MGG
Molec. gen. Genet. 152, 161-165 (1977)
© by Springer-Verlag 1977
Control of Ribosomal RNA Synthesis in Escherichia coli III. Cytoplasmic Factors for Ribosomal RNA Synthesis Akira Muto Department of Biochemistry and Biophysics, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Kasumi-cho, Hiroshima, Japan
Summary. The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the " 4 S " fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation of rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNApolymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acidstarved rel + cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.
tion have been carried out. The results indicate that the 3-5S components (the 4S fraction) in the cytoplasmic fraction contains a stimulatory activity for the frequent initiation of rRNA synthesis. Attempts have also been made to see whether or not the restriction of rRNA synthesis occurs in the nucleoids and the cytoplasmic fraction isolated from the amino acidstarved stringent (rel +) cells, the rRNA synthesis of which is restricted in vivo. No restriction has, however, been demonstrated in this system.
2. Materials and Methods a) Bacteria and Culture Conditions Escherichia coli strain 10B6 (arg-, valS ts, tel +) was used throughout this work. The culture conditions were described in the preceding paper (Muto, 1977).
1. Introduction b) Preparation of Nucleoids and Cytoplasms
In a previous paper (Muto, 1975), it has been reported that RNA synthesis occurs in a system prepared from the non-viscous lysates of Escherichia coli. The in vitro system is composed of the nucleoids (folded-chromosome) and the cytoplasmic fraction. The isolated nucleoids alone, which contain core subunits of DNA-dependent RNA-polymerase (Stonington and Pettijohn, 1971; Worcel and Burgi, 1974), are able to synthesize RNA as an elongation of RNA chains which have been initiated in vivo. The detailed analyses of this process have been described in the preceding paper (Muto, 1977). When the cytoplasmic fraction (the nucleoid-free supernatant fraction of celllysates) is added to the nucleoids, a preferential synthesis of ribosomal RNA (rRNA) due to new chain initiations takes place (Muto, 1975). In the present study, a further analysis of the preferential rRNA synthesis by the nucleoids and the cytoplasmic frac-
The nucleoids and the cytoplasmic fraction were prepared from non-viscous lysates of the exponentially growing and the amino acid-starved (for 10 rain) cells as described previously (Muto, 1975, 1977). The nucleoids were usually preincubated with unlabelled nucleoside triphosphates to complete the RNA synthesis due to the chain elongation initiated in vivo (preincubated necleoids). The preincubation mixture containing 6.0 A260 units (absorbancy at 260 rim) of the nucleoids, 50mM Tris-HCl (pH 7.6 at 30°C), 10 mM MgC12, 1 mM spermidine-sulfate, 1 mM dithiothreitol (DTT), 50 mg sucrose, and 0.5 mM each of nucloside triphosphates (ATP, GTP, UTP and CTP) was incubated at 30°C for 15 rain, and then chilled in an ice bath. To obtain the fraction having a stimulatory activity on RNA synthesis, the cytoplasmic fraction was centrifuged at 50,000 rev./ min for 2.5 h at 4°C in a No. 65 rotor of Beckman-Spinco ultracentrifuge. A 0.35 ml-portion of the resulting ribosome-free cytoplasmic fraction was loaded on 15 to 30% linear sucrose-gradient (total 4.5 ml) in 10 mM Tris-HC1 (pH 7.6) buffer containing 1 M NaC1 and 1 mM DTT, and centrifuged at 35,000 rev./min for 20 h in an SW-39L rotor at 4°C. After centrifugation, fractions (0.3 ml) were collected from bottom of the tube. A 25 gl-portion of each
162 fraction was immediately used for assay for RNA synthesis. Since the stimulatory activity of RNA synthesis was unstable, the fresh preparation was used for each experiment.
