126

b\

1leclicnl Rcscarch Council Bioph)-sits Rcwarch Unit, Riophysics
    D vpartmcnt, King's Collcgc, Uniwrsit\~ of 1,onclon

Nobel Lccturc, Dcccmbcr 1 1. 1962

Suclcic acids arc bnsicnll\~ simple. The)- arc at the root of vet+!- fundu-
mental biological processes, ,grolvth and inheritance. The simplicity of
nUCkiC acid InOlcCUhr ShIlCh2rC and of it5 I-elation 10 junction CxpreSSCS
the unclcrlying simplicit)- of the biological ph~:notncna? clarifies their
nature, and has given rise to the Iirst estcnsi\,c. interpretation of living
proccsscs in terms of t~~acromolccular 5tlWCtUl-(`. Thaw matters ha1.c onlv
become clear 1,). an unpreccclcntrcl combination of biologicd, chcmicla
ant1 ph\~sical stuclics? ran,+ng from g-cnetics to 11).tlrf+y~nboncl stcrcncliem-
istr\-. I shall not discuss all this hrrc but conccntrntc on tltc ficlcl inJvhich
I have ~vorkccl, ant1 sho\v hot\- S-ray clifli-action un;d\~sis has made its
contribution. I shdl tlescribc sonic of the back,qrouncl ot`m\- o\\-n rcwarchcs,
for I suspect I am not done in finding such account5 often mow intcrcsting
than gcncxil re\.ielvs.

I took a physics tlygrcc at Cnnihricl~c in 1938, ivitli wnic training in S-ra)
~t.!-stalloRral~ll~.. This S-ra!- backyrouncl ivns influcxcctl b!, .J. D. R~~rnal,
then at the Ca~~c~nclish. I began research at Birn~in~h;un, unc1cr.J. T. Ran-
dall, S~Uc~~~iIlg lLlmincSCCnCC md hOIv ClCTtrOIlS lii1JW iI cr\TSt:lk. \I!- Con-
tc`tnporarics at Cambridge had tnainly been intcrcstccl in c~lc~mc.tttar~~ par-
ticlcs! but the organisation of the solitl state anti 1hv sp(acial propcrtics
\\hich tlcpcnd~~cl on this organisation intcrcstcd mr nnorc. This ina\. bar-c>
bcvti a fi)rct-unncr of ni!- intcrcst in biolo+cal rnacrotiicrl~~~.~~l~s ant1 ho1v
tlicir structure rclatctl to their highly- specific- propc`rtics \I-liiclt so largcl)
dctcrminc the. processes of lift.


127

During the war I took part in making the atomic bomb. When the
l\`ar was ending, I, like many others, cast around for a new field of research.
i'artly on account of the bomb, I had lost some interest in physics. I was
therefore very interested when I read Schriidinger's book %`hat is Life"
and was struck by the concept of a highly complex molecular structure
lvhich controlled living processes. Research on such matters seemed more
ambitious than solid-state physics. At that time many leading physicists
such as Massey, Oliphant,and Randall (and later I learned that Bohr shared
their view) believed that physics would contribute significantly to biology;
their advice encouraged me to move into biology.

I went to work in the Physics Department at St. Andrews, Scotland,
where Randall had invited me to join a biophysics project he had begun.
Stimulated by Mu&r's experimental modification, by means of X-radia-
tion, of genetic substance, I thought it might be interesting to investigate
the effects of ultrasonics; but the results were not very encouraging,

The biophysics work then moved to King's College, London, where
Randall took the Wheatstone Chair of Physics and built up, with the help
of the R/Zedical Research Council, an unusual laboratory for a Physics
Department, where biologists, biochemists and others worked with the
physicists. He suggested I might take over some ultra-violet microscope
studies of the quantities of nucleic acids in cells. This work followed that
of Caspersson, but made use of the achromatism of reflecting microscopes.
By this time, the work of Caspersson (194lj and Brachet (1941) had made
the scientific world generally aware that nucleic acids had important
biological roles which were connected with protein synthesis. The idea
that DNA might itself be the genetic substance was, however, barely
hinted at. Its function in chromosomes was supposed to be associated
with replication of the protein chromosome thread. The work of -Avery,
MacLeod and McCarty, showing that bacteria could be genetically trans-
formed by DNA, was published in 1944, but even in 1946 seemed almost
unknown, or if known its significance was often belittled.

It was fascinating to look through microscopes at chromosomes in cells,
but I began to feel that as a physicist I might contribute more to biology
by studying macromolecules isolated from cells. I was encouraged in this
by Gerald Oster who came from Stanley's virus laboratory and interested
me in particles of tobacco mosaic virus. As Caspersson had shown, ultra-
violet microscopes could be used to find the orientation of ultra-violet
absorbing groups in molecules as well as to measure quantities of nucleic


