volume 3 no.3 March 1976
Nucleic Acids Research
Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli.
Ralph M.Hecht and David E.Itettijohn
Department of Biophysics and Genetics, University of Colorado Medical Center,
4200 East Ninth Avenue, Denver, CO 80220, USA.
Received 30 January 1976
ABSTRACT
Methods are developed for studying RNA molecules bound directly to
DNA in bacterial nucleoids. It is found that among the 1000-3000 nascent
RNA chains that normally are attached to the DNA via their associated RNA
polymerase molecules, 71 ± 14 chains per nucleoid can be bound differently.
These chains unlike the other nascent RNAs remained bound to the DNA after
the chromosome was deproteinized and sheared. Sensitive assays using
radioactive labels detected no RNA polymerase involved in the RNA-DNA
linkage. The linkage was stable at low temperatures, but the RNA separated
from the DNA at high temperature. The bound RNA molecules were heterodisperse (weight average length 1200 bases). Pulse-chase experiments and
studies of the fate of these RNA molecules in rifampicin treated cells
demonstrated that they are nascent RNAs, degraded or released from the DNA
in_ vivo with kinetics similar to that of the total nascent RNA. Hybridization analyses showed that the chains are composed at least in part of
nascent rRNA and known mRNA molecules. Some, but not more than 5% of the
bound chains, contained sequences of about 300 nucleotides in length,
bound to the DNA in an RNase resistant form.
INTRODUCTION
The DNA in nucleoids isolated from Escherichia coli is condensed in a
compact conformation and has associated with it certain RNA and protein
components of the cell. ~
The proteins bound to the so called "membrane
free nucleoid", in its most highly purified state, make up about 10% by
mass of the structure and are composed primarily of core RNA polymerase
molecules.
It is known that most of the RNA chains associated with
the membrane-free nucleoid are nascent RNA molecules bound to the DNA in
ternary complexes with the RNA polymerase molecules.
Nucleoids isolated
using the most gentle conditions have attached all but a small fraction of
7 8
It is
the 1-3 thousand nascent, pulse labeled RNA chains of the cell. '
not clear at this time whether there are other mechanisms by which RNA
molecules can be attached to the DNA of the nucleoid or if there are RNA
species bound post-transcriptionally.
© Information Retrieval Limited 1 Fatconberg Court London W 1 V 5 F G England
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Nucleic Acids Research
Here we describe the results of a search for RNA molecules bound to
the nucleoid DNA by mechanisms not requiring RNA polymerase or other
proteins.
Sensitive methods are developed for detecting minor amounts of
the putative RNA and the properties of the observed RNA-DNA complexes are
described.
One of the experiments described here was published in a
preliminary form e a r l i e r .
MATERIALS AND METHODS
N u c l e i c a c i d s and n u c l e o i d s :
These were i s o l a t e d from Escherichia
s t r a i n D-10 grown a s p r e v i o u s l y d e s c r i b e d .
v a r i o u s t i m e s ( s e e t e x t ) with [
coli
The c e l l s were l a b e l e d f o r
C-methyl] thymidine and/or [5-H ] u r i d i n e
( o b t a i n e d from Schwarz/Mann) used i n t h e growth medium at r e s p e c t i v e l y
0.5 t o 3 . 0 p C i / m l , 20-60 tnCi/mM and 2 t o 25 uc/ml, 18-50 Ci/mM. Labeling
32
with [ P] orthophosphate (obtained from ICN) was in a special low phosphate media
containing 5-25 yCi [ P] per ml.
Membrane-free nucleoids
were isolated by a procedure similar to that described previously.
Purification of DNA bound RNA: The DNA bound RNA molecules were either
purified from isolated nucleoids or directly from bacterial cell lysates
containing the nucleoids.
In both cases the amount of bound RNA per unit
of DNA and the types of RNA (characterized by hybridization) were similar.
When purified from the isolated nucleoid, the release of RNA chains
attached to the DNA only via RNA polymerase was obtained by incubating the
nucleoids with sodium dodecyl sulfate (SDS), 0.5% v/v final concentration
at 37 C for 20 minutes (see Figure 2 legend).
Separation of DNA bound RNA
was as described below. When purified directly from cell lysates, the
39
lysate was made as usual for purification of nucleoids ' , except that at
the step where detergents are added a 2X volume of a solution containing
0.01 M Tris (pH 7.6), 1 mM EDTA, with 0.75% SDS was added.
The viscous
lysate cleared immediately and was left to incubate for 20 minutes at 30°C.
The viscosity was reduced by gently passing the lysate through a 22 gauge
needle three times.
An equal volume of water saturated phenol (equili-
brated with Tris base to pH 7.6) was added and the mixture was vortexed to
form an emulsion.
The aqueous phase, clarified by centrifugation, was
removed and the nucleic acids were precipitated with 2*5 volumes of ethanol
at -20 C.
The precipitate was collected by centrifugation, redissolved in
a solution containing 0.1 M NaCl, 0.01 M Tris (pH 7.6), 1 mM EDTA and
separated into DNA and 'free' RNA fractions as described below.
Three
different methods have been successful for the fractionation of the DNA
with its associated bound RNA from the RNA released by SDS treatment.
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Nucleic Acids Research
Procedure (iii) as described below and in text is the method of choice.
3
(i) Our earliest studies used sucrose gradient sedimentation to
separate high molecular weight DNA from the slower sedimenting free RNA
as discussed in text for Figure 2a.
Although this procedure is the most
rapid it is limited to small amounts of nucleic acids.
Overloaded
gradients containing high molecular weight DNA preparations were not
reproducible.
(ii)
CsCl density gradient equilibrium centrifugation allowed DNA to
band close to the top of a CsCl gradient, while free RNA remained at the
bottom, at the highest density of the gradient.
adjusted to 1.71-1.75 gms/cm
The density of CsCl was
depending on the experiment.
