Proceedings of the National Academy of Sciences
Vol. 68, No. 1, pp. 6-9, January 1971
The Folded Genome of Escherichia coli Isolated
in a Protein-DNA-RNA Complex
0. GORDON STONINGTON AND DAVID E. PETTIJOHN
Department of Biophysics, University of Colorado Medical Center, Denver, Colorado 80220
Communicated by Theodore T. Puck, October 8, 1970
ABSTRACT The DNA of Escherichia coli has been iso-
lated in a compact structure containing small amounts of
protein and RNA and having a sedimentation coefficient
of approximately 3200 S. The molecular weight of the DNA
in the complex is very large (probably higher than 109); the
protein is predominantly core RNA polymerase; the RNA
is chiefly nascent messenger and ribosomal chains. Solu-
tions containing the complex have low vicosities; this plus
its sedimentation rate suggest that the DNA is in a tightly
folded conformation. The DNA unfolds after exposure to
RNase or heat; this indicates that an RNA component of
the complex is involved in stabilizing the structure..
The DNA in bacterial cells as seen under the electron micro-
scope is confined in regions of the cell called "nuclear bodies,"
but no nuclear membrane is observed (see, for example, refs.
1 and 2). Yet these chromosome-like structures have defined
boundaries, which reorganize during the nuclear segregation
that accompanies cell division (3). This implies that DNA
folding within the nuclear body is maintained-at least
partly--by internal interactions between the elements or re-
gions of the nuclear body. The tertiary conformation of DNA
within this body is unknown; however, studies of the rates at
which different operons can be transcribed and regulated sug-
gest that a considerable portion (perhaps all) of the genome is
available for free interaction with other macromolecules (for
examples see refs. 4-7). The packaging of the DNA must
also be compatible with the requirements of semiconservative
DNA replication and the eventual segregation of daughter
double-helices (8, 9). These considerations imply that the-,
tertiary structure of DNA within the cell is organized; how-
ever, this structure and the interactions that stabilize it are
still to be determined.
We describe here the isolation from E. coli of a DNA com-
plex in which the DNA genome remains folded into a struc-
ture which is compact and nearly homogeneous in sedimenta-
tion. The complex is approximately 80% DNA by weight; it
includes a small amount of protein and also contains nascent
RNA. At least some of the RNA of the complex is essential
for stabilizing the packaged structure, since the DNA unfolds
as a result of RNase treatment.
MATERIALS AND METHODS
The E. coli strain D-10 (RNase I-, ref. 10) was used in these
studies. The bacteria were grown into the log phase in M9
media supplemented with 0.2% casamino acids.
Contribution No. 425 from the Department of Biophysics,
Florence R. Sabin Laboratories, University of Colorado Medical
Center.
Abbreviation: SDS, sodium dodecylsulfate.
6
Viscosities of solutions of the DNA complex were measured
at 250C in a falling-ball viscometer.
Proteins were obtained from a DNA complex that had been
purified by a scaled-up modification of the procedure described
in the legend to Fig. 1. The isolated complex was collected by
centrifugation at 23,000 rpm (SW 50 rotor) for 45 min. The
pellet was solubilized with pancreatic DNase and the pro-
teins were desalted on a Biogel P4 column, lyophilized, and
redissolved in 0.1% sodium dodecylsulfate (SDS)-0.14 M
.3-mercaptoethanol-0.01 M sodium phosphate, pH 7.2.
This solution was heated for 10 min at 650C and cooled to
20'C; 5-20 ,ug of the reduced protein was electrophoresed for
20 volt-hr per cm on 5% polyacrylamide gels as described by
Shapiro et al. (11).
RESULTS
Isolation and sedimentation of DNA complex
When E. coli cells are gently opened in solutions of high ionic
strength, DNA is released from the cells but the solution does
not become viscous. A DNA complex, having a nearly homo-
geneous sedimentation profile, can be isolated from the bulk of
the ultraviolet-absorbing material of the lysate (Fig. 1; see also
ref. 12). This complex contains: nearly all of the DNA of the
cell-free lysate (the radioactivity remaining at the top of the
sucrose gradient of Fig. 1 is due predominantly to free [8H]-
thymidine); most of the DNA-bound RNA polymerase
activity of the lysate (12); the nascent RNA of the bacteria
(12, 13); and less than 1% of the DNA polymerase activity
(14) of the lysate (unpublished observations). Speed is essen-
tial in purifying the complex, since it is unstable in the crude
lysate and probably also unstable in nongrowing cells. Once
purified, it retains its sedimentation and viscosity characteris-
tics for a few days when stored at 0C.
The DNA complex has been isolated from bacteria grown
for several generations with a mixture of 3H-labeled-amino
acids. Less than 1% of the incorporated radioactivity co-
sedimented with the complex when it was purified as in Fig. 1.
After determining the specific radioactivity of the total [3H1-
protein, it was estimated that the complex contained less than
10% protein by weight (assuming that proteins on the com-
plex have the amino acid composition of average E. coli pro-
tein).
The sedimentation rate of the complex, determined by com-
parison with that of a T4 phage marker (15), was about 3200 S.
This value has been corrected for the inconstant rate of sedi-
mentation in the sucrose gradient due to variations in field,
viscosity, and solvent density, using a recently devised com-
puter program (McConnell, J., to be published). The figure
 Vol. 68, 1971 Folded Genome of E. coli 7

