J. Hoi. Biol. (1961) 3, 71-S

The Molecular Structure of Polyadenylic Acid
               I---
             ALEXANDER RICH

Department of Biology, 3Iassachusetts Institute of Technology, Cambridge, MCLSS., U.S.A.
         DAVID R~A~ES

Jational Institute of Mental HeaEth, Bethesda, Maryland, U.S.A.
       F. H. cc) `%&

Medical Resewch Cou-ncil Unit for Molecular Biology, Cavendish Laboratory, Cambridge,
                 England               -.
            J. D.%ATSO~

Department of Biology, Harvard University, Cambridge, Mass., U.S.A.

(Received 28 September 1960)

The structure of fibers of polyadenylic acid at acid pH has been studied by
S-ray diffraction. A model is proposed consisting of two parallel intertwined
helical chains, each having a screw of 3.8 .k and 45" and related to each other by a
+-ad asis parallel to the fiber axis. Coordinates, bond distances and angles and
the calculated Fourier transform are given for this model.

Reasons are given why the quite different model of lKorgan & Bear is thought
to be wrong.

1. Introduction

The isolation of polynucleotide phosphorylase by Grunberg-Manago, Or& & Ochoa
(1955) has made possible the synthesis of a variety of ribonucleotide polymers all
having the same covalent backbone as natural RNA. Physical and chemical studies on
these polymers have been widespread and have led to a better understanding of the
general principles underlying nucleic acid structures. Among the first of these poly-
mers to be investigated by the technique of X-ray diffraction was polyadenylic acid.
In this paper we describe our studies of this material and give the precise coordinates
for a molecular model which is in excellent agreement with all the experimental facts.

Preliminary accounts of this work have appeared previously (Watson, 1957;
Rich, 1957a; Crick, 1957). Since that time additional information has been obtained
from the physical chemical investigations of Beers $ Steiner (1957), Fresco & Doty
(1957) and Fresco (1959), and the results of these investigations will be incorporated
into t.he present discussion.

2. Materials and Methods

The polyadenylic acid originally used in this investigation was kindly supplied by S.
Ochoa. Later preparations were synthesized by methods already described. A large number
of preparations was investigated and the results were very similar in all cases except that
some specimens of low molecular weight could not be oriented.
Fibers were pulled from concentrated solutions of polyadenylic acid and X-ray diffrac-
Con photographs of these fibers were obtained with fiber micro-Gamer=. The humidity of
                        71


72  -1. RICH, D. R. DAVIES, P. H. C. CRICK AND J. D. WATSOS
t IIP lwlium i~tmosphere surrounding the fibers was controlled by bubbling the helium
throupll nppropri&c saturated salt solutions befog it entored the camera. Both the micro-
focus S-ray t.ube (Hilger, London) and the rotating anode tube which has been developed
t&t the (`aventiish Laboratory, Cambridge, were used as high intensity X-ray sources. The
intensities of tho diffraction maxima on the photographs were measured with a Joyce-
Lo&l recording microdensitomoter.

The structural investigation was carried out with the aid of molecular models built from
1% in brass rod to a scale of 5 cm = 1 A. Xodels were built of plausible helical structures
;mtl from these models a set of coordinatee could be obtained for the repeat unit of the
hclis. III the early stages the cl&action pattern was obtained using an optical transform
machine. Laxer, IBM computers were used to calculate from the coordinates the cylindric-
allv a~rsgecl Fourier transform of the model. !&is calculation has been described pre-
viously (Davies & Rich, 1959). Comparison between observed and calculnted diffraction
patterns was then mado, and from this a decision could be reached as to the acceptability
of the model. The coordinates of the fmal model were then r&n4 so that, nil distances
anti angles w'cro stereochemically acceptable.

3. Results

Well oriented fibers of polyadenylic acid are negatively birefringent with values up
to 1)~ = - 0.10. Some less well oriented fibers produced smaller values of the bire-
fringence but always of negative sign. Some preparations produced well oriented
samples while others yielded poor orientation. The degree of orientation was best,
correlated to t,he molecular length of the polymer, and the pH of the material. If the
dampie had been prepared near neutral pH, it was very dif%cult to get a well oriented
sample; however, lowering the pH usually resulted in a sample with more orient&ion.
An S-ray diffraction photograph of polyadenylic acid is shown in Plate I. This is a