The CsCl
solution also contained 5 mM EDTA and 0.01 M Tris (pH 7.6). The gradient
was overlayered with liquid paraffin and centrifuged as described in
Figure 2b.
Most gradients were collected from the top so that the crystal
of precipitated CsCl and the free RNA did not disturb or contaminate the
DNA band.
The DNA was usually recentrifuged in a second CsCl gradient or
purified further by procedure (iii) in order to remove any low molecular
weight free RNA that trailed into the DNA band.
(iii)
Chromatography on agarose columns:
the preparations in a
volume of 0.5 ml were applied to a 50 x 1.5 cm column of agarose A150
(Bio Rad) equilibrated with a solution containing 0.1 M NaCl, 0.01 M Tris
(pH 7.6), 1 mM EDTA and 0.02% sodium azide.
0.05-0.15 ml per minute.
The flow rate was about
A typical separation of DNA from free RNA is
shown in Figure 3.
Alkaline hydrolysis of RNA:
distinguish
An alkaline hydrolysis assay was developed to
H labeled RNA from
H-DNA.
This was necessary because a
small fraction of the total [5- H] uridine incorporated into nucleic acids
labeled dCMP residues in DNA.
Also, if the uridine contained minor
amounts of tritium in positions other than the 5- position, dTMP residues
in DNA became labeled via the deoxynridylate pathway.
Although the amount
of DNA labeled in this manner was less than 1-2% of the total incorporated
label in the experiments described here, it became significant when DNA
was purified free of all but a small fraction of the total RNA.
Alkaline
treatments that degrade RNA to mono-nucleotides also cause substantial
3
3
amounts of the label in DNA (labeled by
exchange.
'
H "crossover"), to undergo
H
This exchange which confuses labeled DNA with RNA when
precipitation methods are used, can be avoided by employing more gentle
alkaline treatments.
In Figure 1 it is shown that in 20-30 minutes at
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Nucleic Acids Research
67°C, RNA in a low salt buffer and 0.1 N NaOH was sufficiently hydrolyzed
to become >99% trichloroacetic acid (TCA) soluble.
If the RNA was in CsCl
and treated with alkali at the same temperature, the RNA was hydrolyzed
much more rapidly.
When the alkaline CsCl RNA mixture was treated at 30°C,
the kinetics of hydrolysis was similar to the low salt RNA mixture.
Under
these conditions in which RNA became nearly 100% TCA soluble, no more than
0.5% of
H radioactivity in DNA underwent
H exchange.
In a typical assay,
aliquots were treated with l/10th volume of 1.0 N NaOH for a given time
interval and temperature as shown in Fig. 1.
Reactions were chilled and
neutralized by the addition of 1.0 N HC1 and 1.0 M Tris buffer.
Two
micrograms of salmon sperm DNA were added as carrier and the total mix was
made 5.0% w/v with TCA by the addition of 50% TCA.
After holding at 0°C
for at least 15 minutes, the samples (about 0.5 ml) were filtered through
a 13 mm Millipore filter, 0.45 p pore size and washed three times with
0.5 ml aliquots of 1% TCA.
The filtrate and washes containing the degraded
PNA were collected and radioactivity was counted in 15 ml of an aqueous
scintillator reagent.
The filter containing the DNA was washed with
ethanol, dried and also counted.
o
-a
o
<
Z
0.4
2
0.2
10
20
Time (min)
Figure 1. The rate of RNA hydrolysis in_ OA^ N NaOH. Total £. coli RNA
purified from cells which were pulse labeled with f5- H] uridine was incubated at the indicated temperatures in 0.1 N NaOH. At later times aliquots
were removed, neutralized and treated with TCA to determine the fraction'of
acid soluble RNA. Solvent contained 0.1 M NaCl, 1 mM EDTA and 10 mM Tris
incubated at 67°C, 0
0; the same solvent at 67°C plus CsCl, 1.72 gm/ml,
D
• ; the same solvent at 30°C plus CsCl, 1.72 gm/ml, •
•.
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RNA-DNA hybridization competition:
Denatured DNA adsorbed on nitrocellu-
lose filters was hybridized to RNA using methods described previously
modified by the use of 50% formamide and 53 C reaction temperatures.
The competition experiments used the "blocking" procedure previously
8 14
described '
in which the filters containing denatured DNA were first
reacted with varied amounts of non-radioactive competitor RNA, treated with
RNAase, washed and then reacted with constant amounts of labeled RNA.
After incubation for 24-48 hrs, as indicated in text, the filters were
washed and treated with RNAase (10 yg/ml for 15 min at 30 C ) , washed again,
dried and radioactivity was counted.
Blank filters lacking DNA were simi-
larly processed for background correction which in no case amounted to more
than 25 cts/min.
Hybridization efficiencies were calculated from the ratio
of the RNAase resistant radioactive material on a DNA filter to the total
radioactive material available for hybridization.
The latter figure was
determined from a trichloroacetic acid (TCA) precipitate of an equal amount
of the labeled RNA deposited on a nitrocellulose filter with 2 pg of nonp
radioactive carrier DNA.
RESULTS
3
Membrane-free nucleoids with
DNA were isolated (see Methods).
14
H-labeled RNA components and
C-labeled
The DNA was unfolded by treatment with
sodium dodecyl sulfate (SDS) and sedimented on sucrose gradients to separate
the high molecular weight DNA from the released nascent RNA chains (Fig.
2a).
Most of the RNA of the nucleoid sedimented more slowly than the DNA;
however, about 4% of the labeled RNA cosedimented with the DNA.
shown below that the
It will be
H label associated with the DNA resides predominantly
in RNA and is not due to crossover labeling into DNA.