A. Muto : Control of Ribosomal RNA Synthesis in Escherichia coli
3I%
I6
2~
x
c) In vitro RNA Synthesis"
E 13
The reaction mixture (1 ml) contained 0.6 A260 unit of the preincubated or non-preincubated nucleoids, 50 mM Tris-HC1 (pH 7.6), 10 mM MgCI2, 0.1 M NaC1 and/or KC1, 1 mM spermidine-sulfate, 1 mM DTT, 0.5 mM K2HPO~, 50 mg sucrose, 0.1 mM each of nucleoside triphosphates (ATP, GTP, UTP and CTP), and 2 gCi[3H]UTP (10.5 Ci/m-mole; The Radiochemical Centre, Amersham). Appropriate amounts of the cytoplasmic fraction or its subfraction (2(~ 100 ~l/ml reaction mixture) were also added in most of the experiments. In some experiments, purified E. coli RNA-polymerase (holoenzyme) or sigma subunits, kindly provided by Dr. Ishihama of Kyoto University, was added at a final concentration of 15 gg/ml or 3 pg/ml, respectively. The reaction mixture was incubated at 30° C. The RNA synthesis was measured by counting cold trichloroacetic acid (TCA)-insoluble radioactivity as described previously (Muto, 1975). For the preparation of the in vitro synthesized [3H]RNA, a 0.5 ml-reaction mixture containing 10 gCi[3H]UTP was incubated at 30° C for 10 min. The preparation of [3H] RNA from the reaction mixture was performed as described in the preceding paper (Muto, 1977). The measurement of rRNA contents in the in vitro synthesized [3H]RNA by hybridization with bacteriophage do8Od3 DNA, and the sedimentation analyses of RNA were carried out as described (Muto, 1977).
3. Results a) RNA Synthesis in vitro As reported in the previous paper, the RNA synthesis due to new chain initiations occurs in the nucleoids system in the presence of the cytoplasmic fraction (Muto, 1975). It is, however, not clear from this experiment whether this process is promoted by some factor(s) stimulating the RNA chain initiation, or whether free RNA-polymerase in the cytoplasmic fraction is responsible for it. To distinguish between these two possibilities, the following experiments were performed. The cytoplasmic fraction prepared from lysates of the growing cells was fractionated through sucrose-gradient centrifugation, and the distribution of the stimulatory activity of RNA synthesis was measured for each fraction by incubating with the preincubated nucleoids. As shown in Figure 1, the stimulatory activity was predominantly found in the region of 3 5S (hereafter called the 4S fraction). The material in this region was clearly different from the free RNApolymerase sedimenting at about 15S, which was detectable by using bacteriophage T4 DNA as a template instead of the preincubated nucleoids. The low RNA-synthesizing activity in the RNA-polymerase region with the preincubated nucleoids may be due to the lower template activity of the nucleoids corn-
~0"-
10 15 Tube No. Fig. 1. Zonal centrifugation of the cytoplasmic fraction. A 0.35 ml of the cytoplasmic fraction from the growing cells was centrifuged through 15 to 30% sucrose-gradient (total 4.5 ml) in 10 mM TrisHC1 (pH 7.6) buffer containing 1 M NaCI and 1 mM DTT at 35,000 rev./min for 20 h at 4°C. Fractions (0.3 ml) were collected, and a 25 gl-portion of each fraction was incubated with the preincubated nucleoids in a 0.25 ml reaction mixture as described in Material and Methods ( o - - o). The RNA-polymerase activity was measured by incubating the reaction mixture containing 25 Bl-portion of each fraction and 10 gg bacteriophage T4 DNA ( © - - o )
j,"
%3
y
,,,,,""
x
~2
"1i
/"
,///
0
10
2O 30 Min Fig. 2. Time course of in vitro RNA synthesis. The reaction mixtures (0.5 mi) were incubated at 30° C. Preincubated nucleoids alone preincubated nucleoids and the 4S fraction (50 ~1 from the contents of tube No. 12, 13 and 14 in Fig. 1) ( o - - o ) ; preincubated nucleoids and sigma subunits (1.5 gg) ( (D-- o) ; preincubated nucleoids and RNA-polymerase holoenzyme (7.5 ~g) (. . . . ). A 0.1 ml-portion of each reaction mixture was taken at intervals, and cold TCA-insoluble radioactivity was measured