TABLE 1. Effect of various agents on the relative viscosity of the TABLE 2. NaCl concentration and the viscosity
DNA complex in solution of the purified DNA complex
,q/o0 * n7/710 '1/"lo
Pancreatic ribonuclease After 50'C
Incubation alone 1.0 14.7, 13.5, NaCl (M) Before heat for 5 min
4 jg/ml 12.3
Pronase 100 Ag/ml 1.0 Above followed by de- 0.9 1.0 1.0 16.0
oxyribonuclease 1 jig/ 0.5 0.9 -
ml 0.2 1.0 -
Trypsin 10 .ug/ml 1. 1 Sodium dodecylsulfate 14.1 0.07 1.0 9.0
(SDS) 0.5%
Diethylpyrocar- Heat 50'C for 5 min 11.2, 10.3,
bonate 0. 1% 1.0 8.5 that of T4 phage (16). In making this estimation it was as-
,8-Mercaptoethanol sumed, as above, that the complex had the same partial
1.0 M 1.2 specific volume as T4 phage and that the molecular weight of
Each sample contained the same amount of DNA (approxi- the complex was equal to 1.4 E. coli genomes plus 20% due to
mately 8 jig/ml) and was incubated at 250C for 3 min in the pres- RNA and protein (see below). With the assumption that the
ence of the listed agent prior to the viscosity measurement. complex sediments as a sphere, the diffusion constant was
* x1o is the viscosity of the buffer-sucrose solution in which the applied to the Stokes equation r = kT/6Tr-D to calculate a
sample was tested (the same buffer as in the sucrose gradient of radius of 0.14 ,um. The average DNA density in this particle
Fig. 1, except that it contained 0.2 M NaCl and 20% sucrose).
Values are not corrected for non-Newtonian effects.

was not corrected for the possible difference in partial specific

volumes of T4 and the complex.
Viscosity properties of purified DNA complex
As shown in Table 1, the purified DNA complex does not add
significantly to the viscosity of a buffered sucrose solution in
which it was suspended. However, after treatment with RNase,
sodium dodecylsulfate, or heat, there was a dramatic increase
in its viscosity. Manipulation and repeated measurements of
the viscous, high molecular weight DNA resulted in shearing;
the changes described in Table 1 are therefore minimum
estimates.
The high salt concentration present during lysis and puri-
fication of the complex was required to maintain its conforma-
tion. At lower ionic strengths the lysate became viscous, and
separations by sedimentation were difficult except at very low
DNA concentration; the DNA then sedimented much more
-slowly than in Fig. 1 and with a broad, heterogeneous profile.
However, after the complex is purified in the presence of 1.0 M
5 10
salt, it retains a high sedimentation constant and low viscosity Fraction Number
even in solutions of reduced ionic strength (Table 2). These
data were obtained by diluting purified complex to a final FIG. 1. Sedimentation of the DNA complex on a sucrose
concentration of 8 jAg of DNA per ml into a buffer-sucrose gradient. A 20-ml culture of D-10 (approximately 2 X 108 cells/
mixture similar to that described in Table 1 except that the ml) was incubated for 30 min at 370C with 50 iCi of [3H]thymi-
dine (8 Ci/mmol); the culture was chilled to 00C in a dry-ice-
NaCl concentration was adjusted to give the listed values. acetone bath; the bacteria were quickly harvested by centrifuga-
We have not examined in detail the effects of ionic strengths tion, resuspended in 0.2 ml of a cold solution containing 0.01 M
lower than those of Table 2, but we have seen that the complex Tris (pH = 8.1 at 4VC), 10 mM sodium azide, 20% w/v sucrose,
becomes viscous when diluted into a large excess of distilled and 0.10 M NaCl; then 0.05 ml of a solution containing 50 mM
water. EDTA, 4 mg of lysozyme per ml, 0.12 M Tris, pH = 8.1 was
added. After 30 sec at 00C, 0.25 ml of 1% Brij 58-0.4% deoxy-
DNA packaging cholate-2.0 M NaCl-10 mM EDTA was added. The mixture was
The sedimentation and viscosity properties of the DNA com- held at 0C for several minutes until it had cleared considerably,
plex, combined with the knowledge that it contains only but had not become more viscous. The lysate was then centri-
minor amounts of RNA and protein, suggest that the DNA fuged for 5 min at 4000 X g and 4VC to remove debris. The super-
in the complex is folded into a compact structure that is natant fraction was layered on a 10-30% w/v sucrose gradient
stabilized, at least in part, by RNA molecules. We have containing 0.01 M Tris (pH = 8.1), 1.0 M NaCl, 1 mM EDTA,
and 1 mM f-mercaptoethanol, and centrifuged for 9 min at
estimated the density at which the DNA is packaged in the 17,000 rpm and 4VC in an SW-50 rotor of a Beckman ultra-
complex by the following procedure. Applying the sedimenta- centrifuge. In a separate but identical gradient, marker T4 phage
tion equation s = MD(1-po/p)/RT to the data of Fig. 1, it (s = 1025 S, ref. 15) was run at the same time. 0-*, absorbance
was possible to estimate the diffusion constant, D, relative to at 260 nm; O-O, 3H radioactivity; -0, T4 phage.
8 Biochemistry: Stonington and Pettijohn Proc. Nat. Acad. Sci. USA