Another aliquot of
the same preparation was centrifuged to equilibrium in a CsCl gradient
(Fig. 2b) and again a small fraction of the
More than 90% of the
H-RNA banded with the DNA.
H-RNA from the isolated nucleoid banded toward the
bottom or densest part of the CsCl gradient where free RNA is expected.
The RNA:DNA ratios at the peaks of the DNA bands, as indicated by the 3H:11+C
ratios, were 1.5 for the sucrose gradient band and 1.6 for the CsCl band.
Thus, two methods which depend on different physical chemical properties for
separating RNA from DNA yield similar DNA-RNA complexes. These separations
were done at very low DNA concentrations (initial concentration <0.5 ug/ml)
to reduce the possibility that the high molecular weight DNA physically
traps RNA.
No evidence for such trapping was found when isolated
P
pulse labeled E. coli RNA was equilibrated with the nucleoids before
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Nucleic Acids Research
unfolding the DNA and subsequent fractionation on sucrose gradients.3
Another method for preparing the DNA bound RNA which we have found
more convenient employs columns of agarose.
Larger quantities of DNA can
be processed and high molecular weight DNA is not essential for this method
of separation.
The experiment in Figure 3 depicts the separation of DNA
and its bound RNA from free RNA, as well as a similar separation of nucleic
acids obtained from the same cells after growth with rifampicin.
rifampicin results will be discussed below).
All the
(The
C-labeled DNA was
excluded on the agarose column and about 1-2% of the total pulse labeled
H-RNA eluted with the DNA.
This fraction is less than that observed in
Figure 2. Separation of free RNA from DNA which contains a_ bound RNA
fractionT (a) Bacteria" were grown for one generation time in the presence
of U'tcJ thymidine and then labeled for 1 minute with [5-3H] uridine (see
Methods). The cells were harvested and nucleoids were isolated. An aliquot
of the purified nucleoids was diluted into a solution containing 0.1 M NaCl,
0.01 M Tris (pH 7.6), 1 mM EDTA and SDS was added to a final concentration
of 0.5%; the mixture was incubated 1 hour at 3U°C and layered with a large
bore pipette onto a 5-30% sucrose gradient containing 0.1 M NaCl, 0.01 M
Tris (pH 7.6), 1 mM EDTA and 0.5% SDS. Centrifugation was for 6.5 hours at
20,000 rpm in an SW 25.3 rotor at 22°C.
(b) Another aliquot of the nucleoid preparation was diluted and
treated with SDS as above. The nucleic acids were precipitated with etha^
nol, redissolved in CsCl solution (see Methods) and the mixture was centrifuged 50 hours at 36,000 rpra in an SW 50 rotor at 22°C: 3H-RNA, 0
0;
t.
and 1 4 C-DNA, t
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Nucleic Acids Research
Figure 2, where the RNA-DNA complex was fractionated from isolated
nucleoids.
When the nucleic acids were prepared directly from cell lysates
containing the nucleoids (as in Fig. 3 ) , they include pulse labeled RNA
which was free of the nucleoid.
It is noteworthy to mention here that a
pulse of 10 seconds or less was required to insure that more than 80% of
the total pulse labeled RNA was associated with the nucleoid (see Ref. 7 ) .
Dissociation of the bound RNA by heating.
The isolated DNA bound RNA can
be stored frozen or at room temperature for days without detectable dissociation from the DNA.
When an aliquot of the complex purified on an aga-
rose column was centrifuged to equilibrium in a CsCl density gradient, more
than 95% of the RNA was associated with the DNA band (Figs. 4a £ c ) .
After
a similar aliquot of DNA and its bound RNA was heated to 96°C, all detectable RNA was released and banded free of the DNA (Fig. 4b). Thus, it
(o)
t
n
D
C
i
r
6
>
4
11
I
9
O
r»
(b)
D
16
M
i-i:
^
8
2
4
3
K
:
_l
20
f
o
40
60
Ft
Figure 3. Separation of free RNA from DNA by agarose column chromatography.
A culture of strain D-10 was incubated with~P-4cJ thymidine as in Fig. 2 and
then half of the culture was transferred to a fresh flask containing a final
concentration of 200 yg/ml of rifampicin and incubated for 3.5 minutes.
Both cultures were pulsed with 25 uC/ml [5-3H] uridine (28 C/mM) for 30
seconds before harvest. As outlined in Methods, the nucleic acids were
purified from cell lysates and then applied to agarose columns.
(a) Untreated culture, (b) Rifampicin treated culture. TCA insoluble
•; and 3H-radioactivity, 0 — 0 .
radioactivity in 11+C-DNA, •
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Nucleic Acids Research
appears that the bound RNA was not attached to DNA by a covalent linkage.
Aliquots of DNA with its bound RNA were heated at different temperatures
and then banded in CsCl density gradients as above.
of 50
Temperatures in excess
were required to dissociate RNA from the DNA (Fig. 5 ) . In a similar
solvent, E_. coli DNA does not denature until temperatures in excess of 90°C
are reached.
Above the T m of 55-6O°C a fraction of the RNAs required
higher temperatures for their release, suggesting some heterogeneity in the
In these experiments the 3 H -
association of the DNA bound RNAs with DNA.
radioactivity in both RNA and DNA was carefully monitored while taking care
to avoid
H-exchange that can occur during alkaline hydrolysis (see
Methods).