t.ypical fiber diffraction photograph characterized by a series of off-equatorial streaks
rather than a number of discrete spots such ae one would expect for a crystalline
material. The diffraction photograph is dominated by a sharp, very intense meridional
reflcsion at 340 d on the fourth layer line. The 0rst layer line in the photograph has
a spacing of 15.2 & and has a medium to weak reflexion near the meridian; further out
there is a moderately strong reflexion at a Bragg spacing of 5.2 A. The latter reflexion
.?hows some arcing and is not confined entirely to the first layer line, but merges with
a rcflesion on the equator. It is of interest to note that a similar reflexion also occurs
in the diffraction pattern of natural ribonucleie acid fibers (Rich & Watson, 1954).
The second layer line has nothing while the third layer line has a reflexion which is
moderately weal; and is located just off the meridian. Sometimes this layer line can be
broken up into two reflexions, one of which is closer to the meridian than the other.
The fourth layer line has, in addition to the very intense meridional reflexion, 5~
suggestion of much weaker components which are further away from the meridian
nut1 blurred. The fifth layer line has a reftexion of moderate,intensity which is off
nx~riclional but arcs across it.

111 aclditiou to the above reflexions which can be seen with untilted fibers, there are
two layer line reflesions which can only be seen by tipping the specimens. An example
of ~1 tipped diffraction pattern is shown in Plate II. As Can be seen, there are two near-
meridional reflesions which occur at G Bragg spacing of 1.90 A and 1.68 .A. It, should
Ire uotcd that t.he 140 -1 reflexion is most intense on the meridian while the outer
1 .IjS cl retlesion covers a somewhat wider arc.

Difl'r:lction photographs were taken of polyadenylic acid at a variety of relative
h~unitliries in order to determine the influence of water on t,he structure. One of the


PLATE I. An S-ray diffraction photograph of polyadenylic acid. The fiber axis is vertical and the
relative humiditr is 780' The weak reflexion on the meridian just inside the intense 3-8 H arc is
                10'
due to Ii/3 radiniion.                              [!I'0 face page i2


PLATE II. An S-rax diffraction photograph of polyadenylic acid obtained with the fiber axis
;erticctl. but tipped about 20" into the beam. Two additional strong reflexions are seen as shown
7~ the arrows.


