffraction data as well as the WatsonCrick model, but one could rely on the
fiber diffraction data only to a limited
extent. The question remained: what was
the structure of a double helix?
The first single crystal structures of a
double helix were solved in my laboratory
in 1973. This was before it was possible to
obtain oligonucleotides in quantities suitable for crystallographic experiments.
However, we succeeded in crystallizing
and solving two dinucleoside phosphates,
the RNA oligomers GpC15 and ApU16.
Furthermore, the resolution of the diffraction pattern was 0.8 Å. At atomic resolution we could visualize not only the
sugar phosphate backbone in the form of
a double helix but also the position of ions
and water molecules. By extending the
structure using the symmetry of the two
base pairs, it was possible to generate RNA
double helices that were quite similar to
the structures that had been deduced
from studies of double-helical fibers of
RNA.
The GpC structure had the anticipated
base pairs connected by three hydrogen
bonds. However, the ApU structure
showed for the first time that WatsonCrick base pairs were formed when the
molecule was constrained in a double
helix, as opposed to the Hoogsteen base
pairs that were favored in single
nucleotide complexes. The significance
of the double helix at atomic resolution
was recognized by the editors of Nature,
who, in their News and Views commentary, called this the “missing link” and
recognized “the many pearls offered” to
help resolve one of the major uncertainties in nucleic acid structure (Nature 243,
114; 1973). These structures capped the
effort that I had started some 20 years
earlier which had been initiated in
earnest with the recognition that poly(A)
and poly(U) would form a double helix.
High-resolution crystallographic analyses of larger fragments of the double
helix (DNA or RNA) did not emerge for
another six to seven years with the
availability of chemically synthesized
oligonucleotides.
The complex manner in which the RNA
double helix can fold into novel conformations was first visualized in the structure of tRNA. The rate-limiting step was
obtaining a crystal of sufficient resolution.
In 1971, we found that the addition of
spermine to yeast tRNAPhe led to a crystal
which diffracted to 2.3 Å resolution17.
This was the first crystal of a macromolecular nucleic acid, and heavy atom derivatives as well as methods to identify the end
of the molecule had yet to be discovered.
This was before the days of area detectors
or cryo-crystallography, and computer
programs were also quite primitive.
However, by 1973, at 4 Å resolution, the
architecture of the polynucleotide chain
could be traced through visualization of
the electron dense phosphate groups. This
revealed an unusual L-shaped structure in
which double helical segments were organized to form the arms of the L18. The
detailed nature of the complexity was
visualized a year later at 3 Å resolution in
which a number of interactions were
found beyond the Watson-Crick base
pairs in the double helical segments19,20.
Although the nucleotides were predominantly in the C3′ endo ribose conformation, there were places where the chain
had to span a longer distance. In one segment, the chain was lengthened through
two nucleotides adopting the C2′ endo
conformation. This was a good illustration of the manner in which an otherwise
inflexible RNA strand would adopt the
less favorable conformation locally to
yield an overall stable structure. This field
of complex RNA structure has now
exploded with ribozymes and, most
recently, the ribosome. Careful inspection
of those structures reveals changes in
sugar ring pucker at selected positions,
usually outside of the double helical segments.
DNA single crystals and left-handed
Z-DNA
The advent of DNA single crystal analysis
awaited the development of chemical
methods for synthesizing oligonucleotides. In the late 1970s these methods were becoming successful. At about
nature structural biology • volume 10 number 4 • april 2003
© 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology
history
this time, I established a collaboration
with a Dutch nucleotide chemist, Jacques
van Boom, on a DNA single crystal study.
Since G-C base pairs were more stable, I
suggested a simple sequence d(CGC
GCG) that might be induced to crystallize. To our delight, it crystallized and diffracted to 0.9 Å resolution. Here, at last,
we could see the DNA duplex at atomic
resolution. To our surprise, what emerged
was an unusual left-handed form of the
double helix: two antiparallel chains still
held together by Watson-Crick base pairs
but in an entirely different conformation
than that seen for B-DNA21. It was a
somewhat elongated and thinner molecule that had only one groove; it was
related to right-handed B-DNA by flipping the bases upside down so that the
upper surface of a base pair in B-DNA
becomes the lower surface of the base pair
when it changes to Z-DNA. This inversion results in rotating the purine bases
about their C-N glycosyl bond (Box 1), so
that it assumes the syn conformation. But
most interesting was the fact that it also
changed the pucker of the purine ribose
ring so it adopted the C3′ endo pucker
found in RNA molecules. In progressing
along the polynucleotide chain the bases
had an alternation of anti and syn conformations and also an alternation of C2′
endo and C3′ endo sugar puckers. The
molecule was a surprising hybrid in terms
of ring pucker. This led to a zig-zag backbone, hence the name Z-DNA. Although
there had been indications of a conformational change in poly(dG-dC)22, we were
quite unprepared for accommodating
this alternative conformation into our
picture of DNA biology. This conformation was unstable relative to B-DNA
because the phosphate–phosphate distances were somewhat shorter, and the
electrostatic repulsion is less in the
B conformation. However, it was later
found that negative supercoiling would
stabilize the Z conformation23. This
discovery led to the recognition of
transient Z-DNA formation in biological
systems in segments that were subjected
to negative torsional strain, especially
during transcription.
Other sequences crystallized as B-DNA,
the most important of which was the
Dickerson dodecamer24. It served as the
basis for many different studies of the
effect of a sequence on conformation.
More recently, the development of NMR
methods for solution studies has made it
possible to define the structure of
oligonucleotides free of lattice constraints.
This has greatly enhanced our knowledge
of the structural biology of DNA molecules.
Today, a great many complex DNA
structures are visualized, including fourstranded conformations. Many DNA–
protein structures are seen, such as the
intricate folding of B-DNA around the
nucleosome core, studies of DNA undergoing replication or transcription or with
significant changes in topology as, for
example, due to the TATA box–binding
protein. In all of these studies, attention is
paid to the ring pucker of the DNA molecule since changes from the simple,
unperturbed double-helical B-DNA con-
nature structural biology • volume 10 number 4 • april 2003
formation are usually associated with
changes in the pucker, utilizing the elastic
element inherent in the polynucleotide
backbone.
Alexander Rich is in the Department of
Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts
02139, USA. Tel: 617-253-4715; Fax: 617253-8699.
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