0
6e
x x

E E

E
-C
-z-

5 a
Fraction Number -0
a-
FIG. 2. Sedimentation of unfolded DNA from the complex. 4
The DNA complex, labeled with [3H]thymidine, was isolated as
described in Fig. 1 except that 0.1% diethylpyrocarbonate was
present during lysis to protect against nucleases. An aliquot taken
from the peak fraction was diluted into 0.2 ml of a solution con-
taining 0.2 M NaCl, 0.01 M Tris (pH = 8.1), 1 mM EDTA, and
14C-labeled T4 DNA (about 3 ,g/ml). This solution was layered
orn a 5-20% sucrose gradient containing 0.5% SDS, 0.1 M NaCl,
1 mM EDTA, and 0.01 M Tris. Centrifugation was first at 5000
rpm for 5 min to introduce the complex into the SDS-containing
gradient, then for 165 min at 20,000 rpm at 220C in the SW-50
rotor. The [3H]DNA of the complex was applied at <0.3 .g/ml
and recovery in the gradient was >80%. The shoulder on the T4
DNA profile, which was observed consistently, is attributed to a
fragmentation product of the T4 DNA. O-O, ['4C1T4-DNA;
0-*, [3H]DNA.

Displacement
(Right) FIG. 3. Polyacrylamide gel electrophoresis of proteins isolated from the purified DNA complex. After electrophoresis (as de-
scribed in Methods) the gels were stained with Coomassie blue, destained with trichloroacetic acid, and scanned at 500 nm on a recording
Gilford spectrophotometer. Tracings of the actual scans are shown above. The 3 and ,' bands, which were well resolved visually, are only
partly resolved by the spectrometer. (a) RNA polymerase of E. coli prepared by the double glycerol gradient method of Burgess (24);
(b) proteins of the purified complex prepared as described in Methods; (c) pancreatic DNase marker.
was then about 0.4 g/cm3, a value roughly one-third the cules were sedimented directly from SDS lysates of E. coli
density at which DNA is packaged in the head of T4 phage made on sucrose gradients.
(17). At ionic strengths less than 1.0 the density is probably The proteins of the complex have been isolated and analyzed
smaller, since the sedimentation rate of the complex is some- by electrophoresis on polyacrylamide gels containing SDS.
what reduced from that shown in Fig. 1 (unpublished observa- As shown in Fig. 3, the protein is predominantly composed of
tions). the RNA polymerase subunits j, j3', and a (21), which is con-
sistent with previous observations (12) that most of the RNA
of
knalysis components of the complex polymerase activity of the lysate co-sediments with DNA com-
plexes. Little or no a- factor was observed, a result agreeing
The DNA of the complex, unfolded by interaction with SDS, with other indications (22, 23) that a is released from the core
sediments at a rate less than one-tenth that of the complex RNA polymerase as it initiates RNA synthesis. In the data
(Fig. 2). On the assumption that the empirical Burgi-Hershey of Fig. 3a the smaller bands between a- and a and between ,
relationship (18) can be extrapolated to these higher molecular and a- are contaminants in the RNA polymerase preparation.
weights and these conditions of sedimentation, a molecular In Fig. 3b the small band immediately to the right of a is due
weight of 1.5 X 109 was calculated for the DNA in the main to the DNase added to degrade the complex, while the next
sedimentation band of Fig. 2. How the sedimentation rate of band is an unknown protein that has repeatedly been found
this DNA varies with concentration is unknown except for one in these preparations. Other very minor bands, not attribut-
measurement made at 10 times this concentration, when the able to RNA polymerase, are also seen in the tracing of Fig.
['H]DNA sedimented 10% faster than in Fig. 2. The molec- 3b; whether these proteins are part of the complex or merely
ular weight estimated here does not differ greatly from the contaminants is not clear. Few, if any, of the DNA-binding
figure expected for the intact E. coli genome, namely 2.5 X proteins of E. coli (other than RNA polymerase) would be
10' (19). The observed sedimentation rates are also compatible expected to remain associated with the DNA of the complex
