ARCHIVES
OF
BIOCHEMISTRY
Stimulatory
AND
BIOPHYSICS
Biology
123-130
(1977)
Effect of a Factor Extracted from Mouse Myeloma on Preinitiation Process of RNA Synthesis SETSUNOSUKE
Molecular
183,
Laboratory,
IHARA
School
of Medicine, Kanagawa, Received
MASAYA
AND
Kitasato Japan
September
Nuclei
KAWAKAMI
University,
I, Asamizodai,
Sagamihara,
3, 1976
A flow-through fraction (fraction DE-O) obtained by diethylaminoethyl-cellulose chromatography of an extract from nuclei of mouse myeloma cells, MOPC 31C, was found to stimulate the activity of a homologous RNA polymerase (enzyme II) when native DNA was used as template. A remarkable enhancement of RNA synthesis resulted from preincubation of enzyme II and DNA with the fraction DE-O at 37” followed by incubation with substrate ribonucleotides and rifamycin AFI013. The effect of the preincubation was time- and temperature-dependent. The rate of RNA synthesis was reduced greatly if any one of the enzyme, DNA, and the fraction was omitted from the reaction mixture during preincubation. RNA synthesis was prevented completely by adding 0.3 M ammonium sulfate at any time during preincubation even if fraction DE-O was present. These results indicate that a factor in fraction DE-O participates in a step(s) of the preinitiation process in transcription by enzyme II.
There have been many reports indicating the presence of factors capable of stimulating eucaryotic RNA polymerase activity (l-9). These factors result in an increase in size or number of product RNA molecules in cell-free transcription, suggesting that they have an effect upon elongation rate (1, 5) or initiation frequency (4, 8, 9) of RNA synthesis. However, no evidence has been presented to answer the question of which step of transcription the factor acts upon directly. It is of interest to investigate the direct influence of the factor on individual steps in the transcription process. Recent extensive studies on eucaryotic transcription mechanisms (10, 11) have shed light on a process, called preinitiation, which involves a series of steps preceding the initiation step’ of transcription. It has been known that a rifamycin derivative, AF/013, blocks certain steps in the preinitiation process in which form II RNA polymerase participates, and that a DNA-enzyme complex formed at 37°C be’ The term initiation step is used in this paper to mean two reactions occurring successively, initiation complex formation and first phosphodiester bond formation. Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
fore addition of AF/013 is no longer dissociated by AF/013 (10). We applied such characteristics of AFl013 to the assay of the influence of stimulation factor on reactions in the preinitiation process. When preincubations of enzyme and DNA under different conditions, for example, in the presence and absence of the factor, are followed by incubations of these mixtures with the substrates and AFl013 under constant conditions, the difference in the amount of RNA synthesized should represent the difference in the rate of overall reaction in the preinitiation process, since nothing but preinitiation reactions take place during preincubation, while only chain growth proceeds during incubation. Using this method, we present evidence that there is a factor in mouse myeloma cells which enhances cell-free transcription by form II RNA polymerase of the same origin during a step(s) in the preinitiation process. MATERIALS
AND
METHODS
Biochemicals, buffers, and cells. chemicals were used in the present beled ribonucleoside triphosphates 123
The following studies: unla(Miles, Kan-
ISSN
0003-9861
124
IHARA
AND