128

acids in cells. Bill Seeds and I studied DNA, proteins, tobacco mosaic
\-irus, vitamin B12, etc. \!llile csarnining oriented films of 1)X:\ preparetl
fill  ultraviolet dichroism studies, I salv in the polnrising microscopr
cxtrcmcl~- uniform fibrcs givin, cr clear cstinction bct\vecn crossed nice],.
1 round the fibres had been produced unx\-ittingll- Tvhile I leas mal,i-
pulntin,q DS.\ gel. Each time that I touched the gel \\ith a glass rclc1
and removed the rod, a thin and almost invisible fibrc of DS,4 ~v;\s
dra\\-n out like a filament of spider's \vcb. The perfection and uniformit)
of the fibrcs suggcstccl that the molecules in them T\vc regularly arrangec1.
I immediarel>- thought the fibrcs might be excellent objects to study b!. X-
ra\. diffraction anal!.sis. I took them to Raymond Gosling, echo had 0111`
only S-ra!- equipment imadc from lvar-surplus racliography parts) a]:(1
\vho ~vas using it to obtain diffraction photographs from heads of ram
spermatozoa. This research Ivas directed by- Randall, xvho had been trained
under 5V. L. Bragg and had worked with X-ray diffraction. A4lmosr:
immcdiatel>-, Gosling obtained ver\- encouraging diffraction patterns
ircc fig. 1). One reason for this success i\-as that ~vc kept the fibres
moist. \Vc remembered that, to obtain clctailccl X-ray patterns from pro-
teiIls, Bernal had kept protein cr!.stals in their mother liquor. It sccn-ic~d
likcl>- that the configuration of all kinds of water-wlublc biolo+cnl
macromolecules ~voulcl depend on their aqueous environment. \\-c ob-
tained ,gootl tliffraction patterns Ivith DS.\ made b!r Signer and Sch\\~anclcr
ll949), jvliicli Signer brought to London to a Faraday Socic%ty meeting
on nucleic acids and x\.hich he gcncrously clistributccl so that all xvorkcrs,
using their \-arious techniques, could stud>- it.

Bet\\-wn 19% ant1 1950 Inan!- lines oft\-idcnce \vcrc unto\-crud intlic,ating
that the genetic substance \\-a~ DS:\, not protein or n~lc~c~~~~l`(Jt~iIl. For
instance, it \vas found that the D&S.-l content of a set of chromosomes \vas
constant? and that DSA from a given species had a constant composition
although the nut-lcotidr sequence in DN:l tnolcculcs \vas complex. It leas
su~:gcstccl that genetic information 1va.s carried in the poly~uclcotide chain
in a complicated sequence of the four nuclcotides. The great significance
of bacterial transformation now became generally rccognised, and the


129

Fig. I. One of the first X-ray diffraction photographs of DNA taken in our laboratory.
This may be compared with the later photograph in Fig. 9. (photograph with R. Gos-
ling; DNA by R. Signer).

demonstration by Hershey and Chase (1952) that bacteriophage DNA
carried the viral genetic information from parent to progeny helped to
complete what was a fairly considerable revolution in thought.

The prospects of elucidating genetic function in terms of molecular
structure were greatly improved when it was known that the genetic sub-
stance was DNA, which had a well defined chemical structure, rather
than an iIl-defined nucleoprotein. There were many indications of sim-

%-63oI5e


130

plicit!- and regularity in DN;\ structure.. The chemists had sholvn (I~:,,
DS.1 ~\`as a pal>-mcr in lvhich the phosphate ancl dcos)-rihosc parts (,i`
the n~olc~c~dc I\-erc ~-cq~lm~ly rcpcatcd in a polynucleotidc chain \\-ith :j'.-
5' linkages. Chargaff ~1950) discovered an important regularity : allhougl,
the sequence of bases alon& r the polynuclc-otide chains \\`a~ complcs ;lntl
the bnsc composition of diffcrcnt DNA's Ixriccl considcrahly, the numl)crs
of adeninc and thymine groups were al\\-a!-~ equal, and SO svere the nuln-
hers of guanine and c)tosinc. In the electron microscope, DSX was seen
as a uniform unbranchccl thread of diameter about 20 11. Signer, Caspcrs-
son and Hammarsten ! 1938) shelved by floli--birefringcnce measurements
that the bases in DSA lay with their plane5 roughl!- perpendicular to tllc
length of the thread-like molecule. Their ultra-violet dichroism measure-
ments gave the same results and shelved marked parallelism of the bases
in the DINX in heads of spermatozoa. Earlier Schmidt { 1937) and Pattri
(1932) had studied optically the remarkable ordering of the genetic ma-
tcrial in sperm heads. &Astbury i 1947) made pioneer X-w)- diffraction
studies of D?;A fibres and found evidence of considerable regularity in
DS.1; he correctly interprctcd the strong 3.4 ;I reflection as being due
to plm~lr bases StaCkCd on each other. The electro-titromc&c study by
Gullnnd and Jordan j ( 1947) shorvcd that the bases Jvcre h\-cll-ogcn-bonded
together, and indeed Gulland (1947) suggested that the pol!xuclcotide
chains might be linked b>- these hyclrogcn bonds to form multi-chain
miccllcs.

Thus the remarkable conclusion that a pure chemical substance was
in\-cstecl with a clecpl!- significant biological activit!. coincided \vith a
considcrablc grol\.th of many-siclccl knolvleclgc of the natuw of the sub-
stance. Xlcam~hilc \vc began to obtain tlctailcd X-ray dilliaction data
from DSi\. This ~vas the only type of data that could provitlc an adequate
description of the 3-climcnsional configuration ol` tlic: i~iolccul~.