3IOLECULXR STRUCTURE OF POLYADEXYLIC ACID       73

!Ilost, unusual features of polyadenylic acid is the fact that the diffraction pattern
~~~~tnains intact even if the photograph is taken with the specimen in an evacuated
,.,\nlera nt zero relative humidity, so that almost all the labile water molecules are
,,,~lno\~ed. This stability is in marked contrast to deoxyribonucleic acid and most of
the other synthetic polyribonucleotides, in which the diffraction pattern becomes
,~~~~orphous or blurred when the fiber is put in a vacuum. The variations in relative
l~umiclity have only a small effect on the equatorial diffraction pattern of polyadenylic
;lcitl. The first reflexion on the equator, 100, is very strong and occurs at a spacing
between 16 and 15 ,&. The lOOppacing is shown for polyadenyiic acid as a function of
l,elative humidity in Fig. 1, and it can be seen that there is only a small change of less

Relotrve humidity (%I

Frc:. I. The change in the 100 reflexion with C~ELI.I@E in relative humidity. Zero &ativo hnmidit.y
uxs produced by taking the diffraction photograph under vacuum.

than :! -$ in this spacing over the whole range of relative humidity. In the two-
stranded molecule of polyadenylic acid plus polyuridylio acid, the 100 spacing
increases by over S Ai when the relative humidity is changed from near zero to 98%
(Rich. l%ib).
The reflexions on the equator are not particularly well defined and a degree of un-
certainty must be attached to their spacings. They were measured with the Joyce-
Loebl recording microdensitometer from twelve films which were the product of 10
*eparate photographs at different relative humidities. In general, only two reflexions
could be seen easily, having a spacing of about 1'7 A and about 7.6 A. On several
films a reflesion of spacing near S-6 L% could be seen, and on two films a fourth faint
reflesion could be observed, but not measured.
The first two reflexions may be tentatively indexed as the 100 and 210 reflexions
from a square net of spacing 17 8. The third would then correspond to 200 and the
fourth to 110. It seems to us most unlikely that these could be the result of diffraction
from R hesagonal net, but in view of the paucity of the equatorial reflexions, and the
rnthrr diffuse nature of those which are observed, their assignment to a tetragonal cell
is not entirely certain.

4. Interpretation of the DiBraction Pattern

Thr strong negatire birefringenoe of the fibers of polyadenylic acid suggested that
the purine bases are oriented more or lese at right angles to the fiber axis. This inter-
p(~tnticm was reinforced by the extremeIy strong meridional reflexion at 3.8 A.


74  A. RICH, D. R. DAVIES, F. H. C. CRICK AND J. D. WATSOS
However, since the t.hickness of the purine bases is 3-4 A, it is clear that they must be
tilted a little in order to explain the observed 3.8 A axial spacing.
The distribution of meridional and off-meridional reflexions clearly suggests that the
molecule is helical in character. The simplest helix that is compatible with this dis.
tribution would be one in which there are four residues per turn with a pitch of 15.2 A.
Accordingly, we spent some time investigating configurations of a variety of single-
stranded polyadenylic acid models, since we were at that time uncertain of the dia-
meter of the molecule. We tried to find a natural explanation for the 3-8 d reflexion,
and in addition to normal stereochemistry, we looked for a reasonable system of
hydrogen bonding to hold the structure together. After considerable effort in this
direction, we concluded that it was impossible with a single-stranded molecule.
Shortly after this we obtained somewhat more reliable data on the equatorial re-
flexions of polyadenylic acid which indicated that there was a larger unit cell having
an edge about 16 to 17 A long. It was clear that a single strand of polyadenylic acid
could not easily 6ll a volume of this sort and accordingly we then began to investigate
multi-stranded helical models.
In general, there are two different classes of multi-stranded structures: those in
which the purine residues are located in the center of the molecule, and those in which
they are located on the outside of the molecule. Investigations of the configurations
possible in the latter class led us to the conclusion that there were none that were
plausible. This point will be discussed in greater detail when we consider the model
advanced by Morgan & Bear (1958).
Investigation of multi-stranded models with the bases on the inside showed that
the ribose-phosphate backbone is not long enough to cover the distance required for a
translation of 3.8 A and a rotation of 90" which was indicated by the strong meri-
dional reflexion on the 4th layer line. However, two-stranded models can be built with
a twofold rotation axis runnin g down the helix axis. The effect of this symmetry axis
is to reduce the unit rotation to 45", and within this framework there are two struc-
tures in which the adenines of opposite chains are joined together in pairs by hydrogen
bonds.
In the first of these structures the 6-amino group is hydrogen bonded to N, of the
opposite purine ring. Models of this type were rejected by us because they require a
large radial separation between phosphates of opposite chains. This would lead to
strong reflexions near the meridian on the first layer line, contrary to what is observed.
They were examined in more detail by Morgan & Bear (1958), and rejected for similar