It should be noted that H-radioactivity that labeled the DNA,
14
^
co-banded with the
C-labeled DNA. By contrast, the H-radioactivity'in
the DNA bound RNA (Fig. 4a) was shifted toward a higher density by %-l
lc)22»C
f
.1 J
3
10
I]
20
Froclio
I
10
19
20
Figure 4. CsCl density gradient centrifugation of DNA and its bound RNA
before and after heating. Aliquots of a preparation of DNA and its associated RNA purified by agarose chromatography (see Fig. 3) were placed in
0.25 ml solutions containing 1.0 M NaCl, 60 ug poly U carrier, 30 ug salmon
sperm DNA carrier, 5 mM trisodium citrate and 0.02 M sodium phosphate
(pH 7.5). After a 5 minute incubation at 22°C or 96°C, the untreated (a)
and ( b ) , and the rifampicin treated (c) and (d) preparations were diluted
with CsCl solution and adjusted to a density of 1.75 gm/ml. These solutions were then centrifuged and fractions were collected as described in
Methods. For ease of presentation, the 3H-RNA counts in panel (c) have
been multiplied by five since less DNA was added in comparison to (d).
Each fraction was treated with alkali, neutralized and assayed (see Methods)
for label in DNA and RNA. 3 H-DNA, D
D ; X1|C-DNA, •
1; and 3 H-RNA,
0
0.
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Nucleic Acids Research
fractions.
detectable 3H-label was incorporated into
We note here that no
DNA from the rifampicin treated cells nor was there an observable enrichment
for any RNA covalently bound to the DNA in the manner described by Sugino
et a l . 1 6
1
i
I
I
1
1.0
TJ
M
J 0.8
-
•>
I 0.6
"5
0.4
j
roc
2
u_
0.2
i
20°
i
40°
-
1
1
60°
8 0°
100°
Temperature f °C)
Figure 5j_ The temperature dependence of DNA bound RNA dissociation.
Aliquots of the DNA and its associated bound RNA were heated at the indicated temperatures, chilled and sedimented to equilibrium in CsCl density
gradients as described in Figure 4. The fraction of dissociated RNA was
computed for each temperature.
The amount of bound RNA:
The mass ratio of RNA to DNA was determined in the
RNA-DNA complexes purified by two different procedures. Cells were labeled
32
for five generations with
P to uniformly label nucleic acids and the DNA
bound RNA was isolated both from purified nucleoids and directly from cell
lysates.
In the latter procedure deproteinization with phenol was used (see
Methods).
We routinely isolated the DNA bound RNA from cell lysates in high
ionic strength solvents but in the latter isolation the cells were lysed in
32
The amount of
P-radioactivity in DNA and
the presence of 0.1 H NaCl.
bound RNA was determined both from the differential sensitivity to alkaline
hydrolysis and to RNAase.
Both methods gave similar results (Table 1 ) .
It
should be emphasized that although the ionic conditions during lysis, the
methods of deproteinizing the DNA and the methods for fractionating RNA from
DNA were different in the two purification methods described in Table 1, the
mass ratio of bound RNA to DNA was similar, i.e. 3.4 ± 0.6 x 10
-3
Not only
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Nucleic Acids Research
was the amount of bound RNA similar when isolated from purified nucleoids
or lysates, but as will be described below hybridization analysis suggest
that the RNA species are similar. We therefore assume that either preparation method yields a similar product and the succeeding experiments
described below utilized only the bound RNA molecules isolated from
lysates.
Table 1.
Amount of DNA bound RNA after Isolation by Different Methods
32
Sour
f TWA
P-radioactivity
Ratio:
or uan.
(cts/min) in:
RNA(cts/min):DNA(cts/min)
bound RNA
DNA bound RNA
DNA
(x 10~ 3 )
Isolated nucleoid (i)
Crude lysate (i)
Crude lysate (ii)
5.6 x 10 5
1.3 x 10 s
1.3 x 10 s
1640
445
531
2.9
3.4
4.0
The amount of DNA bound RNA and DNA in isolated complexes was assayed
by measuring label which was acid soluble and insoluble, respectively,
after (i) alkaline hydrolysis as outlined in Methods or (ii) by RNAase
digestion.8 In these assays, the amount of background radioactivity
was determined from controls that lacked the RNAase or alkali treatments. Less than 7% of the radioactivity in RNA as recorded above was
deducted for backgrounds.
Proteins involved in the linkage:
Could the DNA bound RNA be attached to
DNA through residual RNA polymerase molecules or other proteins which
resist dissociation by the SDS and phenol?
It is known that ternary
complexes made in_ vitro or ^n_ vivo which contain nascent RNA, RNA polymerase and DNA, are disrupted with SDS under the conditions used
here.
'
*
To investigate the protein content of the RKA-DNA complex,
protein was labeled to high specific activities with
S and the DNA bound
RNA was isolated using the CsCl and agarose chromatography methods.
than 45 counts per minute were found associated with 11 yg of DNA.
Less
The
specific activity of the total cellular protein was determined to be
4
-5
8.0 x 10
cts/min/ug; therefore, no more than 5.1 x 10
present per ug of DNA.
ug of protein was
The mass ratio of one core polymerase molecule to
one chromosome equivalent of DNA is 16 x 10~ . Assuming that the RNA polymerase has the same specific activity as total cellular protein (the sulfur
content of this enzyme
and total E_. coli protein
is very similar),
no more than 0.30 RNA polymerase molecules per genome equivalent of DNA
remained attached to the DNA bound RNA.
In a similar experiment in which
the proteins were labeled with a mixture of
(the detection limit) of the
776
H-amino acids less than 0.7%
H-protein initially bound to the nucleoid was
Nucleic Acids Research
associated with the isolated RNA-DNA complex (Hecht and PettiJohn,
unpublished result).
Size of the RNA.
The size distribution of the DNA bound RNA molecules was
determined by sedimentation analysis.
The bound RNA, released from the
DNA by heating, was sedimented on sucrose gradients containing formalde21
22
hyde using methods described by Boedtker
and Richardson.
As a control,
total pulse-labeled RNA from cells was purified and analyzed similarly.