Xs soon as good difl'rnction patterns ~vcrc obtninccl from fibws or DS&i,
great intcrcst leas aroused. In our laborator)-, .\lcs Stokes pro\-idcd a theory
of diKraction li-on1 helical DNX. Rosalind 1:ranklin !I\-ho cliccl `;omc vears
later at the peak of her career) made vcr!. ~xluablc contributions to the
X-ray nnal!.sis. In Cambridge, at the Slcdical Research Council laboratory


131

where structures of biological macromolecules were studied, my friends
Francis Crick and Jim Watson were deeply interested in DNA structure.
Watson was a biologist who had gone to Cambridge to study molecular
structure. He had worked on bacteriophage reproduction and was keenly
aware of the great possibilities that might be opened up by finding the
molecular structure of DNA. Crick was working on helical protein struc-
ture and was interested in what controlled protein synthesis. Pauling and
Corey, by their discovery of the protein a-helix, had shown that precise
molecular model-building was a powerful analytical tool in its own right.
The X-ray data from DNA were not so complete that a detailed picture
of DNA structure could be derived without considerable aid from stereo-
chemistry. It was clear that the X-ray studies of DNA needed to be com-
plemented by precise molecular model-building. In our laboratory we
concentrated on amplifying the X-ray data. In Cambridge, Watson and
Crick built molecular models.

The sharpness of the X-ray diffraction patterns of DNA showed that DNA
molecules were highly regular - so regular that DNA could crystallise.
The form of the patterns gave clear indications that the molecule was
helical, the polynucleotide chains in the molecular thread being regularly
twisted. It was known, however, that the purines and pyrimidines of
various dimensions were arranged in irregular sequence along the poly-
nucleotide chains. How could such an irregular arrangement give a highly
regular structure ? This paradox pointed to the solution of the DNA struc-
ture problem and was resolved by the structural hypothesis of Watson
and Crick.

The helical structure of the DflA molecule

The key to DNA molecular structure was the discovery by Watson and
Crick (1953a) that, if the bases in DNA were joined in pairs by hydrogen-
bonding, the overall dimensions of the pairs of adenine and thymine
and of guanine and cytosine were identical. This meant that a DNA mole-
cule containing these pairs could be highly regular in spite of the sequence
of the bases being irregular. Watson and Crick proposed that the DNA
molecule consisted of two polynucleotide chains joined together by base-


                          Me
H          /

   H

Cl

H


133

tween the two chains is the same for both base-pairs and, because the
angle between the bonds and the C,C, line is the same for all bases, the
geometry of the deoxyribose and phosphate parts of the molecule can be
exactly regular.

Watson and Crick built a two-chain molecular model of this kind, the
chains being helical and the main dimensions being as indicated by the
X-ray data. In the model one polynucleotide chain is twisted round the
other and the sequence of atoms in one chain runs in opposite direction
to that in the other. As a result, one chain is identical with the other if
turned upside down, and every nucleotide in the molecule has identical
structure and environment. The only irregularities are in the base se-
quences. The sequence along one chain can vary without restriction, but
base-pairing requires that adenine in one chain be linked to thymine in
the other, and similarly guanine to cytosine. The sequence in one chain
is, therefore, determined by the sequence in the other, and is said to be
complementary to it.

The structure of the DNA molecule in the B configuration is shown in
Fig. 3. The bases are stacked on each other 3.4 A apart and their planes
are almost perpendicular to the helix axis. The flat sides of the bases can-
not bind water molecules; as a result there is attraction between the
bases when DNA is in an aqueous medium. This hydrophobic bonding,
together with the base-pair hydrogen-bonding, stabilises the structure.

The Watson-Crick hy$Jothesi.s of D&A re~l~c~~~~~, and transfer of
  information from one polynucteotide chain to another

It is essential for genetic  material to be able to make exact
copies of itself; otherwise growth would produce disorder, life could not
originate, and favourable forms would not be perpetuated by natural
selection. Base-pairing provides the means of self-replication (Watson and
Crick 1953 b). It also appears to be the basis of information transfer
during various stages in protein synthesis.

Genetic information is written in a four-letter code in the sequence of
the four bases along a polynucleotide chain. This information may be
transferred from one polynucleotide chain to another. A polynucleotide
chain acts as a template on which nucleotides are arranged to build a
new chain. Provided that the two-chain molecule so formed is exactly


134


135

regular, base-pairing ensures that the sequence in the new chain is ex-
:lctly complementary to that in the parent chain. If the two chains then
separate, the new chain can act as a template, and a further chain is for-
Illed; this is identical with the original chain. Most DNA molecules con-
&t of two chains; clearly, the copying process can be used to replicate
such a molecule. It can also be used to transfer information from a DNA
chain to an RNA chain (as is believed to be the case in the formation of
messenger RNA).