(o--e);
pared with T4 DNA. When the active 4S fraction was added to the preincubated nucleoids, the RNA synthesis proceeded for about 20 rain at 30° C (Fig. 2). Since the 4S fraction is free from RNA-polymerase activity, the result suggests the 4S fraction contains the activity responsible for the new RNA chain initiations. It has been demonstrated that high salt concentrations prevent the initiation of RNA synthesis (Richardson, 1966; Hinkle and Chamberlin, 1970). We then studied the effect of salt-concentration on the RNA synthesis by the nucleoid fraction alone or with the addition of the 4S fraction. While the RNA synthesis by chain elongation in the isolated nucleoids took place throughout the wide range of KC1 concentration, the new RNA synthesis supported by the 4S fraction occurred only at low concentrations of
A. Muto: Control of Ribosomal RNA Synthesis in Escherichiacoli KC1 around 0.1 M (Fig. 3). The addition of rifampicin (20 ~ / m l ) prevented the initiation of R N A synthesis in the nucleoids plus the 4S fraction (data not shown). These two observations also suggest the presence of some factor(s) stimulating the initiation of R N A synthesis in the 4S fraction. It has been known that sigma subunits are necessary for the initiation of R N A synthesis so as to recognize the site of initiation on the D N A . Therefore it would be possible that the activity in the 4S fraction is due to sigma subunits. To test this possibility, the pure sigma subunits were added to the nucleoids system to see whether the initiation of R N A synthesis was stimulated. No stimulation was, however, observed (Fig. 2), indicating that the activity in the 4S fraction was not due to sigma subunits alone even if they would be present there, and that some other factor(s) in the fraction was responsible for the initiation of R N A synthesis in this system.
b) Characterization of the RNA Synthesized in vitro The r R N A contents in the total R N A synthesized in various in vitro system (see Table 1) were determined by the specific hybridization of r R N A with transducing phage ~80d3 D N A carrying r R N A genes as described in the preceding paper (Muto, 1977). Increasing amounts of [3H] R N A synthesized in vitro were hybridized with the constant amounts of qb80d3 D N A in the presence or absence of unlabelled competitor r R N A (Fig. 4). The relative amounts of r R N A in the total R N A , calculated from the hybridization curves of Figure 4, are summarized in Table 1. The R N A synthesized by the preiucubated nucleoids and exogenous R N A - p o l y m e r a s e (holoenzyme) contained only 3% r R N A (Fig. 4(b)). A similar result was obtained with the purified E. coli D N A and RNA-polymerase. On the other hand, the preincubated nucleoids and the 4S fraction produced much higher proportion of r R N A (Fig. 4(a)). The amounts varied from 19% to 36% depending on the preparations (Table 1). It is thus clear that the 4S fraction contains, besides the activity responsible for R N A chain initiations, the activity for the frequent initiation of the r R N A gene transcription. The [ a H ] R N A made by the preincubated nucleoids and the 4S fraction was also analyzed by centrifugation through sucrose-gradient. After fractionation, the amounts of total R N A (by cold TCA-insoluble radioactivity) and r R N A (by hybridization with 4)80d3 D N A ) were measured for each fraction. As shown in Figure 5, the [ 3 H ] R N A sedimented at 23S and 16S r R N A regions (see also Muto, 1975), and most of the r R N A synthesized in this system was detected near the 16S and 23S mature r R N A regions.
163
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5
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O
O
9
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I
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15
./~ .