Each RNA sample was preheated to 65°C for 5 minutes in the presence of
formaldehyde before sedimentation.
The DNA pelleted during the centrifu-
gation (data not shown) while the heat dissociated RNA sedimented heterogeneously with a peak and an average sedimentation rate of 9S (Fig. 6b).
In formaldehyde the secondary structure of the RNA is removed and the
sedimentation rate of the 23S, 16S and 4S RNA species becomes 13.9S,
. 0.6
- 0.4
. 0.2
Fraction no.
Figure 6^ Sedimentation of purified nascent RNA and the DNA bound RNA.
DNA bound RNA and the total pulse labeled RNA were purified from the same
cell lysate after the bacteria were labeled for 30 seconds with [5-3H] uridine. Each RNA preparation was treated with formaldehyde 21 ' 22 , by incubating a 0.1 ml solution containing the labeled RNA, plus 80 yg total E_. coli
unlabeled RNA carrier, and 1.1 M formaldehyde at 65°C for 5 minutes, chilled
and then applied to 5-20% sucrose gradients containing 1.1 M formaldehyde,
0.1 M sodium phosphate (pH 7.6). Centrifugation was for 27 hours at 32,000
rpm in an SW 41 rotor at 4°C. (a) Total pulse labeled RNA. (b) Dissociated
DNA bound RNA. Marker RNA was recorded by its absorbancy at 260 nm, •
f;
3
H-RNA, 0 — 0 .
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10.9S and 3.08S respectively.
Although the total pulse-labeled RNA exhi-
bited a similar average sedimentation rate, its peak was at IIS and it sedimented with a broader profile (Fig. 6a) indicating that the DNA bound RNA
was not a random sample of the total pulse-labeled RNA.
In particular, the
DNA bound RNA lacked the low and high molecular weight RNA components which
were common in the total pulse-labeled RNA.
The 9S sedimentation rate
21
extrapolates to a weight average molecular weight of 400,000.
This means
that the number of bound RNA molecules per genome equivalent of DNA ranges
from 18-25 molecules since the total mass of RNA is 7.3 to 10 x 10
daltons
Q
per 2.5 x 10
daltons of DNA (Table 1 ) .
Recently it was shown that the num-
ber of genome equivalents of DNA per singlet and doublet nucleoids was 2.2
23
and 3.5, respectively, when cells were grown under conditions used here.
Thus, the number of DNA bound RNAs per singlet nucleoid ranges from 40 to
55 and per doublet nucleoid 60 to 88.
Stability of the bound RNA in vivo:
To determine whether or not the bound
RNA was stable in its association with the DNA, in vivo, we measured the
<
z
0 05
2. 0.04
~
0.03 .
<
0.02 .
z
3
0.01 .
o
-O
z
o
Figure 7 ^ Amounts or_ labeled DNA bound RNA after intervals of cold' chase.
A culture of D-10 growing at 14°C (generation time of 6-7 hours) was labeled
for 30 minutes with [^C] thymidine followed by a 10-80 second pulse with
[5-3H] uridine. Part of the culture was harvested immediately, while to the
remaining part, non-radioactive thymidine (final concentration 20 ug/ml)
and uridine, cytosine, guanosine and adenine (final concentration 10 ug/ml
each) were added. At the indicated times, cells from these cultures were
harvested and the DNA and its associated bound RNA were purified from cell
lysates by the CsCl gradient procedure. The fractions of total RNA in the
DNA bound RNA were determined.
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Nacleic Acids Research
fraction of total RNA associated with DNA during a pulse-chase experiment.
Cells pulse labeled with [5- H] uridine for less than 0.3% of a generation
time were chased by the addition of excess unlabeled RNA precursors for the
indicated times (Fig. 7 ) . The fraction of total RNA purified as DNA bound
RNA was about 4.5% prior to the chase in agreement with the data of
Figure 2.
During the period of chase the fraction of total labeled RNA
associated with the DNA decreased 10 fold.
If the DNA bound RNAs were
metabolically stable and were stable in their association with the DNA, the
fraction of total label in the bound RNA should have remained constant or
increased during the chase.
This result suggests that the DNA bound RNA is
transiently associated with the DNA, however, it does not distinguish
among the possibilities that it is degraded or released from the DNA
during the chase.
The following experiment demonstrated that the synthesis of the bound
RNA is sensitive to rifampicin.
[
Cells were labeled for one generation with
C] thymidine followed by an incubation with rifampicin to part of the
culture.
The rifampicin treated culture and the untreated control culture
were then pulse labeled with [5- H] uridine.
The data for this experiment
showing the separation of the total DNA and the bound RNA from the total
free RNA was already presented in Figure 3.
The relative amounts of DNA
bound RNA and total RNA were computed by normalizing the
H-radioactivity
in RNA to the 1 4 C label in DNA (Fig. 3 ) . The ratio of total labeled 3H-RNA
14
C-DNA was 134 for the untreated cells, while the analogous ratio for
to
the rifampicin treated cells was 1.48.
Since the specific activity of DNA
was similar in both cultures and neglecting pool effects from degraded RNA
in the rifampicin treated cells, rifampicin inhibited total RNA synthesis
about 99%.
After the DNA and DNA bound RNA was banded in CsCl gradients
(Fig. 4 ) , the computed ratios showed that the amount of bound RNA per unit
of DNA was also reduced 99% from the control.
It is concluded that the
synthesis of the DNA bound RNA is inhibited by rifampicin to the same
extent as total RNA synthesis.
The DNA bound RNA obtained from rifampicin treated cells appears to be
associated with the DNA in a manner similar to the complex from untreated
cells.
As shown in Figure 4 (c and d) the associated RNA was released
after brief incubation at 96°C and banded toward the bottom of the CsCl
gradient.