Base-pairing also enables specific attachments to be made between part
of one polynucleotide chain and a complementary sequence in another.
Such specific interaction may be the means by which amino acids are
attached to the requisite portions of a polynucleotide chain that' has
encoded in it the sequence of amino acids that specifies a protein. In this
case the amino acid is attached to a transfer RNA molecule and part of
the polynucleotide chain in this RNA pairs with the coding chain.
Since the base-pairs were first described by Watson and Crick in 1953,
many new data on purine and pyrimidine dimensions and hydrogen bond
lengths have become available. The most recent refinement of the pairs
(due to S. Arnott) is shown in Fig. 2. We now take the distance between C,
atoms as 10.7 A instead of the value used recently of 11.0 & mainlybecause
new data on N-H . . . N bonds show that this distance is 0.2 A shorter be-
tween ring nitrogen atoms than between atoms that are not in rings. The
linearity of the hydrogen bonds in the base-pairs is excellent and the lengths
of the bonds are the same as those found in crystals (these lengths vary by
about 0.04 A).

The remarkable precision of the base pairs reflects the exactness of
DNA replication. One wonders, however, why the precision is so great,
for the energy required to distort the base-pairs SO that their perfection is
appreciably less, is probably no greater than one quantum of thermal
energy. The explanation may be that replication is a co-operative pheno-
menon involving many base-pairs. In any case, it must be emphasised
that the specificity of the base-pairing depends on the bonds joining the
bases to the deoxyribose groups being correctly placed in relation to each
other. This placing is probably determined by the DNA polymerising
enzyme. Whatever the mechanics of the process are, the exact equivalence
of geometry and environment of every nucleotide in the double-helix
should be conducive to precise replication. Mistakes in the copying pro-
cess will be produced if there are tautomeric shifts of protons involved


136

in the ll~~r~)genbonding, or chemical nltcxxions of the bases. These mi;c.
takes can correspond to mutations.

After our preliminq- X-ray studies had been made, my friend Leonarc
Hamilton sent me human D?iA he and Ralph Barclay had isolated fronl
human leukoq-tes of a patient xt+th chronic myeloid leukemia. He was
studying nucleic acid metabolism in man in relation to cancer and had
prepared the DSA in order to compare the DNA ofnormal and Ienkacmic
leukocytes. The DNA gave a very well-defined X-ray pattern. Thus began
a cqllaboration that has lasted over man). )`ears and in which we have
used Hamilton's DSA, in the form of man); salts, to establish the
correctness of the double helix structure. Hamilton prepared DSA from
a very wide range of species and diverse tissues. Thus it has been shown
that the DXX double helix is present in inert genetic material in sperm
and bacteriophage, and in cells slowly or rapid]\. dividing or secreting
protein (Hamilton et nE. 1959). No difference of structure has brcn found
bct~~ecn DNA from normal and from cancerous tissues, or in calf thymus
DSA separated into fractions ofdifferent base composition b?. m!-collcagcu
Geoffrey Bra\\-n,

\\`c also made a study, in collaboration ivith Harriet Ephrussi-Taylor,
of active transforming principle from pneumococci, and observed the same
DS:Z structure. The only exception to double htiiical DN,2 so [ar found
is in some very small bacteriophages where the DS;\ is single-stranded.
l\`c ha\rc found, hox~.c\~cr, that DN,A, Tvith an unusually high content of
acleninc, or \\.ith glucose attached to h~drr)s~mctll!-lc~tosinc., c*rystrrlliscd
dif~crentlv.

It did not stem enough to study -X-ray cliffi-action rrom DN:\ alone. Ob-
viously one sl~oulcI try to look at genetic material in intact cells. It ma
possibic that the structure of the isolated DS;Z might he diEwent from
that in Gzlo, lvhcrc DSA Ivas in most cases con~t~inecl 14th protein. Tile
opricnl studies inclicatrd that ihrrc \vas marked molecular order in sperm
heads and that they might thrrcf'ore bc good objects for S-ray study,
x~hcreas chromosomes in tnost types of cells NWC complicatccl objects
\virh little sign of ordrred structure. Randall had bwn intcrcstccl in this


137

I';%. 4. X-ray diA`raction pattern of cephalopod sperm. The DN;Z molcctzles in the
>pcrm heads lmvc their axrs x-crtical. The 3.4 .I intrmucleotidc spacing ~orrcspntis to
the strong diffraction at the top and bottom of the pattern. The sharp reflc~ctious in tlw
central part of thr pattern show that the molecules arc: in crystalline army.

mrttter for some years and had started Gosling studying ram sperm. It
seemed that the rocl-shaped cephalopod sperm, found by Schmidt to bc
highly anisotropic optically, ~vould be escellcnt for X-ray investigation.
Rinnc (1933), while making a stud\; of liquid crystals from many branches
of Nature, had rtlrcady t&a diffraction photographs of such sperm; but
presumably his technique was inadequate, fbr hc came to the mistaken con-


Fiq. 5. X-w>- tlillix~tion l~l~oto~rnl~ll ol DS.\ librcs (U conliguratiotl I at high humidity.
`1`11r. lil)rc,b arc \ c,rtical. `I'hc 3.4 .I rrflrction is at the top ant1 bottom. `1'11~ anql~' in the
pr"no""""`l s shq"`, made 11~ the reflections in the central region, corrrspontls to the
con\t;~nl 311ql~. of awcnt of thr pol~xuclrotirle chains in the hclicnl mol(x.ulc*. ' I'tloto-
qrnf)ll \\-irll H. Ii. I\.ilson: l).\.\ by I,. II. Hamilton.