reasons.
The second of these structures has the adenines paired in the same.way as in the
crystal structure of adenine hydrochloride (Broomhead, 1948; Co&ran, 1951),
namely, with hydrogen bonds between the 6-amino nitrogen and N7 of the opposite
ring. A first primitive model of this sort had the bases stacked perpendicular to the
helix axis with a separation of 3.8 A, and no attempt was made to pull in the ribose
phosphate backbones closer to the axis. The calculated diffraction pattern showed
reasonable agreement with the observed pattern, although the intensities of the first
layer line reflexions were reversed, the iirst being strong and the second weak.
However, at the same time a second model of this kind was constructed in which
an additional hydrogen bond was made between the 6-amino nitrogen and a phosphate
oxygen of the opposite chain. This additional hydrogen bond meant that the two
strands were now held together by two purine-purine hydrogen bonds and two


    MOLECULAR STRUCTURE OF POLYADENYLIC ACID       7:
I~~kne-phosphate hydrogen bonds. Formation of this additional hydrogen bond
l.esults in pulling in the phosphates closer to the helix axis with consequent improve-
lllent of the agreement between the diffraction patterns. Furthermore, in order to
ulal;e t.his bond. it is necessary to tip the bases so that they are no longer perpen-
dicular to the helis axis; but inclined by 10" to 11" from the perpendicular. This
tilting provides a natural explanation for the 34 A spacing rather than the expected
rs.4 A spacing.

           TABLE 1
  Coordinates of polyadenylic acid
(The atoms listed are those illustrated in Fig. 5)

Adenine residue
      s,

Ribose-phosphate chain

c',
c',
C'S
C',
C'S
0'1
Of*
0'3

P
05

06
0,

lower 0',

1.X       3.68      0.35      4.08       64.3"
3.14       3.73       0.46      4.88       49.9"
3.92       2.70      0.35      4.76       34.5"
3.24       1.54      0.11      3.59       25.5'
1.89       1.39      - 0.02      2.35       36.3"
I.08       2.52      0.10      2.74       66.7"
I.59       0.08      - 0.27      I.59        2.7"
2.80      - 0.50      - 0.27      2.84      - 10*1o
3.81       0.33      - O-05      3.82        4.9"
0.21       2.55      0.01      2.56       94*7O

5.24       o-00      0.00      5.24        o*o"
5.69       o-29       1.44      5.70        2.9"
5.58      - 1.09      2.09      5.69      - 11.0"
6.04      - 2.02      0.98      6.37      - ls*5o
5.51      - 3.45      I.19      6.51      - 32.1"
5.48      - 1.42     -0.21      5.66  F   - 14.6'
7.03       0.75       1.48      7.08        6.3"
6.46      - 1.22      3.26      6.58      - 10.7"
3.34      - 4.92      0.89      5.95      - 5iwil"
4.07      - 3.53       1.00      5.39      - 40-g*
1.86      - 4.65       o-90      5.01      - 68.2"
3.89      - 5.91       1.89      7.07      - 56*6O

3.71      - 5.45

- 0.54      6.58     - 55.7"

-1 right-handed coordinate system is used for both Cartesian and cylindrical polar coordinates.

Furthermore, tilting the bases alters the distribution of scattering intensity on the
upper layer lines. If the bases were not tilted we would only expect higher orders of
38 A, e.g. t,he 1.9 X reflexion which is observed as moderately strong (Hate II).
However, tilting also throws some diffracted intensity somewhat off the meridian so
that we might expect additional reflexions to occur in that region. We have pointed
out the occurrence of a second strong reflexion at 1.68 A which, as we will see below,
is predicted by this model.

By hydrogen-bonding the phosphate group to the adenine residue of the opposite
chain, t.he molecule tended to become more rounded in cross section, and the helical


70  -1. RICH. D. R. DAVIES, F. H. C. CRICK AND J. D. WATSOX

grooves, which are so marked in DNA, are considerably reduced in polyadenylic acid.
This has the effect of reducing the predicted intensity of the second layer line near the
meridian so that it is in closer agreement with the observed diffraction pattern.

5. Description of the Molecule

Once we were convinced that this model was along the right general lines for pre-
dicting the overall diffraction pattern, we carried out a series of successive adjust-
ments whereby a careful set of atomic coordinatis was obtained which showed that it
is etereochemically feasible to have a model of this sort. Thus, we have a set of atomic

Fro. 2. -1 sketch to show the bond angles and distances of polyadenylic mid. Not to scale.

coordinates which yield an accepted range of values for bond angles, distances, and
van der Weals cont.acts between the atoms. The coordinates for polyadenylic acid are
listed in Table 1, t'he bond distances and angles are shown in Fig. 2 (not to scale), and
the &rtcr van der Waals contacts are marked on the projection of part of the
structure shown in Fig. 3.

The coordinates were finalized before the publication of the papers of Fuller (1959)
and Spencer (1959). They could be revised slightlyzth advantage but all the dis-
tances and angles are acceptable, although the angle N&C$a (106') is strained a little
owing to the formation of the hydrogen bond between N,,, and 0,. The adenine has
been based on Co&en's parameters for adenine hydrochloride (Co&ran, 1951).


    310LECUL.iR STRUCTURE OF POLYADESYLIC ACID      77
The hydrogen bond from N,, to x7 is 6" away from the straight direction and that
fi.om N,, to 0, is 14" away-; that is, the angle O,$IoH is 14". This is on the large side,
[:llt acceptable.