There was no clear evidence for RNA found in covalent linkage
with the DNA
, although a background of alkali labile
H-radioactivity
remained evenly distributed throughout the gradient in the region of the
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DNA band.
Thus, the small amount of 3H-labeled bound RNA which was made
in rifampicin treated cells seems by this criterion to be attributable to
the small fraction of potentially inhibitable RNA polymerase molecules
which randomly escape inactivation.
Hybridization analyses of the bound RNA.
To determine whether or not the
DNA bound RNA chains are unique RNA species, the RNA was analyzed by
hybridization techniques.
The presence of rRNA sequences in the bound RNA
fraction was investigated by hybridizing the isolated RNA to Proteus
mirabilis DNA in competition with purified E_. coli rRNA.
Proteus DNA has
sequences homologous to £. coli rRNA but has very little homology to other
24 25
3
E_. coli RNA species.
'
As shown in Figure 8, a fraction of the
H-
labeled isolated RNA hybridized to the Proteus DNA and was competed in its
hybridization by purified E_. coli rRNA.
At each concentration of unlabeled
competitor rRNA, the fraction of hybridized 3H-RNA competed was equal to
14the fraction of an internal standard,
C-rRNA competed. This shows that
the hybridized H-RNA is competed by the rRNA and not by some minor RNA
contaminant in the competitor rRNA preparation.
Since the
C-rRNA and the
unlabeled rRNA preparations were made by different procedures, it would be
unlikely that the relative concentration of a contaminant RNA would be
identical in both preparations.
This result demonstrates that the DNA
bound RNA species are made up in part by rRNA sequences.
The size of the
rRNA fraction can be estimated from the hybridization data of Figure 8, if
one assumes that the hybridization efficiencies of the H-labeled rRNA
14
sequences and the
C-rRNA sequences are the same. Since the latter
hybridization efficiency was 0.50, and the amount of hybridized H-RNA
was 11% of the total, the amount of rRNA in the bound RNA was about 22%.
A similar result was also obtained when the DNA bound
H-RNA was purified
from isolated nucleoids (Hecht, data not shown).
The DNA bound RNA complexes from which RNA was isolated for the
hybridization analysis were extensively purified (see legend Fig. 8 ) .
The final purification removed all detectable free RNA chains.
Therefore,
the observed rRNA sequences were derived from the RNA-DNA complex and not
from contaminating free RNA.
About M-0% by mass of the total nucleoid associated RNA is nascent rRNA
chains when nucleoids are obtained from cells grown as those in the present
Q
experiments.
The rRNA fraction of the total nucleoid associated RNA is
therefore about twice that of the DNA bound RNA; indicating that the bound
RNA is not a random sample of the total nucleoid RNA.
780
Since the DNA bound
NucPeic Acids Research
o
a
0.3
rRNA Competitor
0.6
(jig )
Figure 8^ Hybridization competition of the DNA bound RNA and rRNA. Hlabeled DNA bound RNA molecules were first purified by isopycnic centrifugation in a CsCl gradient and additionally purified by agarose column
chromatography. The bound RNA was then released and separated from the
DNA (see Methods). Identical aliquots of the isolated 3H-RNA were annealed
in separate vials with nitrocellulose filters containing 10 yg denatured
P_. mirabilis DNA which had been previously annealed with variable amounts
of purified unlabeled 16S and 23S rRNA (mass ratio 23S:16S rRNA = 2.0).
The amounts of unlabeled rRNA which had been reacted with each filter are
given in the abscissa above. Mixed with the 3H-RNA in each vial was
0.006 yg14 C-23S rRNA (4 x 1 0 4 cpm/yg) from E_. coli, added as an internal
control. Each vial contained in a volume of 0.1 ml, 250 ll*C cts/min of
rRNA and 900 3H-RNA cts/min. After incubating 27 hrs the filters were
treated with RNAase, washed and counted. The filter incubated without rRNA
competitor had 125 and 100 cts/min respectively of hybridized ^ C and 3 H
labeled RNA; all other data are normalized with respect to this zero point.
3
H-RNA, 0
0; 14 C-rRNA, t ».
RNA comprises only a small fraction (4%) of the total nucleoid RNA, the
bound rRNA sequences can comprise only a small portion of the total rRNA
chains which were associated with the nucleoid.
By criteria described
above, the bound RNA molecules are nascent RNA chains; moreover, the rRNA
sequences of the isolated membrane-free nucleoid are known to be predomi7 8
nantly if not entirely nascent or immature rRNA chains. ' Thus, it
appears that the bound rRNA molecules are nascent rRNA chains.
This inter-
pretation is also consistent with the size distribution of the isolated
bound RNA species which reveals few RNA chains as large as 23S rRNA species
(see Fig. 6 ) .
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Nucleic Acids Research
In additional hybridization experiments (Hecht, unpublished results),
the bound RNA species were annealed to denatured DNA from phage *80dlac
which has homology for lac mRNA and very limited homology for other E. coli
RNAs.
When the DNA bound
H-RNA was isolated from cells (E. coli strain
E203 grown in a glycerol medium) which had been induced for 8-galactosidase
synthesis with 1 mM isopropyl-B-thiogalactoside, O.S to 1.0% of the labeled
RNA hybridized to excess DNA.
The amount of hybridized RNA was reduced to
less than half of this level when the
cells grown without the inducer.
H-RNA was isolated from the same
The magnitude of the decrease due to
OR
repression was similar to that previously reported in studies
mRNA.
of the lac
This finding suggests that lac mRNA is also present in the DNA bound
RNA species.
Hybridization competition analyses have also been done similar to that
of Figure 8, but using as unlabeled competitor the total "cytoplasmic RNA".