elusion that tllc nuclcoprotcin \vas liquicl-cr)-stallinc. Our S-m)- photo-
grnphs i\\`ilkins nncl Randall 1953) sho~vccl clcnrly that the material in
the slmm hcncls hat1 3-dimcnsionnl order, i. c. it was crystallinc ant1 not
liclLlitl-c~!-stallinc. The clif~raction pattern (Fig. 4) bore ;1 close rcscn~-


139

blance to that of DNA (Fig. 5), thus showing that the structure in fibres
of purified DNA was basically not an artefact. Working at the Stazione
Zoologica in Naples, I found it possible to orient the sperm heads in fibres.
Intact wet spermatophore, being bundles of naturally-oriented sperm,
gave good diffraction patterns. DNA-like patterns were also obtained
from T2 bacteriophage given me by Watson,

X-ray diffraction analysis is the only technique that can give very detailed
information about the configuration of the DNA molecule. Optical tech-
niques, though valuable as being complementary to X-ray analysis, pro-
vide much more limited information - mainly about orientation of bonds
and groups. X-ray data contributed to the deriving of the structure of
DNA at two stages. First, in providing information that helped in
building the Watson-Crick model; and second, in showing that the Wat-
son-Crick proposal was correct in its essentials, which involved readjusting
and refining the model.

The X-ray studies (e. g. Langridge et al. 1960, Wilkins 1961j show
that DNA molecules are remarkable in that they adopt a large number
of different conformations, most of which can exist in several crystal forms.
The main factors determining the molecular conformation and crystal
form are the water and salt contents of the fibres and the cation used to
neutralise the phosphate groups (see Table l),

I shall describe briefly the three main configurations of DNA. In
all cases the diffraction data are satisfactorily accounted for in terms
of the same basic Watson-Crick structure. This is a much more con-
vincing demonstration of the correctness of the structure than if one con-
figuration alone were studied. The basic procedure is to adjust the mole-
cular model until the calculated intensities of diffraction from the model
correspond to those observed (Langridge et al. 1960).

As with most X-ray data, only the intensities, and not the phases, of
the diffracted beams from DNA are available. Therefore the structure
cannot be derived directly. If the resolution of X-ray data is sufficient to
separate most of the atoms in a structure, the structure may be derived
with no stereochemical assumption except that the structure is assumed
to consist of atoms of known average size. With DNA, however, most of


l.i

-


Fig. 6. X-ray pattern of microcrystalline fibres of DNA. The general intensity distribu-
tion is similar to that in Plate 4 but the diffraction is split into sharp reflections, owing
to the regular arrangement of the molecules in the crystals. Sharp reflections extend to
spacings as small as 1.7 .k. (Photograph with N. Chard; DNA by L. D. Hamilton.)


I          CONTOUR INTERVAL  2E ;:
Ii.          /
           ZERO CONTOUR DASHED _

Fig. 7. Fourier sythcsis map (b?- S. .\rnott, sho\ving the distribution of clcctron densir)-
in thr plane of a base-pair in the B conliquration of DS.\. The distribution corresponds
to an a\-erage base-pair. The shape of rhr base-pair appears in the map, but inclixidual
atoms in a base-pair are not rc~~lvcd. (The Fourier synthesis is being rcvisctl and the
map is subjrct to improvcment.1

the atoms cannot bc separately located by the X-rays alone (see Fig. 7).
Thcrcforc, more cstcnsivc stereochomical assumptions arc made : these
take the form of molecular model-building. Thcrc arc no alternatives 10
most of thcsc assumptions; but \vhcre there might bc an altcrnativc, e. g.
in the arranxcmcnt of hydrogen bonds in a base-pair, thr X-1.3). data
should bc used to establish the correctness of the assumption. In other
~vords, it is necessary to establish that the structure proposctl is unique.
.Ilost of our lvorl; in recent J-ears has been of this nnturc. To bc rcnsonably
certain that the DNA structure leas correct, X-ray data, as cstcnsi\.c a~
pwsiblc, had to be collected.

The B Cot&urcrtiott

Fig. 5 sholvs a diKraction pattern of a fibre of DSA at hish humidity
\\-hen the n~olcculcs arc separntccl b!. \\-atcr and, to a large cstent, behave


ind~~pcndcntl~- of` c~lch other. \Vc have not maclc intrnsive stud!. of DSAI
under thcsc conditions. The patterns could bc improvccl, but the), XC
rcasonabl>~ 1vcl1 tlcfinctl, and the sharpness of man). of their fcaturcs
s1~01v.s that the molcculcs 1ta1.e a rc,ylar structure. The confi,quration is
knotIn as B (WC also Fig. 3) ; it is obscr\-ccl in i,iz'u, and there is c\-iclcnce
that it csists ~\-hcn DX,\ is in solution in water. There are 10 nuclcoticlc
pairs lxr hclis turn. There is no obvious structural reason \I-h>- this num-
ber sho~~ld be integral; if it is exactly so, the significance of this is not )-et
appaI`t`Ilt.
PVhrn DS:I crystalliscs, the process of crystnllisation imposts restraints
on the molcculc and can gi1.c it extra regularit)-. AI1so, the pcrioclic arrx~,gr-
mcnt of the tnolcculcs in the microcr)-stals in the fibre causes the diffinc-