M KCt Fig. 3. Effect of KCI concentration on RNA synthesis in vitro. The nucleoids were incubated at 30°C for 15 rain in the presence ( © - - o ) or absence ( e - - o ) of the 4S fraction in 0.1 ml reaction mixture containing different amounts of KC1
Table 1. The relativerRNA content in [3H]RNAsynthesizedin vitro Source of
rRNA (%)
Preincubated nucleoid or DNA
4S fraction
Exp. 1
Exp. 2
Exp. 3
Growing cells Growing cells
Growing cells Starved cells
28 25
36 24
19
Starved cells Starved cells
Growing cells Starved cells
19 22
23 23
18
Growing cells\ E. coli DNA J
RNA polymerase (holoenzyme)
3 5
The nucleoids and the 4S fraction were prepared from the growing and the amino acid-starved E. oli 10B6 (rel+) cells. The RNA was labelled with [3H]UTP for 10 rain in the reaction mixture containing preincubated nucleoids and the 4S fraction of different combinations as indicated. The rRNA contents in the total [3H]RNA synthesized were determined by the hybridization of rRNA to ~80d3 DNA. For details, see the preceding paper (Muto, 1977).
Ct
q cD x
o
? T
N2
/o
R:J
T
0
e.__..~8f@
25
50 75 25 50 75 Input RNA }al Fig. 4a and b. Hybridization between [3H]RNA synthesized in vitro and ~80d3 DNA. Increasing amounts of the [3H]RNA synthesized by the preincubated nucleoids and a the 4S fraction or b exogenous RNA-polymerase holoenzyme were incubated with the constant amounts (10 gg) of ~80d3 DNA in 0.5 ml 6 x SSC containing 10 mM EDTA and 0.05% SDS at 66°C for 18 h. The hybridization assays were performed in the presence (o .... o) or absence ( o - - o ) of unlabelled competitor rRNA (50 gg). The radioactivities of the [3H]RNA samples were respectivelya 20,000 and b 30,000 cts/ min/ml. The RNA concentrations were a 5 and b 0.5 ~/ml
164
A. Muto : Control of Ribosomal RNA Synthesis in Escherichia coli
23S 16S
L
%
x
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8
6 < Z rY
Molec. gen. Genet. 152, 161-165 (1977)
© by Springer-Verlag 1977
Control of Ribosomal RNA Synthesis in Escherichia coli III. Cytoplasmic Factors for Ribosomal RNA Synthesis Akira Muto Department of Biochemistry and Biophysics, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Kasumi-cho, Hiroshima, Japan
Summary. The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the " 4 S " fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation of rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNApolymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acidstarved rel + cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.
tion have been carried out. The results indicate that the 3-5S components (the 4S fraction) in the cytoplasmic fraction contains a stimulatory activity for the frequent initiation of rRNA synthesis. Attempts have also been made to see whether or not the restriction of rRNA synthesis occurs in the nucleoids and the cytoplasmic fraction isolated from the amino acidstarved stringent (rel +) cells, the rRNA synthesis of which is restricted in vivo. No restriction has, however, been demonstrated in this system.
2. Materials and Methods a) Bacteria and Culture Conditions Escherichia coli strain 10B6 (arg-, valS ts, tel +) was used throughout this work. The culture conditions were described in the preceding paper (Muto, 1977).