This competitor RNA is composed of the remaining cellular RNA after the
nucleoid and its bound RNA species were removed from crude lysate by centrifugation.
The cytoplasmic RNA competed with at least 80% of the labeled
DNA bound RNA species (Hecht, unpublished result), suggesting that most of
the DNA bound RNA sequences are also present in the cytoplasmic fraction.
These observations taken together demonstrate that the DNA bound RNA molecules are not unique RNA species, but they are predominantly nascent RNA
chains having known functions in the cell.
DNA bound RNA sequences resistant to RNAase.
To examine the possibility
that certain sequences of the DNA bound RNAs may be attached in an RNAase
resistant state, the isolated RNA-DNA complex was treated exhaustively with
RNAase.
A small fraction of the bound RNA remained associated with the DNA
in an RNAase resistant form (Fig. 9 ) . The results of many experiments such
as that of Figure 9a have shown that 1.0 ± 0.3% by mass of the bound RNA
was resistant to degradation by RNAase.
When the complex was heated prior
to a second incubation with the RNAase, the resistant fraction became sensitive to RNAase (Fig. 9b and c ) .
In Figure 9c the small amount of remnant
H which was solubilized after alkaline treatment of fractions containing
the DNA may be derived from proton exchange of tritium radioactivity in the
DNA (see Methods).
than 15% of the
In any case this remnant solubilized
H amounted to less
H in RNAase resistant RNA seen in Figure 9b.
The agarose column chromatography used to fractionate the RNAase resistant RNA-DNA complex, also separated the RNAase from the complex.
Upon
heating the isolated complex 10 min at 96°C the resistant RNA fragment was
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Nucleic Acids Research
0.08
Figure 9_;_ RNAase resistant RNA sequences in the bound RNA. The DNA bound
RNA complex labeled (40 sec) with %-uridine was isolated on agarose
columns (see Fig. 3 ) . A) The purified complex was incubated 45 min at 30°C
with 30 pg/ml RNAase in a solution containing 0.01 M tris (pH 7.6), 0.3 M
NaCl, 1 mM EDTA and 10 pg unlabeled E_. coli DNA carrier. The mixture was
then applied to an agarose A-50 column and eluted as described in Methods.
B) Peak fractions of an RNAase resistant RNA-DNA complex such as that of A
were pooled and half the preparation was incubated again in the above buffer
salt-mixture with 50 ug/ml RNAase A for 1 hr at 30°C. The mixture was then
passed through a Sephadex G-100 column. C) The other half of the preparation was first heated for 5 min at 96°C, then chilled and incubated with
RNAase as in (B) and finally passed through a Sephadex G-100 column. To
differentiate label in RNA and DNA each fraction was treated with alkali
•;
as described in Methods. ^H-DNA, ° r DNA absorbancy in B and C, •
3
H-RNA, 0
0.
released and it could be separated from the DNA by equilibrium CsCl density
gradient centrifugation (Fig. 10). A small fraction (<10%) of the RNA
banded near the DNA at the density expected for covalently associated RNADNA complexes
; however, the amounts of this fraction were always small
and not reproducible from preparation to preparation.
The sedimentation rate of the released RNAase resistant RNA was determined in a sucrose gradient containing formaldehyde.
The dissociated DNA
pelleted while the RNAase resistant RNA exhibited a weight average sedimentation rate of 5S (Fig. 11) which represents a weight average molecular
5
21
weight of 1 x 10
daltons or about 300 bases.
The sedimentation profile
was broad, suggesting heterogeneity in the size of the RNA.
If each of the
DNA bound RNA molecules having an average length of 1200 bases (Fig. 6)
had a single attachment site containing 1% of its bases in an RNAase
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Nucleic Acids Research
resistant form, the size of the resistant fragments should be about 12
bases.
Since the RNAase resistant RNA is about 300 bases long and is com-
posed of only 1% of the total bound RNA (Fig. 9 ) , we conclude that most of
the bound RNA chains do not have attachment sites resistant to RNAase.
i
1
•
1
A
12 .(o)22° c
8 -
-
CM
1
2
K
°
i
u
u
T
i
i
'(b)96° c
I
I
I
I
<
H-RN
1
X
saaf i n
~12
8
t
i
•
\
}
z
-
oo
C
C
i
Q
4 -
K
I
/
4
-
i
t
\
V
2
6
10
20
30
F faction no.
10
14 18
Fraction no.
Figure 10_ (left). CsCl gradient centrifugation of_ RNAase resistant RNA.
DNA bound RNA was prepared and treated with RNAase as in Figure 9a.
Fractions containing DNA and the purified RNAase resistant RNA were pooled
(1.5 ml) and 100 yg each of salmon sperm DNA and poly U were added.
(A) Half this mixture was incubated at 22°C for 10 min. (B) The other half
was heated to 96°C for 10 min. Saturated CsCl solution was added to both
samples, adjusted to a final density of 1.75 gm/ctn3 and centrifuged for
42 hrs at 20°C in an SW 50.1 Beckman rotor at 35,000 rpm. Fractions were
collected from the gradients, treated with alkali and assayed for 3H-radioactivity in acid soluble RNA, 0
0; and 3H-radioactivity in acid insoluble
DNA, •
«.
Figure 11 (right). Sedimentation of the RNAase resistant RNA after release
from DNA.
The preparation of DNA and its associated RNAase resistant RNA
from Figure 9a was pooled with 70 yg of purified total E_. coli RNA. The
nucleic acids were precipitated with ethanol, collected by centrifugation,
redissolved in phosphate buffer, heated at 67°C in the presence of formaldehyde and sedimented on a sucrose gradient as in Fig. 6. The gradient was
collected from the bottom and the absorbancy at 260 my of each fraction was
recorded to localize the marker rRNAs, the positions of which are indicated
by the arrows. Each fraction was analyzed for alkaline labile RNA and
alkaline resistant DNA as before. 3H-RNA, 0
0; 3H-DNA, fl i.