It should be mentioned here that it is necessary in the determination of molecular
.trllcture from fiber patterns to show that a given molecular model is stereochemically

FIG. 3. Part of the structure projected in the direction of the z axis. The shorter t-an der Waals
,>oilti\cts arc shown b!- tlottrtl lines.

FIN. 1. Fchrluntic view of poll-adenylic acid as seen from the side. The flat ribbons represent
the ribone-phosphate backbones lrhich are parallel to each other. The disks represent the adenine
rings n-hirb arc hydrogen bonded together.

feasible, and to present reasonably exact atomic coordinates. Tbis is not because it is
necessary to compare in great detail the diffraction pattern of this refined model with
what is observed esperimentally, for very often the experimental data on an X-ray


78  A. RICH, D. R. DAVIES, F. H. C. CRICK AXD J. D. WATSOX

fiber diffraction pattern are not of high quali
                              L -  ty. The importance of presenting exact
coordinates rests on the fact that this is a means for ruling out certain structures. It
may be very easy to obtain an approximate set of coordinates, but it may prove to he
impossible to refine the model properly so that it conforms to known values of atomic
parameters as well as have the symmetry demanded by the diffraction pattern.

The helical molecule of polyadenylic acid consists of two polynucleotide chains
organized about a twofold rotation axis. Figure 4 is a schematic view of the molecule
b which the flat ribbons renrc?nmt. t,he rihnnn.nhonnhnte hackhone chains and the disk2

_ =------- ---- ------ =---~~----- -l-__--.-. .

FIG. 6. The two-stranded molecule of polyadenylic acid as viewed down the fiber axis. Each
adenine residue forms three hydrogen bonds. An extra 0,' atom is shown to indicate the backbone
connections. For clarity the hydrogen atoms on the sugar have been omitted.

represent the adenine residues. Successive residues are related by a translation of 3.8 A
and a rotation of 45". Both backbone chains are parallel to each other, as sho\rq `V
arrows. This is in contrast to the two-stranded molecule of deoxyribonucleic acid h
which the twofold rotation axes are perpendicular to the fiber axis, and the two back-
bone chains are anti-parallel. The adenine rings are tilted about 10" from the hori-
zontal, but this is not shown in Fig. 4. Because of the twofold rotation axis, the two
bases which are hydrogen bonded to each other are tipped in opposite directi@!
much like the blades of a propeller.

Figure 5 shows a view of the molecule as seen down the fiber axis, The hydNeD
bonding between the adenine residues is shown, aa well as the hydrogen bon&g


     MOLECULAR STRUCTURE OF POLYADENYLIC ACID      79
l,t+ween the adenine amino group and the phosphate oqgen atom Corn the opposite
&in. Two arrows point to successive 0, atoms of the ribose ring, and that atom is
5110wn twice in order to show how the backbone chain is connected. Nitrogen 1 of the
:,&nine ring is protonated, with a positive charge on the ring. The reasons for this will
be discussed below.
111 order to see the molecule in terms of the space which it occupies, we have made
;1 diagram in which the atoms are represented as spheres drawn with their van der
\\`aals radii. Figure 6 shows this view of the molecule. The fiber and molecular axis is

Polyadtnylic acid

   Adcninc
0  Ribosc carbon
0 Oxygen
a Phosphorus

FIG. ti. \`an der Waals model of polyadenylh acid. Hydrogen atoms am not represented in this
Jin,oram.

~mtical, and it can be seen that the adenine b&s are slightly tilted. The outstanding
feature which this diagram illustrates is the compactness of the molecule. The mole-
cule is a helix, but the helical grooves are not very evident owing to the fact that the
1)hosphate groups are pulled in towards the center of the molecnle in order to hydra-
gen-bond with the adenine amino group.
This compact form is probably responsible for the faot that the helix is intact even
under a vacuum. In addition, the rounded outline is probably responsible for random-
ness in packing, as discussed below.


so  A. RICH, D. R. DAVIES, F. H. C. CRICK AKD J. D. WATSON

6. The Density and Space Group of Polyadenylic Acid

To obtain an approximate estimate of the density to be expected we must estimate
the number of mater molecules that will fit into the unit cell. This can be done approxi-
mately by using the partial specific volume of polyadenylic acid in solution, which is
known to be in the neighbourhood of 064 (R. Haselkorn, personal communication).
If we t,ake a tetragonal cell with dimensions 16% x 16-S x 15-2 & then calculation
shows that there should be room for about eight molecules of water per nucleotide.
This gives a densit,y of about 1.47. If the fiber used contained a small amount of
sodium ion (which occupies very little volume) this figure might be a little higher, say
1.5.

This is rather lower than the observed value of I.53 obtained by flotation. This dis-
crepancy can only be explained by assuming that in the fiber there were less crystalline
regions with a higher density. As the postulated tetragonal cell is a very open one this
is not implausible.

In spite of these uncertainties the density shows clearly t,hat there are two nucleo-
tides for every 3.8 x in the z direction, since three nucleotides would be an impossibly
tight fit (calculated density 1.70) and one nucleotide would give far too low a density
(calculated 1.23).

