THE EFFECT OF SECONDARY STRUCTURE ON THE TEMPLATE
      ACTIVITY OF POLYRIBONUCLEOTIDES'

BY MAXINE F. SINGER, OLIVER W. JONES, AND MARSHALL W. NIRENBERG

NATIONAL INSTITUTE OF ARTRafiIS AND METABOLIC DISEASES AND NATIONAL HEART INSTITUTE,
                       BETHEBDA, MARYLAND

Communicated by Rob& J. Huebner, Januaty 16,1963

Current experiments on the synthesis of polynucleotides and proteins seem to us
to emphasize the functional importance of polynucleotide secondary structure.
Thus, for example, the highly ordered double-helical structure of DNA' readily leads
to ideas concerning DNA replication.2. * Although our notions concerning the

VOL. 49, 1963 BIOCHEMISTRY: SINGER ET AL. 393

secondary structure of the various types of RNA are much less precise, it does
appear that RNA molecules are primarily single stranded and contain varying
degrees of helical content.`,  Recently, investigators from two different labora-
tories have proposed similar helical structures for transfer-RNA6* ' and both groups
have discussed the functional significance of the suggested configurations.  It is
therefore of interest to consider the secondary structure of template RNA.  Pre-
vious reports from this IaboratorySS s described the template RNA dependent in-
corporation of amino acids into proteins in a stable cell-free system from E. co2i.
Using this system and randomly mixed polyribonucleotides composed of various
combinations of the four common ribonucleotides, nucleotide codewords corre-
sponding to almost all of the protein amino acids have been determined.1-15
Observations made with this system suggested that the secondary structure of poly-
mers influenced template efficiency.  For example, the ability of poly U to direct
polyphenylalanine synthesis is lost when poly A-poly U double or triple helices are
formed.9. lo

The present report describes certain physical properties of a series of poly UG prep-
arations as well as the e5ciency of these polymers in directing amino acid incor-
poration in the cell-free system.  The data indicate that secondary structure in a
polyribonucleotide limits its efficiency as a template.  This finding is discussed
and a specific functional role for polynucleotide secondary structure in the coding
mechanism is proposed.

A preliminary account of some of these data has been published.1

Materiala and Methals.-The procedures for the synthesis of polymers, determination of base
ratios in the polymers, and measurement of amino acid incorporation have been described pre-
vioualy.s~ 1' The UG polymers are isolated by a modified procedure16 designed to concentrate the
longer chain length polymer and eliminate some of the shorter chain material.  At the end of the
polymerbation the reaction mixture is deproteinized by the method of Sevag.17 The aqueous
solution of polymer ie made 2 M in KCl by addition of an appropriate amount of solid KCl.
Polymer is precipitated by the addition of 0.2 volume of cold absolute alcohol, and collected by
centrifugation. The precipitate is dissolved in a small amount of water.  In several cases the
polymers did not dissolve readily in water unleaa a small amount of EDTA (final concentration
about 5 mM) waa added. Precipitation with KC1 and alcohol is repeated two more times.  The
final precipitate is washed succeseively with So%, 95%, and 100% alcohol and finally with ether
and dried over paraffin.  Polymers were subsequently dissolved and dialyzed against distilled
water before me.

The phosphorolysis of the polymers by polynucleotide phosphorylase was determined by meas-
uring the formation of Patlabeled nucleoside diphosphate in the presence of PP. The procedure
referred to as Assay A by Singer and Guas*8 waa used; the pertinent polymer wae substituted for
the poly A of that method. For the phosphorolysis studies, illicrococcwr lysodeikticus polynucleo-
tide phosphorylase, purified approximately 250-fold (Fraction VIII1e) waa used.

Measurements of the temperature dependence of the spectra of polymers ("melting curves")
were carried out either in a Cary recording, model 14M, or Beckman, model DU, spectropho-
tometer. The Cary instrument waa equipped with a thermostatted cell holder and the temperature
waa measured inside the sealed quartz cuvette by means of a hypodermic needle type thermistor.
The Beckman instrument waa equipped with thermospacers and the temperature waa estimated
in a water-filled, unhealed, blank cuvette.  All solutions were gaased with helium just before
filling the cuvettes and the cuvettes were sealed with a coating of General Electric Company
RTV4, silicone rubber compound.%

The chain lengths of polymers were determined by measuring the ratio of total organic phos-
phate to phosphate removable by E. coli alkaline phosphatase (Worthington Biochemical Cor-
poration). The procedure outlined by Heppel and co-workers*l waa followed.