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Nucleic Acids Research
There are 74 + 14 bound RNA molecules per doublet nucleoid or 18-25 per
genome equivalent of DNA.
If one out of 25 of the RNA molecules had a
tract of 300 bases bound in an RNAase form, it would account for the
observed 1% RNAase resistance.
Thus, the results imply that there are only
a few RNA molecules per genome equivalent of DNA having RNAase resistant
tracts and that most bound RNAs do not have RNAase resistant attachment
sites.
DISCUSSION
Previous studies have shown that the condensed state of folded DNA in
isolated nucleoids is stabilized by RNA molecules bound to the
1-3 27 28
nucleoid.
' '
Digestion of the nucleoid bound RNA with RNAase causes
the DNA to unfold and acquire properties more similar to that of extended,
double-helical DNA.
Also, the DNA spontaneously unfolds when one attempts
to isolate nucleoids from cells grown for a few minutes with rifampicin or
3 4 27 29
other inhibitors of RNA synthesis. ' ' '
Nucleoid bound RNA molecules
seem in addition to be involved in segregating the chromosome into separate
domains of supercoiling.
Partial hydrolysis of the nucleoid RNA reduces
the number of domains and an exhaustive hydrolysis permits a complete
27 28
'
These earlier results suggested that
relaxation of the supercoiling.
certain unknown RNA-DNA interactions in the nucleoid restrain the rotation
and extension of the DNA.
It is not clear whether all of the nascent RNAs
of the nucleoid, a selected fraction of them, or a special class of as yet
undiscovered RNA is involved in stabilizing the nucleoid.
Nor has the
chemical basis of the critical RNA-DNA interaction been established.
For
example, the possibility cannot be ruled out that the stabilization is
attributable to some fortuitous association or tangling of the nascent RNA
with the densely packaged DNA, occurring during isolation of the nucleoid.
Whatever the basis of the interaction it seems likely that each stabilizing
RNA molecule would be attached to at least two separate sites on the DNA.
The major purpose of the research described here was to determine if
there are any RNA molecules in the nucleoid bound directly to the DNA
independently of the ternary complex which normally binds nascent RNA to
the DNA.
It was demonstrated that after the ternary complexes of the
nucleoid are disrupted, about 75 RNA chains per nucleoid equivalent of DNA
remained bound to the DNA.
No remnant RNA polymerase could be detected in
this RNA-DNA association.
The detection limits were such that there can be
no more than 0.02 RNA polymerase molecules per bound RNA chain.
It also
appears that the bound RNA molecules are predominantly, if not exclusively
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Nucleic Acids Research
nascent RNA chains since:
i) the relative amount of radioactive label
incorporated into the bound RNA chains was maximal in the shortest period
of labeling.
If the synthesis of a chain 1200 bases in length had to be
completed before it was attached to the DNA, a lag in attaining maximal
labeling of the bound RNA would have been expected,
ii) the bound RNA
molecules were almost completely eliminated if cells were grown with
rifarapicin to eliminate nascent RNA.
We conclude that these nascent RNA
chains are bound directly to the DNA and do not require the ternary complex
for stability.
The hybridization analyses showed that these chains are not a unique
class of RNA.
rRNA sequences.
Rather they are composed at least in part of known mRNA and
The possibility has not been ruled out that a small '
fraction of the bound RNA molecules could be unique to the nucleoid.
At
present the hybridization experiments lack the sensitivity to resolve very
small RNA fractions existing only in the nucleoid.
The bound RNA molecules
isolated by this procedure comprise only a small portion (4%) of the total
RNA of the nucleoid.
Apparently a few nascent RNA chains are associated or
can become associated with the DNA of the nucleoid differently than the
majority of these molecules, so that they remain attached even after the
DNA is unfolded and the bound RNA polymerase molecules are removed.
Earlier studies of the transcription in_ vitro and in_ vivo of small
supercoiled viral DNAs have shown that nascent RNA chains can remain associated as a hybrid with the supercoiled DNA template after the RNA poly29 31
merase is removed by treatment with SDS and phenol. '
The site of
attachment is exclusively at the 3' end of the nascent RNA and involves
only the terminal 50 nucleotides.
Recently, Richardson
has found that at
low RNA polymerase:DNA ratios the disruption of ternary complexes can lead
to extensive hybrid formation (up to 600 bases) between a nascent RNA and
its supercoiled DNA template.
It is possible that some of tha RNA-DNA
complexes observed here are derived from a similar interaction between
certain nascent RNAs and the supercoiled nucleoid DNA.
However, there
appear to be some differences, since preliminary experiments have indicated
that these RNA chains are not bound to the DNA preferentially at their 3'
ends (Hecht, unpublished results).
A few of the bound RNA chains have
extensive sequences (ca. 300 bases) associated with the DNA in an RNAase
resistant form.
This finding is consistent with the idea that at least some
of the RNA chains are bound to the DNA via a hybrid-like structure.
Since
the DNA in these RNAase resistant complexes was extensively sheared, it is
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Nucleic Acids Research
apparent that their RNAase resistance is not dependent on maintenance of
the supercoiled state of the DNA.
It should be emphasized that there is no evidence demonstrating that
the RNA-DNA complexes observed here are involved in stabilizing the isolated nucleoid.
ACKNOWLEDGEMENTS
This research was supported by U. S. Public Health Service Grant
No. GM18243 and by U. S. National Science Foundation Grant No. 43358.
We
wish to thank Mr. A. Koop for assistance with the experiment of Figure 5.
This is contribution No. 635 from the Department of Biophysics and Genetics,
University of Colorado Medical Center.
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