144

tion pnttcrn to 1)~ split into sharp rcfkctions cowrsponcling to tllv various
crystal plane5 i Fig. 6:I. Car f 1
            c II mcasurcmcnt of the positions trf the re-
flc>c,tions and clcduction of the cr!3tal lattice cnablcs the clircctions of tllc
rcllcctions to bc iclcntificd in three climcnsions. Diffraction patterns from
most fibrous substances rcscmblc Fig. 5 in that the diffraction data xc


145

"-dimensional. In contrast, the crystalline fibres of DNA give fairly com-
plete 3-dimensional data. These data give information about the ap-
pearance of the molecule when viewed from all angles, and are compar-
able with those from single crystals. Techniques such as 3-dimensional
Fourier synthesis (see Fig. 7) can be used and the structure determina-
tion made reasonably reliable.

The A conjguration

In this conformation, the molecule has 11 nucleotide pairs per helix turn;
the helix pitch is 28 A. The relative positions and orientations of the base,
and of the deoxyribose and phosphate parts of the nucleotides differ
considerably from those in the B form; in particular the base-pairs are
tilted 20" from perpendicular to the helix axis (Fig. 8).

The A form of DNA was the first crystalline form to be observed (Fig.
1). Although it has not been observed in U&D, it is of special interest be-
cause helical RNA adopts a very similar configuration. A full account of
A DNA will shortly be available. A good photograph of the A pattern is
shown in Fig. 9.

The C conjiguration

This form may be regarded as an artefact formed by partial drying. The
helix is non-integral, with about 9 l/3 nucleotide pairs per turn. The
helices pack together to form a semicrystalline structure : there is no special
relation between the position of one nucleotide in a molecule and that in
another. The conformation of an individual nucleotide is very similar to
that in the B form. The differences between the B and C diffraction pat-
terns are accounted for by the different position of the nucleotides in the
helix. Comparison of the forms provides further confirmation of the cor-
rectness of the structures. In a way, the problem is like trying to deduce
the structure of a folding chair by observing its shadow: if the conforma-
tion of the chair is altered slightly, its structure becomes more evident.

The helical structure of RNA molecules

In contrast to DNA, RNA gave poor diffraction patterns, in spite of much
effort by various workers including ourselves. There were many indica-
tions that RNA contained helical regions, e. g. optical properties of RNA

lo--630156


146

solutions strongly suggested (e. g. Doty 1961) that parts of RNA molt-
cules resembled DS;\ in that the bases \vcrc stacked on each other ancl
the structure ~v;t`j helical; and S-ray studies of synthetic polyihonuclco-
tides suggcstcd that R_SX rescmblrd DNA (Rich 1959). The diffraction
patterns of RS,A (Rich and Watson 1954) bore a general resemblance
to those of DSX, but the nature of the pattern could not be clearly dis-
tinguished because of disorientation and diffuseness. An important dif-
ficult)- \vas that there appeared to be strong meridional reflections at 3.3 ;I
and 4 pi. It ~vas not possible to interpret these in terms of one helical
structure.

In earl!- work, man)- RSA preparations were very. heterogeneous. \,Vc
thought that the much more homogeneous plant virus RSA might gi1.c
better patterns, but this was not so. Ho\vexer, Mhen preparations of ribo-
somal RSA and `soluble' RNA became available, rvc felt the prospects
of structure anal+s were improved. M'e decidecl to concentrate on `soluble'
RSA largeI>- because Geoffrey Brown in our laboratory ~\-as preparing
large quantities of a highly-purified transfer RSA component of soluble
RS,\ for his ph!-sical and chemical studies, and bccauw he was frac-
tionating it into various transfer RSA'l's specific for incorporation ofpar-
titular amino acids into proteins. This RS;1 was attracti1.c for other rea-
sons: the molecule \vas unusuall~~ small for a nucleic acid, thcrc lz'crc in-
dications that it might have a regular structure, its biochcrnical role was
important, and in many wa!-s its functioning \vas understood.

F\.c found it \-cry. difftcult to orient transfer RS;\ in librcs. Han-ever,
by carefully stretching RNA gels in a dry atmosphrrc unclcr a clissccting
microscopc~ I ~ou~~cl that fibrcs \\-ith bircfringcncc as higlt as that ofDS.A
coultl bc mndc. But thcsc fibrcs gave patterns no lxttcr tlran those ob-
taincd l\itll other t!.pcs of R?;L\, and the molcculcs tlisoricntctl I\-hen the
tvatcr content of the fihrcs 1va.s raised. 11'atson Fuller, Alichacl Spencer,
and m)sclf ivorkccl for many months trying to make hcttc.1. 5pccimcns for
X-1-a? sludy. 1Yc made little progress until Spencer fo~~ntl a specimen
that gave some faint but sharp diffraction rings in addition to the usual
diftilsc RS,\ pattern. This spccimcn consisted of RS.\ gel tllnt had been
scaled for ,X-l-a!- stud!- in a small ccl& and hc found that it had dried
slol\-1~ o\\-ing to a leak. The dill'raction rings xvcre so sharp that 1t.c were
almost certain that the)- Lverc spurious diffraction clue to crystalline im-
purit). - this being common in X-ray studies of biochemical preparations.
-4 specimen of RS.\ had gi\cn x-cry similar rings clue to DS;\ impurity.


