Reprinted froill tlir Proceedings of the NATlON.\L Acauen~ OF SCIENCEH

7'01. 48, No. 8, pp. 1466-1472.

August, 1962.

THE IDENTIFICA TION OF THE RIBOSOMAL RNA CISTRON BY


   COMPETITIVI3 INTERAC7'ION AT THE RNA C'ISTRON

SB&UEN('E COII.I/'LE'All~NTARITY,* II.

SATURATION OF AND

     BY s. A. \r4NKoFhKYt .\ND s. SPIEGELM4N

1)EPARTiMENT OF MICRORIOI,OC.Y, LTNIVERSITY OF II,I~INOIS, tJRBANA, ILLINOIS

      Coininunzcated by H S. Gutowsky, May `7, 1962

The presence of a sequence in DIKA complementary to homologous ribosomal
RXA is indicated by experiments described in a preceding paper.'  The data showed
that a hybrid complex resistant to RXAase is formed when mixtures of ribosomal
RXA and denatured DSA from the same cell are heated and then subjected to
a slow co01.~  When DNA from a heterologous source is substituted, no such com-
plex is observed.  The specific pairing of ribosomal RNA with a complementary
sequence in DXA leads to the following two predictions:  (I) The ratio of RXA
to Dn'A in the specific complex should approach a maximum value at levels indicat-
ing the involvement of a minor fraction of the DNA; (2)  Nonribosomal RNA
should not compete for the same site.

                                         It is the intent
of the present paper to describe the results obtained and discuss their implications.
The data are consistent with the existence of a specific sequence in I)XL4 capable of
complexing with homologous ribosomal RR`A and not occupiable by nonribosomal
RXA from the same organism.

                                       E. coli strains RR
and A-155, a uracilless derivative of B were used in the present study.  The methodb
of growth, labeling, counting, CsCl density gradient centrifugation, and the prepa-
ration of the following materials are detailed in the earlier       heat-denatured
DXA, free of RNAase;  P31 and H3-labeled 23s ltSA free of "informational"
RXA.  The methods of assaying for RKAase activity arid sensitivity of RTVA-Dn`A
complexes to nuvlease degradation are the same as used in the earlier' study.

                               The procedure
used is essentially that of Hayashi and Spiegelman.3  Logarithmically grom-ing
E'. coli A-1.55 cells in nutrient broth were collected by cmtrifiigation, washed twice

Experiments relevant to these two issues have been performed.

Materials and Methods.-Strain and preparation of materials:

"Step-down" labcling of cells for inj`ormational RNA pro parationu *

VOL. 48, lW2 BIOCHEMISTRY: l'ANROFKSI' AND SPIEGELMAA' 146i

in sc minimal broth, and resuspended in the minimal medium to an 0.n.660 of
0.360.  The cells were aerated for 10 min at 37C; then 1.48 pg/ml of H3-uridinc
was added (New England Xiidear Corporation, 3.28 c/m;\l) and aeration at, 37C
continued for 10 min. Isotope inrorporation was halted by pouring the cultures into
1/8 their volume of frozen, crushed minimal medium.  The cells were collected by
centrifugation, then concentrated 20-fold in ThI buffer.  The ItXA was purified
and fractionated in linear sucrose gradients by the methods described previously.',
The fractions corresponding to 8-12s were pooled, concentrated, and used in thc
experiments described.

Experimental Results.-A. Saturation curup (d the ribosomal RNA-DNA complc .r:
The detertion of a saturation plateau requires the performance of experiments in-
volving the following steps:  (1) LIixtures containing fixed amounts of heat-de-
natured DNA and varying amounts of labeled ribosomal RKA are exposed to a slow
cool from 55C to 30C; (2) The resulting products arc then subj
librium density centrifugations in CsCl solutions 1 o separate free KKA from that
which is complexed to DNA.  The sorts of hybrids observed are exemplified by thc
two cases in Figure 1.  It will be seen that at the lower level of inpiit, most of the
RNA in the reaction mixtiire is complexed to the DXA.  The proportion of the
input fixed decreases as the concentration of RYA is increased.

CsCl EQUILIBRIUM GRADIENTS

.1_ , I 1 , , -,- 7 7 1-7

'",-(031,1 235 Coll RNA X Coli DNA
40 k

fi

20 L

ot

MI -

FIG. l.-CsCl equilibrium derlsity gradient profiles.