The molecule of polyadenylic acid in the helical form described has a great deal of
symmetry. In addition to the twofold rotation axis along the helix axis, there is also
an eightfold screw axis, which can be used crystallographically as a fourfold screw
axis. Hence the space group may well be P4, where a c-axis is 15.2 a and the asym-
metric unit consists of two adjoining nucleotides. The helix is covalently linked in a
right-handed fashion but this is not specified by the space group symmetry.

   7. Comparison of Calculated and Observed Intensity Data
From the final set of coordinates the continuous Fourier transform was calculated
using the computer program already mentioned. This program computes a cylindric-
ally averaged Fourier t,ransform for helical molecules. This is the most appropriate
form of comparison to be used for polyadenylic acid since there is little evidence for
crystallinity or sampling of the transform with one or two exceptions cited above.
The program computes the cylindrically averaged intensity I(R, Z/c), where

I(R,I/c) =~{~`Jn(2nqR)~os~(;-+,)+~}]~
+ ~$.Jn(?m;R)Sill(n(~-#j) +?}I*}

where j = total number of atoms in the asymmetric unit; rj, $, and z, are the cylin-
drical polar atom coordinates, n = order of Bessel functions suitable for the helical
symmetry. We have used four orders of Bessel function for each layer line; additional
contributions could be neglected. In this computation we have not used a temperatm
factor.

Intensity data were collected from diffraction photographs obtained with the,
multiple film technique. The intensities of the diffraction maxima were measured wit\
the recording microdensitometer by taking traces which went at right angles to the;'
layer lines as well as along t.hem. This was done because, as a result of the lack O{


MOLECULAR STRUCTURE OF POLYADENYLIC ACID      81

(Son$ete orientation of the fiber molecules, many of the reflexions were arced, with
;Ippreciable intensity off the layer lines. Because of this arcing of the re%eions, and
Irecause of the limited integrating ability of the recording densitometer, we regard
the intensity data obtained as only semi-quantitative. We have, therefore, not
Laonsidered it worthwhile to correct the recorded intensities for the usual cyli&-ical
& polarization factors. These corrections, if applied, would have the principal
efect of reducing the recorded intensities near the meridian.

It was possible to measure the relative intensities of the two reflexions which occur
ilt 1438 $ and 1.90 A, but no attempt ha.4 been made to relate these accurately to the
rest of the diffraction pattern.

Rti-`)

FIG. i. The observed and calculated intensities for polyadenylio acid. The cmlculeted intensities
are indicated by the continuous curves. The observed data are plotted in the shaded areas, the
apex indicating the position of maximum intensity. The height of the apex indicatea intensity, but
the measurements may be in error by as much as 50%. The observed reflexions on layer lines 8 and
9 are not on the same scale as the other observed intensities.
plot.ted at one-fifth the actual values.            *Intensities on this layer line are

The observed and calculated intensities on the various layer lines are shown in
Fig. 7. It can be seen that there is moderately good but not perfect agreement. Note
particularly t.hat the model predicts strong intensity at a spacing of l-90 A (second
order of 3.8 +i) as well as a strong reflexion at 1.68 B which should be just off the meri-
dian. This latter reflexion which can be seen in Plate II has an intensity distribution
compatible with its being off-meridional.

The observed intensity data are plotted in Fig. 7 in such a way as to indicate its
semi-quantitative nature. The most intense part of the diffracted intensity on a layer
line was located by projecting the diffraction pattern onto an appropriate Bernal
chart. Tllis point was used to determine the position of the apex of the pentagonal
   G


8'1   -4. RICH, D. R. DAVIES, F. H. C. CRICK AND J. D. WATSON
shaded areas which indicate observed intensity. This could be done unambiguously in
most cases. However, the medium intense reflexion on the 5th layer line never split
into its two off-meridional components. Hence ita position on the layer line wa
determined by assuming that the arc originated on that layer line; this uniquely
determines the cylindrical radius R for the reflexion. The same technique was used for
the 9th layer line reflexion. The height of the shaded intensity figure was determined
by the densitometer tracings, while the width was estimated using both the tracings
and visual appraisal. The straight edges on the pentagonal intensity @n-es do not, of
course, represent the shape of the diffraoted X-ray beams. In Fig. 7, the observed
and calculated intensities for the 4th layer line (3.8 A) are plotted on a scale O-2 times
that of the rest of the diagram, as indicated by an asterisk. Only the outermost
equatorial re0exions are plotted in Fig. 7, since the inner reflexions are discrete and
are handled separately.

F 0

0.04    0.08    0.12    O.l6    0.20
      R tk',

FIG. 8. Observed and calculated structure faotom for the equator of polyadenylic acid. The
dashed line represents the structure fa&or for the molecule minus the struoture faotor for a
cylinder of mater. The observed date are hdi08ti by vertical linm which cross the horizontal
axis. The positions of the reflexions at extreme vslues of the relative humidity am indicated by