Sedimentation velocity studies were performed at 56,100 rpm and 20C using the Spinco

394 BIOCHEMISTRY: SINGER ET AL. PROC. N. A. S.

Model E Ultracentrifuge equipped with W absorption optics.  The solutions contained ap-
proximately 0.03 mg of polymer per ml of 0.05 M cacodylate and were 0.1 M in NaC1.  For
measurements at neutral pH the cacodylate served as a buffer at pH 7.2; for alkaline measure-
ments the solutions were made 0.01 M in KOH (pH 12).  Tracings were obtained from photo-
graphic images of the cell using the Joyce-Loeble Recording Microdensitometer.  The sedimenta-
tion coefficient was calculated** from the rate of change of position of the 50% point of the bound-
ary.

Optical rotation was measured on a Rudolph Polarimeter using the mercury line at 365 mp and
a 1 decimeter light path.

Results.-Dependence of amino acid incorporation on the base ratio in poly UG:
Table 1 shows the incorporation of phenylalanine, valine, leucine, and tryptophan
into protein, measured with a series of poly UG preparations of increasing G con-
tent.  The relative amounts of the four amino acids incorporated with any partic-
ular polymer are clearly related to the proportion of U and G.  The data show that
efficient incorporation of tryptophan requires a greater proportion of G in the
polymer than is necessary for incorporation of valine or leucine.  These data, there-
fore, confirm the earlier assignment of codewords UUG, UUG, and UGG to valine,
leucine, and tryptophan, respectively.ll- 12, l4

It is evident that the efficiency of a polymer as template RNA (Table 1) for any
of the amino acids shown decreases sharply when the U/G ratio becomes less than 1.
For example, the incorporation of phenylalanine with the polymer of U/G ratio
6.7/1 is about 60-fold greater than that obtained with the polymer of U/G ratio,
0.58/1.  Although the decreased ability of the latter polymer to direct phenylala-
nine incorporation is expected, the lack of activity in coding for leucine, valine, and
tryptophan is surprising.  In an attempt to understand this observation various
properties of the polymers were investigated and the results of these studies are
presented below.

Chain length of polymers:  Each of the polymers shown in Table 1 had a chain
length in excess of 300 nucIeotide units but probably not greater than 500 units.
Because of the large amounts of polymer required for the chain length determina-
tion, more accurate measurements were not made.

                         The sedimentation coefficients of the
polymers described in Table 1 are given in Table 2.  At pH 7.2 the Sp0 of the poly-

Sedimentation characteristics of polymers:

TABLE 1

STIMULATION OF

AMINO ACID INCORPORATION BY POLY UG

Pol mer base ratio

Nucleoside diphosphate

(t;/G)*           &?/I 3.3/1
ratio (U/G)t         8/ 1 5/1

1.6/1 0.58/1

                Control minus
3/1 1/1 polynucleotide

Incorporation above Controlr
     ppmoles

Cl' amino acid

Phenylalanine      901 1708        1067        15 45

Tryptophan        38 210        276        10 60

Valine 287 1036 1050 61 13
Leucine 267 834 856 35 64

* Base ratio of polymer determined as previously described."
r Figures represent incorporation of Cia amino acid above the basal incorporation obtained in the absence
of added polymer.

Reaction mixtures (0.5 ml) contained 0.1 M Tris buffer, pH 7.8. 0.01 M magnesium acetate; 0.05 M KC1:
6 X 10-8 M8-mercaptoethanol. 1 X 10-8 M ATP. 5 X IO-' M potassium  hosphoenol yruvate; 10 pg crystal-
line phosphoenolpyruvate kinade (CalBiocbem): 2' X lo-! M of each of 19 Lamino aci& lacking the 04 amino
acid; 0.8 X 10-4 M 04 amino acid; 5 pg of polynucleotide. and preincubated, dialyzed a30 E. coli extract.'
Each aspay was erformed in duplicate. The s ecific radiohvitiea of the amino acids varied 2-6 millicuries
per millimole. A1 reaction mixtures were incutated at 37O for 90 min. These incorporation data therefore
represent total incorporation of C"-smino acids into protein rather than rate of incorporation.

Ratio of UDP to GDP used in polymer synthesis.

Basal incorporation is given in the last column.