with the results of other experiments (20) in which DNA mole- in the presence of 1.0 M NaCl (25).
 Vol. 68, 1971
The RNA of the complex consists primarily of nascent
messenger and ribosomal RNA chains, the latter composing
nearly half of this total population. The RNA analysis has
been reported earlier (12, 13) in connection with our studies
of rRNA synthesis.
DISCUSSION
There are at least two fundamentally different kinds of molec-
ular interactions that may organize and stabilize DNA pack-
aging in bacteria: (a) The DNA folding may be an inherent
property of the macromolecule. That is, the tertiary structure
of DNA, like that of many proteins, could be determined by
intramolecular interactions between the monomeric units of
the polymer, mediated by rather nonspecific constraints of
the solvent environment. (b) The DNA folding could be
stabilized by other molecules or macromolecules that interact
specifically with the DNA. In this case the DNA, like RNA
as it is coiled in certain viral capsids, could be constrained into
a compact structure by intramolecular bridges connecting
specific sites on the same DNA molecule. As an example of
the first alternative, it has been recently reported (26) that
the DNAs of several bacteriophages, in solutions containing
high concentrations of certain neutral polymers and univalent
salts, fold into compact structures. This reversible conforma-
tion change was attributed to repulsive interactions between
DNA and the polymer. In other theoretical work, it has been
proposed (27) that DNA supercoiling, attributable to over-
winding of the double helix, is responsible for its compact
structure in the cell.
The results described here support the second alternative,
and implicate an RNA species as the hypothetical bridge. At
this point, speculations about the relevant RNA-DNA inter-
actions do not seem justified, except to note that there is
ample prior evidence for highly specific, stable coupling be-
tween these nucleic acids (28). The effect of SDS on the com-
plex suggests that a protein may be involved in stabilizing the
conformation, but there is no other evidence as yet to support
this idea.
None of our findings rules out the participation of solvent-
mediated interactions in organizing and stabilizing DNA fold-
ing. It is possible, then, that certain features of both the alter-
natives described above may be important in DNA packing.
Certainly there is a requirement for counter-ions to shield
against the charge repulsions that would otherwise destabilize
tightly folded DNA. Indeed, the instability of the DNA com-
plex in solutions of very low ionic strength is probably at-
tributable to ionic repulsions within the complex. In the cell
(as well as in phage heads) polyamines, simple cations, and
certain basic proteins may provide the necessary ionic shield
(16,29,30).
It is not clear why the salt concentrations required to
stabilize the DNA complex are greater during isolation than
after the complex has been purified. Possibly, the high ionic
strengths, in addition to having counter-ion effects, also
inhibit RNase activities.
Folded Genome of E. coli 9
The DNA complex described here has potential application
in studies of the template function of DNA. It has been rec-
ognized for some time that certain aspects of DNA replica-
tion, transcription, and genetic control may be related to the
tertiary structure of the genome in the cell (31). Difficulties
of manipulating large, fragile DNA molecules are avoided
with this complex, since the compact structure of the DNA
reduces its sensitivity to shear. We have observed (12) that
the complex is an excellent template for either its endogenous
RNA polymerase or added enzyme.
This work was supported in part by a research grant from the
U.S. Public Health Service no. GM14989 and by Public Health
Service Training grant no. GM00781, from the National Insti-
tutes of General Medical Sciences. One of us (0. G. S.) is a pre-
doctoral trainee of the U.S. Public Health Service.
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