1. Introduction b) Preparation of Nucleoids and Cytoplasms
In a previous paper (Muto, 1975), it has been reported that RNA synthesis occurs in a system prepared from the non-viscous lysates of Escherichia coli. The in vitro system is composed of the nucleoids (folded-chromosome) and the cytoplasmic fraction. The isolated nucleoids alone, which contain core subunits of DNA-dependent RNA-polymerase (Stonington and Pettijohn, 1971; Worcel and Burgi, 1974), are able to synthesize RNA as an elongation of RNA chains which have been initiated in vivo. The detailed analyses of this process have been described in the preceding paper (Muto, 1977). When the cytoplasmic fraction (the nucleoid-free supernatant fraction of celllysates) is added to the nucleoids, a preferential synthesis of ribosomal RNA (rRNA) due to new chain initiations takes place (Muto, 1975). In the present study, a further analysis of the preferential rRNA synthesis by the nucleoids and the cytoplasmic frac-
The nucleoids and the cytoplasmic fraction were prepared from non-viscous lysates of the exponentially growing and the amino acid-starved (for 10 rain) cells as described previously (Muto, 1975, 1977). The nucleoids were usually preincubated with unlabelled nucleoside triphosphates to complete the RNA synthesis due to the chain elongation initiated in vivo (preincubated necleoids). The preincubation mixture containing 6.0 A260 units (absorbancy at 260 rim) of the nucleoids, 50mM Tris-HCl (pH 7.6 at 30°C), 10 mM MgC12, 1 mM spermidine-sulfate, 1 mM dithiothreitol (DTT), 50 mg sucrose, and 0.5 mM each of nucloside triphosphates (ATP, GTP, UTP and CTP) was incubated at 30°C for 15 rain, and then chilled in an ice bath. To obtain the fraction having a stimulatory activity on RNA synthesis, the cytoplasmic fraction was centrifuged at 50,000 rev./ min for 2.5 h at 4°C in a No. 65 rotor of Beckman-Spinco ultracentrifuge. A 0.35 ml-portion of the resulting ribosome-free cytoplasmic fraction was loaded on 15 to 30% linear sucrose-gradient (total 4.5 ml) in 10 mM Tris-HC1 (pH 7.6) buffer containing 1 M NaC1 and 1 mM DTT, and centrifuged at 35,000 rev./min for 20 h in an SW-39L rotor at 4°C. After centrifugation, fractions (0.3 ml) were collected from bottom of the tube. A 25 gl-portion of each
162 fraction was immediately used for assay for RNA synthesis. Since the stimulatory activity of RNA synthesis was unstable, the fresh preparation was used for each experiment.
A. Muto : Control of Ribosomal RNA Synthesis in Escherichia coli
3I%
I6
2~
x
c) In vitro RNA Synthesis"
E 13
The reaction mixture (1 ml) contained 0.6 A260 unit of the preincubated or non-preincubated nucleoids, 50 mM Tris-HC1 (pH 7.6), 10 mM MgCI2, 0.1 M NaC1 and/or KC1, 1 mM spermidine-sulfate, 1 mM DTT, 0.5 mM K2HPO~, 50 mg sucrose, 0.1 mM each of nucleoside triphosphates (ATP, GTP, UTP and CTP), and 2 gCi[3H]UTP (10.5 Ci/m-mole; The Radiochemical Centre, Amersham). Appropriate amounts of the cytoplasmic fraction or its subfraction (2(~ 100 ~l/ml reaction mixture) were also added in most of the experiments. In some experiments, purified E. coli RNA-polymerase (holoenzyme) or sigma subunits, kindly provided by Dr. Ishihama of Kyoto University, was added at a final concentration of 15 gg/ml or 3 pg/ml, respectively. The reaction mixture was incubated at 30° C. The RNA synthesis was measured by counting cold trichloroacetic acid (TCA)-insoluble radioactivity as described previously (Muto, 1975). For the preparation of the in vitro synthesized [3H]RNA, a 0.5 ml-reaction mixture containing 10 gCi[3H]UTP was incubated at 30° C for 10 min. The preparation of [3H] RNA from the reaction mixture was performed as described in the preceding paper (Muto, 1977). The measurement of rRNA contents in the in vitro synthesized [3H]RNA by hybridization with bacteriophage do8Od3 DNA, and the sedimentation analyses of RNA were carried out as described (Muto, 1977).