VOL. 49,1963

BIOCHEMISTRY: SINGER ET AL.

395

TABLE 2

SEDIMENTATION COEFFICIENTS OF POLY UG PEEPARATIONS

Polymer base ntio c Sd

W/G)                 pH 7.2 ` pH 12
6.7/1                  5.9 5.2
3.3/1                  3.1 2.3
1.6/1                  5.7 3.3
0.58/1                 11.0 2.1

o Inepeetion of the densitometer tracingmindicated relatively little or no breakdown of the
polymen during medimentation at  H 12. Nonoedimenting nnterial amounted to sip-
roximately 575, at pH 7 and 12 wit[ al1,the polymen except the I& (U/G. 0.58/1!, when
8 WM about zO% at pH 12. Fdrthermore, in each cam the medimenting material wan more
homogeneous at pH 12 than at pH 7 and thin WM most striking with the last polymer

(U/G. O.aS/I).

mer containing 70 per cent G is considerably higher than the S, values of the others.
However, the decrease in &, noted for all polymers at pH 12 is much greater for
this high G containing polymer.
Phosphorolysis of polymers by polynucleotide phosphotylase:  The susceptibility
of the poly UG preparations to phosphorolytic cleavage by M. Zysodeilcticus poly-
nucleotide phosphorylase was studied and the results are presented in Table 3.

                TABLE 3
PHOSPHOROLYSIS OF POLY UG BY POLYNUCIXITIDE PHOSPHORYLASE

Phoaphorolyais Rate
(m~molem/ll min.)

Phosphorolysis Ratet
Rate with poly A

... 6.4
22.6 3.2
25.9
11.6

1.1

3.3ji

0.58/1

1.6/1

3.7
1.7
0.16

o Experiment carried out at a separate time.
t The numben in the laat column repraent the rate of phaphorolynia of the given polymer relative to that of
he inoubation mixtures conhined approximately one mole of polymer phwphate per ml (we rection on Ma-

PO$ A.

Larid. and MetU for details).

The rate of phosphorolysis (estimated with a measurement at a single time when the
reaction rate is hown to be linear with homopolymers) depends on the relative con-
tent of uracil and guanine.  In particular, when the polymer has more guanine
than uracil the rate falls off very sharply.  This effect is not the result of the de-
creasing uracil content since the members of an analogous series of poly UC prep-
arations were all phosphorolyzed at similar rates.  Thus, when the ratio of uracil
to cytosine was 4/1, 3.3/1, 1.7/1, and 0.6/1, the rates of phosphorolysis relative
to poly A were 3.3, 4.0, 3.7, and 2.2, respectively.  The slow phosphwolysis of
poly UG containing more guanine than uracil appears to result from the high guanine
content.

E#& of temperature on the ultramhlet absorption spectra of poly UG preparations:
Thomass' and Doty and co-workera41 ti have discussed the use of hypochromicity
as an indicator of secondary structure in polynucleotides.  We have determined
so-called "melting curved' for polymers described in Table 1.  Polymers having
ratios of uracil to guanine greater than 2/ 1 showed essentially no change in absorp
tion between 200 and 300 mp between mom temperature and 85'.  With poly
UG having a uracil to guanine ratio of 1.6 to 1, we observed a slow increase in the
absorption at 250,260,270, and 280 mp upon increasing the temperature from 22'
to 82O; however, the total increase at 270 mp was only about 5 per cent (Fig. 1).

396

BIOCHEMISTRY: SINGER ET AL.

PROC. N. A. S.

+o 1.20

+

0

0

ti
Is
a

\
4
t

0

e `.`O

N

v)

9

1.00

30 40 50 60 70 80 90
       TEMPERATURE ("C)

FIG. 1.-Changes in ultraviolet absorption of poly UG as a function of temperature.
The ordinate is the ratio between the absorption at 270 mp at the indicated temperature
and the absorption at 270 mp at the first temperature measured.  --e--, poly UG. U/G
ratio equal to 0.5811, in 0.1 M KCI, pH 7.8; -0- poly UG, U/G ratio equal to 0.58/1,
in 0.01 M potassium phosphate, pH 7.0; -A-, poly UG, U/G ratio equal to 1.6/1 in
0.01 M potassium phosphate, pH 7.0.