                   Buffer used was TMHV (0.05 '21
                     Both containrd 71 pg per
        Upper cwntairied 0.3 I pg aiid the lower 0.62 pg of P31-
            Reaction mixtures slow (ca. 13 hr) cooled from
Mixtures were then brought to a density of 1.73 with CsCl and a total

tris, pH 7.3; 0.001 A!/ MgC12; 0.3 M NaCl 0.005 M EIYI'A.
rnl of heat-denatured E. coli DNA.
238 RXA (6.7 X 10' cpm/pg) of E. coli.
55C to 33C.
volume of 3 ml.

They were centrifuged fur 52 hr at 33,000 rprn and 25C.

1468 RIOCEIEMISTIZ)': ).'A h-KOFSRY ASD SPIEGELMAN PROC. N. A. H.

I\"----- 14961

Free RNA Control

OLo MINUTES

FIG. %.-l-XAast~ resistanre of ItSA\ -1)NA
complexes.  109 pg/ml of heat-denatured E. roll
115.4 were slow-rooled in TMSV with the in-
dicated roncentrations of E' rolz 23S-P3L-RNA
(6.7 x 10' rpni/pg). After separation of
hybrids by centrifugation as in Figure 1, sensi-
tivity to RNAasc was examined as described
previously.

235 E Coli RNA X Colt DNA

w 0 004
(L

Tofu1 RNA

X'

/'

/

0 01 02 03 04 05

INPUT RNAIDNA

FIG. :J.-Saturation curves of total and
llS A ase-resistant counts in 1 )NI\ region
of CsCl equilibrium gradients.  Details of
incubations and centrifugation are as
noted in Figures I and 2.

From our previous experience'
with ribosomal REA, it might be
expected that nonspecific binding
involving incidental coincidences
over small regions will inevitably
complicate such experiments. Ad-
ventitious complexing of this sort
could become quantitatively serious
at high levels of RNA input.  For-
tunately, as has been shown,' sen-
sitivity to RNAase permits a ready
distinction between nonspecific and
specific complexes. This same de-
i ire was used in the present study.
Fractions in the hyhrid regions of
the density gradients were pooled,
dialyyed, and exposed to RNAase.'
The iesiilts obtained for four levels
of RXA are detailed in Figure 2.
In each case, free P32-laheled RNA
was included as an internal control
in the reaction mixture. The be-
havior of only one control is de-
picted since they were all identical.
It mill be noted that the free RKA
is almost completely solubilized in
5 min, whereas the H3-labeled RNA
from the hybrid regions exhibit
RNAase-resistant residues of vary-
ing percentages. The data of
Figure 2 are in agreement with what
would have been expected from in-
creasing amounts of nonspecific com-
plexing at higher concentration
levels of RKA.  The greater the in-
piit of RNA, the larger is the propor-
tion of the counts in the hybrid re-
gion which are foimd to be sensitive

to RKAase.

  composite summary of these and similar experiments are plotted in Figure 3
We ha1.e here a comparison of the total RKA found in the DNA density region with
that which resists degradation by RNAase.  The behavior of these two are strik-
ingly different.  The total RKA shows no signs of saturation, whereas the resistant
residue clearly approaches a plat eau, corresponding approximately to an RNA :
DXA ratio of 0.001.5.  The existence of this maximal level is consistent with the
1 iew that the DSA contains a restricted region capable of specifically complexing
with ribosomal RSA.

H. Comprtitive arid noncompetitiw intrmctzon in thr coursc 1J' hyhrad formation:
Competition experiments can also be used to revoal the existence of :L lovalized
specific interaction between ribosomal RXA and its homologous DNA.  In the
present system this can be realized by using two identifying isotopic labels on dif-
ferent RNA preparations.  If the two RXA molecules are competing for the same
site and the total concentration is at or near the saturation level, one label will
displace the other as its proportion is increased in the reaction mixture.  If they
do not compete for the same site, fixation to the DSA of one will be essentially in-
different to the presence of the other.