short vertical lines.

When considering the agreement between the observed and calculated intensity
diskibutions in Fig. 7 it should be emphasized that no allowance has been made for
the effect of bound water molecules on the diffraction pattern. Polyadenylic acid
takes up a small amount of water and holda it Grmly, and it is quite likely that this
                                                                   -.
water will modify the diffraction pattern slightly.

The water surrounding the molecule may have a substantial effect on the equatorial
diffraction pattern. We have accordingly computed the equatorial intensities by,;
taking this water environment into account. This calculation was catied out by
assuming that the molecule is cylindrical in outline (cf. I!& 6), with an outer radius';
r,, and is suspended in a sea of water which extends from r, to infinity. The equated
intensities or structure factors are then the difference between the contribution due t6"
the molecule itself and that due to a cylinder of water of radius r,,. Thus, for tb:
equator we have plotted in Fig. 8 the structure &&or for the molecule itself, th<'
cylinder of water and the difference betwe$;n these quantities (dashed line). That is:!


MOLECULAR STRUCTURE OF POLYADENYLIC ACID
     P = F (poly A) - F (water cylinder)

83

,, here E = height of the cylinder of water (34 A for 2 nucleotides);

p = electron density of water = 0.33 electronslA3;
r. = 7.5 A.
It can be seen that the structure factor passes through zero very near the pint at
,\hich the 110 reflexion should appear in the square lattice. Hence this accounts for
the failure to observe this reflexion. The fact that 210 reflexion is readily observed
,Vliereas 200 is frequently very weak may be partially, but not entirely, accounted
for by the higher multiplicity of 210. Observed data are plotted in Fig. 8 for the
rcflexions 100,200 and 210 at 20% and 9So/o relative humidity. These structure factor
,iiea.surements should be regarded as accurate only to approximately f 30%, due to
tlic diffuseness of the reflexions. However, it can be seen that there is rough agreement
l&k the calculated structure factor curve.

The diffuseness of these equatorial reflexions may arise from several sources. It is
quite likely that the molecules tend to crystallize in the tetragonal cell because of the
4~" rotation between adjoining nucleotide residues in the chain. However, there are
cpportunities for various types of randomness. The chains are directional, and so they
can be oriented up and down at random. In addition, because the helix is very com-
pact and rounded, rotational disorder can be present. These may have the effect of
ljroducing an equatorial packing domain which is small, and leads to broad diffuse
equatorial reflexions. This randomness is also responsible for the smeared, non-
discrete nature of the off-equatorial reflexions, which come closer to representing the
continuous Fourier transform than a sampling at discrete points.
We have spent aome time studying the packing between molecules m the tetragonal
lattice at various equatorial spacings. There are several possibilities, but we shall not
discuss them because the di.Eraction data are not sufficiently precise.

8. The Model of Morgan & Bear

A paper was recently published by Morgan & Bear (1958) which described two
possible structures for the molecule of polyadenylic acid. We believe both of these
structures to be incorrect. Since these authors reject their second StNCtUre on the
grounds that it does not fit the diffraction data, we need not consider it further here.
Their structure I is very unusual in that the two polynucleotide chains are wrapped
around each other in anti-parallel fashion with each chain containing four residues per
turn. The ribose-phosphate chains are located in the center of the molecule with the
adenine residues projecting out radially from the center core. The two chains are related
by dyad axes, perpendicular to the fiber axis, going through each pair of hydrogen-
bonded ribose rings. Although it has some provocative features, this model can be
ruled out for several reasons which we shall proceed to outline.

In the first place it is not clear to us that the structure can be built satisfactorily
along the lines the authors have described. Whereas most of the bond distrtnces in
their model are acceptable, many of the angles are not. In Table 2 we list the bond
angles that occur in their structure together with the standard values. It can be seen
that distortions are present as large aa 20" to 30", and these are quite unallowable.
Furthermore, the authors have neglected to consider some of the unfavorable van der


s4  -1. RICH. D. R. DAVIES, F. H. C. CRICK AND J. D. WATSOS
\Ynals contacts bet,ween various units. Thus, they have the two charged phosphate
groups "in contact" with each other. However, the phosphorus atoms are 3.89 A
apart and the oxygen8 are 246 L% apart, both of these values being considerably below
the accepted values. We think it is unlikely that these coordinates can be refined tc
standard values. This is probably an example of the situation discussed above, in that
a preliminary and uncritical examination of a potential molecular configuration looks
promising but, on closer examination, it turns out that the model is not feasible
stereochemically.

TABLE 2

Bond angles in the model of Morgan & Bear

Angle

Vnlue in structure I    Standard value

Distortion

O,`C,`C,
S,C,`O,'
C,`C,`O,'