Under the same conditions (0.01 M phosphate buffer) poly UG having a uracil to
guanine ratio of 0.58 to 1 gave a total increase in absorption at 270 mp of 12 per cent
on raising the temperature from 30 to 85O (Fig. 1).  The data are given for 270
mp since the hypochromicity of G containing structures appears to be optimal at
that ~avelength.2~  Figure 1 shows two curves for poly UG (U/G, 0.58/1);  one
was obtained in 0.1 M KC1, pH 7.8, the other in 0.01 M phosphate buffer, pH 7.0.
The curves are similar although the start in the increase in absorption is somewhat
delayed in 0.01 M phosphate buffer, pH 7.0.  This observation is consistent with
the findingz5 that the "T," of d-pGpGpGpG is lower in 0.2 M NaCl than in phos-
phate buffer alone.  The experiment in 0.1 M KC1 was carried out in the Cary in-
strument and a temperature dependent shift in the wavelength of maximum ab-
sorption from 255.5 mp to 253 mp was also observed.  This shift took place at
about 90.

Optical rotation of poly UG preparations:  As an independent measure of poly-
nucleotide helical content4 the optical rotations of several polymers shown in Table
1 were measured.  Determinations were carried out at room temperature in 0.01
M potassium phosphate buffer, pH 7.0, using the mercury line at 365 mp.  The
specific rotations of the polymers having U/G ratios of 6.7/1, 3.3/1, and 0.58/1,
were +99, +74, and +399, respectively.

                                The relative amounts of
phenylalanine, valine, leucine, and tryptophan directed into protein by the poly
UG preparations (Table 1) is clearly related to the base ratio of the polymer and

Discussion.-Amino acid incorporation into protein:

VOL. 49, 1963 BIOCHEMISTRY: SINGER ET AL. 397

these data provide strong support for earlier codeword assignments.", 12, 14  How-
ever, several properties of the poly UG preparations used indicate that meaningful
comparisons between theoretical frequencies of doublets or triplets and relative
amino acid incorporations cannot be made.  One such factor is the secondary struc-
ture of the polymers and is discussed in detail below.  Another factor is the pos-
sibility that the polymers may be nonrandom.  Since G is incorporated into poly-
mer preferentially (Table 1) the ratio of UDP to GDP changes during polymer syn-
thesis.  Thus, the base ratio determined by analysis could represent the average
base ratio rather than that of any particular polynucleotide region or molecule.
Preferential incorporation of G into polymers has been noted pre~iouSly,~~~
and similar observations have been made by Bretscher and Grunberg-Manago, 26
using A. agilis polynucleotide phosphorylase.  The mechanism of this concentra-
tion phenomenon is currently under investigation."

Egect of secondary structure on the template activity of messenger RNA:  Several
lines of evidence presented above indicate that copolymers of U and G contain a
high degree of secondary structure when the relative content of G is high, and this
interpretation is consistent with recent reports on the secondary structure of poly
G itself.24. 25*   (1) Although the four UG preparations described have approxi-
mately the same chain lengths the sedimentation coefficient of poly UG (0.58/1) at
neutral pH is markedly higher than that of the others.  This increase in S value
may result from greater aggregation (due to hydrogen bonding) with increasing G
content for, at pH 12, where the secondary structure of poly G collapses,~s~ 28 all
the polymers have similar sedimentation characteristics.  (2) The sharp drop in
phosphorolysis rate observed on going from a poly UG with a U/G ratio of 1.6/1
to one with a U/G ratio of 0.58/1 can also be explained by an increase in secondary
structure.  O~hoa~~ and Grunberg-Manago*2 have shown that polyribonucleotides
having ordered secondary structures are phosphorolyzed much less rapidly than
polymers existing as random coils.  Poly G, for example, is completely resistant to
phosphorolysis by polynucleotide phosphoryla~e.'~* a2  (3) Only polymers contain-
ing relatively large amounts of G (U/G, 1.6/1, and 0.58/1) showed any increase in
ultraviolet absorption at raised temperatures, and the increase is greater the higher
the G content.  (4) The relatively high optical rotation of poly UG (U/G, 0.58/1)
also indicates ordered secondary ~tructure.~

Thus, the first three polynucleotides listed in Table 1 contain only moderate
amounts of secondary structure and direct amino acid into protein with high effi-
ciency.  The last polymer, which contains almost 70 per cent G, exhibits a great
deal of secondary structure and a markedly decreased ability to serve as a template
for protein synthesis.  This correlation between template efficiency and secondary
structure is consistent with our earlier observation that the ability of poly U to
direct polyphenylalanine synthesis is lost when poly A-poly U helices are f~rmed.~
Furthermore, we have foundlo that the extent of inhibition of polyphenylalanine
synthesis by oligoadenylic acid preparations increases with the length of the oliio-
nucleotide chain and can be correlated with the stability of oligoadenylic-poly U
helices.