147

We were therefore not very hopeful about the rings. However, after
several weeks Spencer eliminated all other possibilities: it seemed clear
that the rings were due to RNA itself. By controlled slow drying, he pro-
duced stronger rings; and, with the refined devices we had developed for
stretching RNA and with gels slowly concentrated by Brown, Fuller orien-
ted the RNA without destroying its crystallinity. These fibres gave clearly
defined diffraction patterns, and the orientation did not disappear when
the fibres were hydrated. It appeared that the methods I had been using
earlier, of stretching the fibres as much as possible, destroyed the crystal-
linity. If instead, the material was first allowed to crystallise slowly, stretch-
ing oriented the microcrystals and the RNA molecules in them. Single
molecules were too small to be oriented well unless aggregated by crystal-
lisation. It was rather unexpected that, of all the different types of RNA
we had tried, transfer RNA which had the lowest molecular weight,
oriented best.

The diffraction patterns of transfer RNA were clearly defined and well
oriented (Spencer, Fuller, Wilkins and Brown, 1962). These improve-
ments revealed a striking resemblance between the patterns of RNA and
il DNA (Fig. 10). The difficulty of the two reflections at 3.3 f% and 4 A
was resolved (Fig. 11) : in the RNA pattern the positions of reflections on
three layer-lines differed from those in DNA; as a result, when the pat-
terns were poorly-oriented, the three reflections overlapped and gave the
impression of two. There was no doubt that the RNA had a regular helical
structure almost identical with that of A DNA. The differences between the
RNA and DNA patterns could be accounted for in terms of small dif-
ferences between the two structures.

An important consequence of the close resemblance of the RNA struc-
ture to that of DNA is that the RNA must contain base sequences that
are largely or entirely complementary. The number of nucleotides in the
molecule is about 80. The simplest structure compatible with the X-ray
results consists of a single polynucleotide chain folded back on itself, one
half of the chain being joined to the other by base-pairing. This structure
is shown in Fig. 12. While we are certain the helical structure is correct,
it must be emphasised that we do not know whether the two ends of the
chain are at the end of the molecule. The chain might be folded at both
ends of the molecule with the ends of the chain somewhere along the
helix, It is known that the amino acid attaches to the end. of the chain
terminated by the base sequence cytosine-cytosine-adenine.


Relntiott of the tnolecular stt7lciut.e of R,\~.l to fiinction


Fig. Il. Diffraction pattcrn
of transfer RX.% showing
wsolution of diffraction. in
the regions of 3.3 .A and i .!.
into three layer-lines indicated
I)? the arrolvs and corrrsponcl-
ing to the 1 DS;\ pattern.
!Photograph with F\`. Fuller
and hf. Spencer; KSA b>-
G. L. Brown.~

the way in xvhich the cnztme involved inDXAreplication interacts GthDSA,
or ofother aspects of the mechanics of DSAreplication. The presenceof com-
plcmcntary base sequences in the transfer RSh molecule, suggests that it
might be self-replicating like DSA; but there is at present little evidence
to support this idea. The diffraction patterns of virus and ribosome RSA
show that thcsc molcculcs also contain helical regions; the functions of
these arc uncestain too.

In the case of DS.1, the discovery of its molecular structure led im-
mediately to the replication hypothesis. This was due to the simplicity
of the structure of DS.1. It seems that molecular structure and function
arc in most cases less clirtctl~- rclatccl. Derivation ofthe helical Con~gLlt-~tioll
of RN;\ molecules is n step toT\xrcls interpreting RNA function; but more
complctc structural ini~~r!~l~tion, e. g. dctcr~~in~ltion of base sequences,
and more kno\vlcdge about 1101~ the various kinds of RNA interact in the
ribosomc, v,Al probabl!. be required before an adequate picture of RNA
function cmcrgcs.

Since the bioIogic~~1 specificity of nucleic acids appears to be cntircl? dc-
tcrmincd b)- their base sequcnccs in them, determination of these sequences


1 50

0 AMINO ACID

Fig. 1'. AIolccular moclcl ad diagram of n Lransfcr RX.1 mokcdr.