kakee); 13HlUTP (New England Nuclear, Boston); calf thymus DNA (Sigma, Type I); cY-amanitin (Boehringer, Mannheim). Rifamycin AF/013, a gift from Dr. L. G. Silvestri (Gruppo Lepetit, Milano), was dissolved in dimethyl formamide giving a concentration of 12 mg/ml. An appropriate volume of this solution, or the same volume of dimethyl formamide for the control assay, was added to the reaction mixture for RNA synthesis. Buffer I contained 10 mM Tris-HCl (pH 7.9, 4”C), 5 mM MgCI,, 0.05 mg/ ml of phenylmethylsulfonyl fluoride (PMSF)*, and 0.5 mM dithiothreitol. Buffer II contained 20% (v/v) glycerol, 50 mM Tris-HCl (pH 7.9, 4”C), 1 rnM EDTA, 0.05 mg/ml of PMSF, and 0.5 mM dithiothreitol. The buffer with the double concentration (2x buffer II) was stored and diluted before use. In all cases, dithiothreitol was added immediately before use. Mouse myeloma, MOPC 31C (producing IgG,), was maintained in Balb/c mice by serial transfers. The excised tumors were stored frozen in liquid nitrogen until use. Isolation ofnuclei. All procedures described below were performed between 0 and 4°C unless specified. Ten grams each of thawed tissues was homogenized in 30 ml of 0.25 M sucrose in buffer I using a Teflonglass homogenizer (Dounce type, 15 strokes). The homogenate was filtered through two layers of cheesecloth and centrifuged for 10 min at 2100 rpm. Pooled pellet prepared from 30 g of tissue was resuspended by homogenization (live strokes) in 60 ml of 0.25 M sucrose in buffer I containing 0.1% Triton X100, filtered and centrifuged as above. The pellet was resuspended in 45 ml of the above medium and mixed with 90 ml of 2.2 M sucrose in buffer I. This suspension (45 ml each) was layered onto a lo-ml bed of 2.2 M sucrose in buffer I and centrifuged in an Hitachi RPS-25-2 rotor for 1 h at 22,000 rpm. Gelatinous pellets thus obtained were washed with a small volume of 0.34 M sucrose and used as the nuclear fraction. Extraction of RNA polymerases and stimulation factor. RNA polymerases were solubilized from the nuclear fraction by procedures modified from those of Schwartz et al. (12). The nuclear pellet prepared from 30 g of tissue was suspended in 15 ml of 0.34 M sucrose and mixed with equal volume of 2x buffer II. Ammonium sulfate (4 M, pH 7.6) was added into the suspension to give a final concentration of 0.3 M. Ten milliliters each of the viscous nuclear lysate was sonicated with a Tomy model UR-15OP sonitier (maximum power setting, for a total period of 5 min at intervals). The sheared lysate was centrifuged for 1 h at 50,000 rpm in an Hitachi RP-65 rotor. The supernatant (fraction Sl) was dialyzed overnight against 6.4 vol of buffer II, so that the ammonium 2 Abbreviation used: PMSF, phenylmethylsulfonyl fluoride; DEAE, diethylaminoethyl.
KAWAKAMI sulfate concentration of the dialysate fell to 0.04 M. The sedimentable portion of the dialysate was removed by centrifugation as above. The supernatant (fraction S2) can be stored frozen at -70°C without loss of enzyme activity. Fraction 52 was chromatographed on a DEAEcellulose (Whatman DE-52) column, as shown in Fig. 1. The flow-through fraction (fraction DE-O), which had no RNA polymerase activity but exerted significant stimulatory effect on enzyme II, was stored at -7O”C, at which temperature the stimulatory activity was completely maintained at least for 1 year. Preparation of enzymes I and II. Two forms of RNA polymerase, enzymes I and II, were considerably purified from fractions DE-I and DE-II, respectively (see legend to Fig. 1). A detailed description of the purification procedures will be present elsewhere (S. Ihara and M. Kawakami, manuscript in preparation). In brief, enzyme I was further purified by carboxymethyl-Sephadex chromatography and phosphocellulose chromatography, and enzyme II by DEAE-cellulose rechromatography and two cycles of phosphocellulose chromatography. Both enzymes were dialyzed against 0.04 M ammonium sulfate in buffer II before assay. Enzyme activity was determined by the following procedures (Assay I). Incubation was performed at 37°C for 20 min in 60 ~1 of assay mixture containing
FIG. 1. DEAE-cellulose chromatography of fraction S2. Fraction S2 was applied to a DEAE-cellulose column (Whatman DE-52, 100 ml of bed/1000 mg of protein) equilibrated with 0.04 M ammonium sulfate in buffer II. After a wash with the same solution, adsorbed materials were eluted with a linear gradient of 0.04 to 0.4 M ammonium sulfate in buffer II. RNA polymerase activity in 15 ml each of fractions thus eluted was measured according to Assay I described under Materials and Methods. The reaction mixture contained (0- - -0) or did not contain (0-O) 10m6 M or-amanitan. Pairs of vertical lines 0, I, and II indicate the ranges of pooled fractions DE-O, DE-I, and DE-II, respectively. Concentrations of protein (-) and ammonium sulfate (- - - - -1 are shown.