Thus, single strandedness and lack of extensive intramolecular hydrogen bonding
appear to be requisite for messenger RNA activity in this in vitro system.  Results
obtained with natural RNA preparations are in accord with this conclusion.  For

398 BIOCHEMISTRY: SINGER ET AL. Pmc. N. A. S.

example, TMV-RNA directs protein synthesis with high efEciencye~  in this sys-
tem, compared to the efficiency of other RNA preparations.g  Melting curves for
TMV-RNA'. indicate that a larger per cent of its secondary structure is destroyed
at 37 than is destroyed with ribosomal or transfer RNA preparations.

From an experimental point of view, caution should be used in handling template
RNA to be used in in vitro systems.  In the course of experiments on the melting
of polymers we noted that the change in absorption with temperature is not always
readily reversible.  Thus the storage (for example, freezing and thawing) of poly-
mer solutions may cause significant changes in secondary structure and therefore in
template efficiency.

At present three factors influencing template activity are known-the size of the
polymer chain,14* 15* 13* 35 the nucleotide sequence, and the secondary structure.
Previously we showed that poly U fractions of relatively high molecular weight are
more active as templates than smaller molecules14 and more recent data indicate
that high template activity corresponds to average chain lengths of  100 or
greater.as.   The effect of nucleotide sequence on template efficiency is difficult to
assess.  Recent data show that the code is very degenerate16 but it is not known
whether all nucleotide sequences will code.  Although nonsense sequences may
exist, thus far none have been definitively demonstrated.  As indicated in the pres-
ent report, the secondary structure of a polynucleotide must also be considered in
any evaluation of its ability to serve as template RNA in the in vitro system.

The molecular basis for the genetic information specifying the beginning or end of
a protein is unknown.  Previously we suggested that nonsense nucleotide sequences
might function in this manner." The results of the present study suggest an al-
ternative explanation.  Thus, a limited region of messenger RNA containing a high
degree of secondary structure as a result of intramolecular hydrogen bonding (for ex-
ample, G-G interaction) might also specify the beginning or end of a protein.
Areas of marked secondary structure, which may be likened to knots in a rope,
might separate the messenger molecules containing information for the synthesis
of more than one protein into functional units.  Mechanisms enabling certain pro-
teins to be synthesized in close geographic proximity may be advantageous as, for
example, in the synthesis of proteins containing different subunitsa6*  or for the syn-
thesis of coordinately repressed enzymes.  In a more general sense, we expect that
future studies of the three distinct RNA fractions, transfer RNA, ribosomal RNA,
and messenger RNA, will indicate functional significance for the specific conforma-
tional aspects of their structures.  With regard to messenger RNA, we would sug-
gest that both the nucleotide sequence and secondary structure are relevant to the
decipherment of the genetic code.

Summary.-A series of copolymers containing varying amounts of uracil and
guanine have been studied as templates in a cell-free system for protein synthesis.
Polymers containing relatively large proportions of guanine have low template ac-
tivity.  Evidence indicating that the guanine-rich polymers contain a high degree
of ordered secondary structure is presented.  It is suggested that the existence of
such secondary structure accounts for the poor template activity of these polymers.
The possible functional significance of RNA secondary structure in the coding
mechanism is discussed.

VOL. 49, 1963 BIOCHEMISTRY: SINGER ET AL. 399

It is a pleasure to thank Linda Greenhouse and Helen Maleady for valuable technical assist
ance. We would also like to thank Dr. Samuel Luborsky for his experimental and theoretical FS-
sietance with the sedimentation studies.

* The following abbreviations are used: Poly U, polyuridylic acid; poly A, polyadenylic acid;
poly C, polycytidylic acid; poly G, polyguanylic acid; poly UC, copolymer of uridylic and
cytidylic acids; poly UG, copolymer of uridylic and guanylic acids; G, guanylic acid; U, uridylic
acid; A, adenylic acid; C, cytidylic acid; TMV, tobacco mosaic virus.  All other abbreviatione
conform to those acceptable to the Journal of Biological Chemistry.

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