3. Results a) RNA Synthesis in vitro As reported in the previous paper, the RNA synthesis due to new chain initiations occurs in the nucleoids system in the presence of the cytoplasmic fraction (Muto, 1975). It is, however, not clear from this experiment whether this process is promoted by some factor(s) stimulating the RNA chain initiation, or whether free RNA-polymerase in the cytoplasmic fraction is responsible for it. To distinguish between these two possibilities, the following experiments were performed. The cytoplasmic fraction prepared from lysates of the growing cells was fractionated through sucrose-gradient centrifugation, and the distribution of the stimulatory activity of RNA synthesis was measured for each fraction by incubating with the preincubated nucleoids. As shown in Figure 1, the stimulatory activity was predominantly found in the region of 3 5S (hereafter called the 4S fraction). The material in this region was clearly different from the free RNApolymerase sedimenting at about 15S, which was detectable by using bacteriophage T4 DNA as a template instead of the preincubated nucleoids. The low RNA-synthesizing activity in the RNA-polymerase region with the preincubated nucleoids may be due to the lower template activity of the nucleoids corn-
~0"-
10 15 Tube No. Fig. 1. Zonal centrifugation of the cytoplasmic fraction. A 0.35 ml of the cytoplasmic fraction from the growing cells was centrifuged through 15 to 30% sucrose-gradient (total 4.5 ml) in 10 mM TrisHC1 (pH 7.6) buffer containing 1 M NaCI and 1 mM DTT at 35,000 rev./min for 20 h at 4°C. Fractions (0.3 ml) were collected, and a 25 gl-portion of each fraction was incubated with the preincubated nucleoids in a 0.25 ml reaction mixture as described in Material and Methods ( o - - o). The RNA-polymerase activity was measured by incubating the reaction mixture containing 25 Bl-portion of each fraction and 10 gg bacteriophage T4 DNA ( © - - o )
j,"
%3
y
,,,,,""
x
~2
"1i
/"
,///
0
10
2O 30 Min Fig. 2. Time course of in vitro RNA synthesis. The reaction mixtures (0.5 mi) were incubated at 30° C. Preincubated nucleoids alone preincubated nucleoids and the 4S fraction (50 ~1 from the contents of tube No. 12, 13 and 14 in Fig. 1) ( o - - o ) ; preincubated nucleoids and sigma subunits (1.5 gg) ( (D-- o) ; preincubated nucleoids and RNA-polymerase holoenzyme (7.5 ~g) (. . . . ). A 0.1 ml-portion of each reaction mixture was taken at intervals, and cold TCA-insoluble radioactivity was measured
(o--e);
pared with T4 DNA. When the active 4S fraction was added to the preincubated nucleoids, the RNA synthesis proceeded for about 20 rain at 30° C (Fig. 2). Since the 4S fraction is free from RNA-polymerase activity, the result suggests the 4S fraction contains the activity responsible for the new RNA chain initiations. It has been demonstrated that high salt concentrations prevent the initiation of RNA synthesis (Richardson, 1966; Hinkle and Chamberlin, 1970). We then studied the effect of salt-concentration on the RNA synthesis by the nucleoid fraction alone or with the addition of the 4S fraction. While the RNA synthesis by chain elongation in the isolated nucleoids took place throughout the wide range of KC1 concentration, the new RNA synthesis supported by the 4S fraction occurred only at low concentrations of
A. Muto: Control of Ribosomal RNA Synthesis in Escherichiacoli KC1 around 0.1 M (Fig. 3). The addition of rifampicin (20 ~ / m l ) prevented the initiation of R N A synthesis in the nucleoids plus the 4S fraction (data not shown). These two observations also suggest the presence of some factor(s) stimulating the initiation of R N A synthesis in the 4S fraction. It has been known that sigma subunits are necessary for the initiation of R N A synthesis so as to recognize the site of initiation on the D N A . Therefore it would be possible that the activity in the 4S fraction is due to sigma subunits. To test this possibility, the pure sigma subunits were added to the nucleoids system to see whether the initiation of R N A synthesis was stimulated. No stimulation was, however, observed (Fig. 2), indicating that the activity in the 4S fraction was not due to sigma subunits alone even if they would be present there, and that some other factor(s) in the fraction was responsible for the initiation of R N A synthesis in this system.