C,`C,`O?'
PO,`C,
O,`PO,

139"
132'
124"
120"
102"
77"

    lloO
    110"
    110o
    110"
about 120'
    110"

    29"
    22"
    14O
     9"
about 18"
    33"

Secondly, the structure looks energetically unfavorable. It is held together by one
weak hydrogen bond between the sugars, and furthermore, the negatively charged
phosphate groups are brought close together so that the molecule would tend to fly
apart in solution, whereas it has been shown experimentally by Fresco (1959) that
                                                                   1.
polyadenylic acid retains its helical cotiguration in solution.

It has been established by Fresco & Doty (1957) that the helical form of poly"
adenylic acid is stable at pH 5, but not at pH 7. The atructnre of Morgan $ Bear does"
not explain this. Nor does it explain in a natural way the spacing of 3.8 A between the
bases.

Further support for models having the bases on the inside arises from chemical
work concerning the action of concentrated nitrous acid on the adenine residue in
ribonucleotide chains. In 4 M-NaNO, at pH 4.3 adenine residues are usually oxidized:
to hypoxanthine, since the amino group is replaced by an oxygen. However, poly-,,.
adenylic acid does not react under these conditions when left at room temperature for_
four days. (A. Bendich & H. Rosenkranz, personal communication.) This result:;
nould not be understandable if the adenine residues project away from the molecular&,
ask into the solution; however, it is naturally explained by the fact that the adenintC:'
residues are buried in the center of the molecule and are hence unreactive to nitrouri-
acid.

Finally, it should be mentioned that a cylindrically averaged transform calculati$i
carried out with the Morgan-Bear coordinates does not yield a diffraction pattern in;.
agreement with the observed data. The second strongest reflexion on the calculas
pattern is on the 3rd layer line, while the near-meridional part of the 2nd layer @&
is more intense than the contribution further out. In short, we'feel there is very litgtl
chance that this model is correct.


MOLECULAR STRUCTURE OF POLYADEXYLIC ACID      85

9. Discussion

We arrived at our interpretation for the structure of polyadenylic acid before the
effect of hydrogen ion concentration was noted. Beers & Steiner (1957) pointed out
that an abrupt shift occurred in the ultraviolet absorption spectrum of polyadenylic
acid -when the pH was lowered to about pH 5. They correlated the shift in the spectrum
with the absorption of one proton per nucleotide base. Shortly after that, Fresco &
Doty (1957) showed that, at the lowered pH, the molecule had a rigid form composed
of variable numbers of polyadenylic acid molecules in contrast to the flexible ran-
domly coiled form which it exhibited at pH 7. Thus we were faced with the question
of how the additional proton was absorbed in polyadenylic acid. The answer to this
question is quite clear in the case of adenine hydrochloride in crystalline form.
Co&ran (1951) has made a detailed analysis of the crystal structure of the protonated
adenine residue and his results show that the additional hydrogen is located on N1
of the purine ring. We therefore considered the effects of adding a proton at this position
on the structure which we had postulated for polyadenylic acid. The results are shown
in Fig. 5 where a positive charge is shown on N1 in close proximity to the negatively
charged phosphate group of the opposite ribose-phosphate backbone. In this position
it is clear that the additional charge on the adenine residue stabilizes the molecule
electrostatically. This might be described as an "inner salt," since the polymer no
longer has an overall cha,rge, even though there are isolated charges on different parts
of the helix.

In this form the molecule has considerable stability. It haa been shown by Fresco
S; Klemperer (1959) that the "melting temperature," T, (the mean of the tempera-
t.ure ra.nge over which the material changes from a helical to a random coil form) of a
soIution of polyadenylic acid at pH 4-25 and ionic strength 0.15 is about 90oC. This is
roughly the same as the T,,, of calf thymus DNA at pH 7-O and similar ionic strength,
and is considerably higher than the Tm's of polyinosinic acid, polyinosinic plus poly-
cytidylic acid and polyadenylic p&z polyuridylic acid (Doty, Boedtker, Fresco,
Haselkorn & Litt, 1959).

Polyadenylio acid, in addition to combining with itself to form this two-stranded
molecule, can also combine with polyuridylic acid to form two- and three-stranded
structures (Rich & Davies, 1956; Felsenfeld, Davies & Rich, 1957). Similar structures
are formed with polyinosinic acid (Rich, 1957u) and with polyribothymidylic acid
(A. Rich, unpublished results). It should be noted that these structures are all formed
at neutral pH where polyadenylic acid exists as a flexible single chain. All of these
structures exhibit diffraction patterns that are quite different from that shown in
Plate I. However they all have in common a configuration in which the charged
ribose-phosphate chains are helically arranged on the outside of the molecule sur-
rounding the hydrogen bonded purine or pyrimidine residues.

We would like t.o ecknomledge assistance by Leslie Barnett, and also by Dr. S. Benzer
in obtaining the final coordix~ates.

REFERENCES

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Broomhead, J. (1948). A& Cryst. 1, 324.
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