i< prOh;lbl!- the most f~tnd~~~n~~~td problem it1 ~LIC~C~C acid XYSGWC~ todav.
`l'hc number of bases in a DNA molcculc is t-oo lnrgc for dctc~-ruination of
~nse scqucnce by X-ray difrraction to bc fcnsiblc. Ho\vever, in transfer
;:SA the number of bases is not too lar~c. The possibility of compkte
.tructurc analysis of transfer RX&-1 by means of X-rays is indicatccl by tlvo
c>lxer\-arions. First, ux haxx~ obscrwd (Fig. 13), in X-ray patterns of
transfer RN;!, scparntc spots, each corresponclin~ to a siniyle cr)-stal of
I<N;L Il'c estimated their size to he about 10 +G ancl have confirmed this
cxstitl~atc b!; observing, in the polarising microscope, bircfringcnt regions
lhat probably are the crystals. It should not be too clifEcuIt to grow' crystals
-c\-era1 times Lwgcr, tvhich is large enough fk single-crystal X-ray anal-
i-sis.
The second encouraging obscrxxtion is that the X-ray data from DKA
have restricted t-rsolution almost entirely on account of disorientation of
the microcr>-stals in DSA fihrcs. The DSA intensity data indicate that
the tcmprrawrc factor (B = 4 &i) is the same for DKA as for simple
compounds. It thus appears that DS.4 crystals ha\-c fairly perfect crystal-
linity and that, ifsingle crystals of DSX could bc obtained, the intensity data
I\-nuici bc nclccluxtc fhr precise clctcrminntion of all atomic positions in
DS.1 `apart from the llon"l)~riotli~ base scyucnce).
T\-Y arc inr-csriyrntin~ the possibility of obtaining single crystals of DSI~,
but the mow crciting problem is to obtain single crystals of transfer RNA
\vith crx-stnllinv pwfcction c'clual to that of DS:\, and thereby anal\-se
ba9c scc~~~mcc. -It prcsvnt, the RSA1 cr!xtnls arc much less perfect than
thnst of D-X.1. Ho~s~t-~r, most of our espcrirncnts have been made rvith
RX.1 rhnt is :I mixtuw of RS.\`s specific fc)r dilfkw~t amino acids. \2`c
liar-r wlrlr~~n ttst~l RX.1 that is \.cy. l;tryl?~ spcGfic for one amino acid
Inll\.. \\`I* lqx that ;) wad l~~`~~I~;~~~;~tions of` such RX.1 ma\- lx: obtainccl
conciuting of one t).pc` of tnol~culc only. `ct'v might ~spcct such RX:\ to
term cr\w&i ;1s pcrf=cYY as rhosc of US,\. If` so, tl1cl.c should be no oh-
sravlc to thus clircct anal\% of thr ~vholc structure of the moIcculc, in-
clucliny tlxc cerluvncc of that lxws nnd the fold at the uncl of the hclis.
\Vv tiln!. 1~~ (~\-or-ol)tiulisiic, but thv rcffnt and somcxvliat unespectucl Sue-
ccssc's of S-r:ty dilt'rnction anal) sis in the nucleic xitl and protein ficlcls,
arc cnusc Lor optinlism.


152

Fig. 13. Diffraction pattern
of unoriented transfer RSA,
showing diffraction rings xvith
spots corresponding to reflvc-
tions from single crystals of
RNA. The arro~vs point to
reflections from planes - 6 .i
apart.

During the past tw-elve years, while stud!.ing molecular structure of nu-
cleic acids, I have had so much help from so many people that all could
not he ackno\\-lcdged properly hcrc. I must, however, thank the following:

Sir John Randall, for his long-standing help and cncouragcmcnt, and
for his vision and energy in creating and directing a unique laboratory;

all m)- co-I\-orkcrs at various times over the past twxlve J-cars; first?
Raymond Gosling, Ales Stokes, Bill Seeds and Hcrbcrt FYilson, then Bob
Langridgc, Clivc Hoopcr, Max Fcughelman, Don Marvin and Gcoffrc)
Zubay, and at present, Michael Spencer, \Vatson Fuller and Struther
Arnott, rvho xvith much ability, skill and pcrsistencc (often through the
night) carried out the X-ray, molecular model-buildiyg, and computing
studies; my late colleague Rosalind Franklin lvho, Ivith great ability and
espcriencc of X-ray diffraction, so much helped the initial investigations
on DNA;

Leonard Hamilton for his constant encouragement and friendly co-
operation, and for supplying us with high-quality DS;1 isolated in man)
forms and from man)- sources; Geoffrey Brolvn for giving me moral and


153

intdlectd support throughout the work and for preparing RSA for X-ray
<tud!r; Harriet Ephrussi-Taylor for the privilcgc of collaborating \I-ith her
II studying cr!stallisation of transforming principle; the laborator)
.echnicians, mechanics and phorographcrs, including P, J, Cooper, N.
chard, .J. Hayward, Mrs. F. Collier, Z. Gabor ancl R. Lcrner, for having
pla);cd a valuable part in the xvork at various r;tages.

I also \vish to thank:

the Medical Research Council for their far-sighted and consistent sup-
port of our work ; King's Callegc far being our base; I. B. M. United
Kingdom Limited and I. B. M. it'orld Trade Corporation and the Lon-
_ton t`niversity Computer Unit for help with computing; The Rockefeller
Foundation and The British Empire Cancer Campaign for financial sup-
post; the Sloan-Kettering Institute, _ Uew York, and the Stazione Zoo-
logica, Naples, for use of facilities.

More generally, I thank:

Francis Crick and Jim t+:atson for stimulating discussion; Norman
Simmons for having refined techniques of isolating DNX and thereb!
helping a groat many workers including ourselves; many other workers
for- supplying us Tvith DNA and RSA; and especially, Er\vin
Chargaff for la!-ing founcl~tions for nucleic acicl structural studies b>
his anal?4cnl t\wk and his discovery of the equality of base contents in
DS.4 and for generously helping us newcomers in the field of nucleic
acids.


154

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