b) Characterization of the RNA Synthesized in vitro The r R N A contents in the total R N A synthesized in various in vitro system (see Table 1) were determined by the specific hybridization of r R N A with transducing phage ~80d3 D N A carrying r R N A genes as described in the preceding paper (Muto, 1977). Increasing amounts of [3H] R N A synthesized in vitro were hybridized with the constant amounts of qb80d3 D N A in the presence or absence of unlabelled competitor r R N A (Fig. 4). The relative amounts of r R N A in the total R N A , calculated from the hybridization curves of Figure 4, are summarized in Table 1. The R N A synthesized by the preiucubated nucleoids and exogenous R N A - p o l y m e r a s e (holoenzyme) contained only 3% r R N A (Fig. 4(b)). A similar result was obtained with the purified E. coli D N A and RNA-polymerase. On the other hand, the preincubated nucleoids and the 4S fraction produced much higher proportion of r R N A (Fig. 4(a)). The amounts varied from 19% to 36% depending on the preparations (Table 1). It is thus clear that the 4S fraction contains, besides the activity responsible for R N A chain initiations, the activity for the frequent initiation of the r R N A gene transcription. The [ a H ] R N A made by the preincubated nucleoids and the 4S fraction was also analyzed by centrifugation through sucrose-gradient. After fractionation, the amounts of total R N A (by cold TCA-insoluble radioactivity) and r R N A (by hybridization with 4)80d3 D N A ) were measured for each fraction. As shown in Figure 5, the [ 3 H ] R N A sedimented at 23S and 16S r R N A regions (see also Muto, 1975), and most of the r R N A synthesized in this system was detected near the 16S and 23S mature r R N A regions.
163
2O ? o
x15 E G1. "T"
5
O
O
O
9
O_ 0
I
I
I
II0
I
i1 .2 .3
•
15
./~ .
M KCt Fig. 3. Effect of KCI concentration on RNA synthesis in vitro. The nucleoids were incubated at 30°C for 15 rain in the presence ( © - - o ) or absence ( e - - o ) of the 4S fraction in 0.1 ml reaction mixture containing different amounts of KC1
Table 1. The relativerRNA content in [3H]RNAsynthesizedin vitro Source of
rRNA (%)
Preincubated nucleoid or DNA
4S fraction
Exp. 1
Exp. 2
Exp. 3
Growing cells Growing cells
Growing cells Starved cells
28 25
36 24
19
Starved cells Starved cells
Growing cells Starved cells
19 22
23 23
18
Growing cells\ E. coli DNA J
RNA polymerase (holoenzyme)
3 5
The nucleoids and the 4S fraction were prepared from the growing and the amino acid-starved E. oli 10B6 (rel+) cells. The RNA was labelled with [3H]UTP for 10 rain in the reaction mixture containing preincubated nucleoids and the 4S fraction of different combinations as indicated. The rRNA contents in the total [3H]RNA synthesized were determined by the hybridization of rRNA to ~80d3 DNA. For details, see the preceding paper (Muto, 1977).
Ct
q cD x
o
? T
N2
/o
R:J
T
0
e.__..~8f@
25
50 75 25 50 75 Input RNA }al Fig. 4a and b. Hybridization between [3H]RNA synthesized in vitro and ~80d3 DNA. Increasing amounts of the [3H]RNA synthesized by the preincubated nucleoids and a the 4S fraction or b exogenous RNA-polymerase holoenzyme were incubated with the constant amounts (10 gg) of ~80d3 DNA in 0.5 ml 6 x SSC containing 10 mM EDTA and 0.05% SDS at 66°C for 18 h. The hybridization assays were performed in the presence (o .... o) or absence ( o - - o ) of unlabelled competitor rRNA (50 gg). The radioactivities of the [3H]RNA samples were respectivelya 20,000 and b 30,000 cts/ min/ml. The RNA concentrations were a 5 and b 0.5 ~/ml
164
A. Muto : Control of Ribosomal RNA Synthesis in Escherichia coli
23S 16S
L
%
x
IO x E
E8
8
6 < Z rY