To examine questions of this nature, the following types of experiments were
performed with E. coli REA and DKA.  Mixtures containing fixed levels of P32-
23s RKA, heat-denatured DKA, and varying amounts of H3-238 RXA mere incii-
bated arid then centrifuged in CsCl.             and H3-labeled RNA fixed
in the DNA and resistant to enzyme were then determined.  $'or purposes of com-
parison, similar experiments were carried out, with H3-informational RNA (8-
128) replacing the H3-2:3S RNA in the mixtures.  The informational variety was
chosen since it was known to complex well with DSA arid thus provides a test for
the specificity of the ribosomal combination with DYL4.

Figure 1 gives two examples of the hybrid regions observed in CsCl gradients.

The amounts of

HYBRID REG/ON, H3- /NF RNA, P32-23S RNA

40- 1600

I

A1200

- 800 2

-

-E


   a
-0
- 400

rnl rnl

FIG. 4.--Hvhrid regions in CsCl gradients of mixtiires contain-
ing ribosomal and informational RNA  Both incuhstion mix-
tures contain 63 pg/ml of heat-denatured E. colt DN.4 and 2 66
pg/ml of E. colt P3*-23S RNA.  (A) contained in addition 2.9 pg
of H3-informational RXA and (B) 5.7 pg of H3-informational
RNA per ml.  I>etmls of incuhstions and centrifugations are as
in Figures 1 and 2.

Both types of nucleic acids have hybridized with, however, an interesting and con-
sistent difference.  The H3-labeled informational RXA appears to be symmetric.ally
distributed among the denatured strands of the DNA.  This is clearly not the
(xw with the ribosomal complex which is distinctly displaced toward the heavy
side of the mean density of denatured DNA.  The likely significance of this will he
briefly noted in the Discussion.

.\gain, to avoid confiision with irrelevant complexes, the fr:ictions in the hybrid
I egions were pooled :id t he RXAase-resistant residue of complexed radioactivity
determined.                            There is clear evidence
(Fig. 5A) of displacement of the P3?-labeled ribosomal RKA as more H3-labeled
RNA of the same kind is incorporated into the complex.  Further. a saturation

Figure 5 summarizes the resulting data.

I170 RIOCHEJfISl'II1': J7~A.YKOFSKY AiVD SPIEGEIXAN PROC.

N. A. 8.

0.5r l

P

(A) P32-23S Ribosomol RNA lConslont conc I

!- (6)

123456 123456

I

H3-RNA ADDED (pg)

0 0" 051

n

2 02

t

P

A

23s Ribosomal (P3*/ RNA

SH'-lnf RNA

,23S Ribosomal RNA

123456789

TOTAL RNA INPUT (pg)

FIG. 5.-Tests for competitive interactions in mixturps of ribosomal and
informational RNA molecules.  (A) All mixtures rontained 63 pg/ml of heat-
denatured E. rolz 135.4 and 2.66 pg/ml of E. colz P31-23S ribosomal RNA.  Hj-
23s ribosomal RX.4 of E. coli varied as shown.  (B) DNA and P32-RNA same
as (A) except trhat H3-informational RXA was added in the amounts indicated.
(C) The same preparations as (A) and (13).  The plot here is total (P32 + H3)
RXAase stable counts per 100 pg of DNA as a function of total input of RNA.
All incubations during slow cool and equilibrium centrifugations were carried
out as described in Figure 1.

plateau in the H3-RKA complexed is observed within the concentration range tested.
On the other hand, when the H3-label is on informational RNA, its hybridization
with the DKA has no effect on the ability of the DXA to combine with the P3?-
labeled ribosomal material (Fig. TiR).  In addition, no evidence of saturation is
observed with the informational variety within the Concentration range tested.
This difference in saturation behavior is more clearly illustrated in Figure 5C in
which the total (]Id2 + H3) RNA fixed per 100 y of DIVA is plotted against the total
RNA in the mixture.  When the ribosomal varieties alone are present, the sum of

VOL. 4s. 1962 RIOCHEMISTR Y: YANKOFSKY il IVI) SPIEGELMALV 1471

the two labels fixed approaches a plateau--a phenomenon completely absent in
the mixture of informational and ribosomal RXA.
These experiments provide additional evidence supporting the contention that
ribosomal RNA specifically complexes with a restricted region not occupiable hy
nonribosomal RNA from the same organism.
L>zscussion.-The use of double labeling combined with equilibrium centrifuga-
tions in density gradients and enzyme sensitivity permitted the accumulation of
data which !eads to the conclusion that DXA does contain a localized sequence com-
plementary to ribosomal RNA.  The nature of the data obtained in the present
and previous investigation' may be summarized as follows : (1) The RNA complexed
in a hybridization test is ribosomal as demonstrated by the base composition of the
hybridized material.  (2) Complex formation stable to RNAase occurs only with
homologous DNA.  (3) Saturation of homologous DNA with ribosomal RNA in n
cpmplex stable to RKAase ocwrs when approximately 0.2 per cent of the DKA is
occupied.  (4) Competition for a specific site was established by using two identical
ribosomal RNA preparations distinguishable by isotopic labeling.  ITnder the same
conditions, nonribosomal RIVA from t he same organism does not compete.

It will not escape the attention of the reader that certain problems of obvious in-
terest have not received explicit mention.                First, all
the experiments described have cmployed only the 23s ribosomal component.
This raises the obvious question of sequenw similarity with thc 16s variety.
Further, no dctails are reported on hrterologoiix tests among the tjtwteria, :t point
of obvious interest in vicv of the simil:trity of the ritjosomal RXA 1)aw romposi-
tions.  These, and related problems have been the sihject of vontiniiing investiga-
tions by the methods developed in t,he present investigation.  The results will be
reported in separate commnnivations.

We may perhaps briefly allude to one point centering around the significance of
the fact that rihosomal ItXA saturates at a level corresponding to approximately
0.2 per cent of the DIVA in E. coli.  In view of the complicated operations required
to obtain this number, its exact value must not be taken too seriously, and certainly
not until similar determinations are reported with other organisms.  However,
values similar to this have been repeatedly obtained in independent experiments, and
it would seem that it is certainly correct, as to order of magnitude.  This, however,
is about 10 times greater than one mould calculate from the DNA content per nu-
cleus4 and the assumption that there is only one complementary sequence per ge-
nome.  We conclude. therefore, that there must be repeating units of this kind.
They could be similar in sequencc, or identical, depending on whether the popula-
tion of ribosomal RKA molecules arc identical or different.  There is one highly
suggestive observation which leads us to conclude that, whichever is the case, these
repeating units are not scattered throughoilt the genome, hiit, contiguous. A
persistent pecularitiy observed with homologous ribosomal IZXA-DXA hybrids
(Fig. 1 and P2 curves of Figs. 4A and 4R) is the pronounced displacement of the
complex towards the heavy side of the mean density of the heat-denatured DNA.
This displacement is riot observed with the heterologous c-omplexes.  Further-
more, it is not seen with informational RNA-DNA hybrids at this level of RKA in-
put (H3-curves of Figs. 4A and 4B).  The simplest interpretation for this shift is
to assume that the repeating units complementary to ribosomal RNA are contiguous

We may briefly cite a fen-.

md that any IISA st'rand which has one such sequence is likely to have another.
Consequent,ly, t,his selected set of strands complex wit'h several ribosomal Rh-A
molecules, resulting in the observed increase in densit,y.
Summary.-The experiments reported offer further evidence for the presence in
DKA of a sequence complementary tmo rihosomal IIXA.  Saturation experiments
suggest, t'hat' the part,icular region involved corresponds to hetween 0.1 and 0.2
per cent of t,he total genome.  Competition for a restricted DKA region can be
exhibited by t,he use of variously labeled homologous ribosomal IZKA.  Xonribo-
soma1 RSA from thc same organism does not, compete for the same site.  The
amount of ribosomal RNA complexed per unit: of D?JA at saturation suggests a
number of repeating, similar, or ident,ical sequences.  Flirther, the density shift)
of the hybrids suggest,s t,hat t)hese units are not scat'tered but contiguous in the
DNiZ st,ruct>ure.

* This iiivestiyntioii was :tided by gr:Lrit,s in :Lid frcinr t,he 1J.S. Ptiblic Health Service, Xational

t Predoctoral fellow trainee in Molecular (ienetics (USPH 2G-319).

Yankofsky, 8., and S. Spiegelinan, t,hese hoCEEL)INc:S, 48, 1069 (1962).
2 Hall, B. I)., and S. Spiegelman, these PRO
3 Hayashi, M., and S. Spiegelman, these PROCEEDINGS, 47, 1564 (1961).

Science Foundat,ioii, :tnd the Office of Naval Rescarclr.

, 47, 137 (1961).

Barner, H., and 8. S. Cohen, J. Hacteriol., 72, 115 (1956).