FACTOR
FROM
MYELOMA
NUCLEI
2.4 pmol of Tris-HCl (pH 7.9, 4”C), 2.4 pmol of ammonium sulfate, 0.096 pmol of MnCl,, 0.024 pmol of EDTA, 0.24 pmol of dithiothreitol, 0.024 pmol each of ATP, GTP, and CTP, 0.0024 Fmol of 13H]UTP (260 mCi/mmol), 2.5 pg of heat-denatured calf thymus DNA, 20% (v/v) glycerol, and 10 ~1 of enzyme solution. After the incubation, an aliquot of the mixture was removed and applied to a Whatman GF/C disc. The disc was immediately placed in icecold 5% trichloroacetic acid in 0.01 M sodium pyrophosphate, and rinsed three times with ice-cold 2% trichloroacetic acid in 0.01 M sodium pyrophosphate and twice with ethanol. The radioactivity of the dried disc was counted in 5 ml of toluene-based scintillation fluid. One unit of activity represents incorporation of 1 nmol of UMP under these conditions. Specific activities (units per milligram of protein) of our enzyme I and II preparations are 10 and 320, respectively. Assay of stimulation factor (Assay II). Forty microliters of preincubation mixture contained 2 pmol of Tris-HCl (pH 7.9, 4”C), 1.6 pmol of ammonium sulfate, 0.08 pmol of MnCl,, 0.02 pmol of EDTA, 0.2 pmol of dithiothreitol, 1 pg of PMSF, 20% (v/v) glycerol, 10 pg of bovine serum albumin, 5 bg of native calf thymus DNA, and appropriate amounts of enzyme and factor, i.e., unless otherwise noted, 0.014 unit of enzyme I or 0.085 unit of enzyme II and 5 ~1 of fraction DE-O (4.25 pg of protein). After various periods of preincubation at 37 or o”C, incubation at 37°C for RNA synthesis was started by adding 20 ~1 of the solution containing 1 pmol of Tris-HCl (pH 7.9, 4”C), 0.8 pmol of ammonium sulfate, 0.04 pmol of MnCl,, 0.01 pmol of EDTA, 0.1 prnol of dithiothreitol, 20% (v/v) glycerol, 0.012 pmol each of ATP, GTP, and CTP, 0.0012 nmol of ?HlUTP (250 mCi/mmol), and 6 pg of rifamycin AFI 013 which gave a final concentration of 100 pg/ml. After a given period of the incubation, the samples were processed and the radioactivities were counted as described for Assay I. All data presented here are mean values in duplicate assays, corrected by subtracting a mean value in the control assay omitting the incubation. Other procedures. Escherichia coli RNA polymerase (holoenzyme) was purified from E. coli K12 by the method of Burgess (13), and the fraction obtained by the second glycerol sedimentation was used. Salt concentrations of dialysates and chromatographic eluates were determined by conductivity measurement using buffers with known salt concentration as standards. Protein was determined by the method of Lowry etal. (14). In order to remove interfering substances, the sample solution was mixed with trichloroacetic acid to a final concentration of 10% and left for 30 min at 0°C; the resulting precipitate was collected by centrifugation, washed with ice-cold 10% trichloroa-
STIMULATES
RNA
cetic acid and ice-cold orimetry.
125
SYNTHESIS acetone,
and subjected
to col-
RESULTS
Inhibition of RNA Polymerase Activity Rifamycin AFl013
by
We have confirmed in a preliminary experiment that AF/013 inhibits completely the activity of myeloma RNA polymerases, both of enzymes I and II, at concentrations above 50 pglml as long as the drug, DNA, and enzyme are added simultaneously to the reaction mixture for RNA synthesis. In order to investigate the effect of the presence of AFt013 on a course of RNA synthesis before or after a supposed formation of DNA-enzyme complex, AFI 013 was added to the reaction mixture giving a final concentration of 100 pg/ml at the beginning of preincubation of enzyme and DNA or just before the subsequent incubation of the enzyme and DNA with substrate ribonucleotides (Fig. 2). Evidently enzyme I is prevented by AF/013 from RNA synthesis even when it is prein-
[Ls
2 0 0
10 INCUBATION
20 TIME
(ml”)
FIG. 2. Inhibition of RNA polymerase activity by rifamycin AF/013. Enzyme I (a) or enzyme II (b) was preincubated with DNA for 10 min; then substrates (ATP, GTP, CTP, and 13HlUTP) were added and the incubation was performed at 37°C for the indicated period. AF/013 was added to the reaction mixture at the beginning of preincubation at 37°C (x1, at the beginning of incubation after preincubation at 37°C (U-0) or 0°C (a---a), or not added throughout preincubation at 37°C and incubation (0-O).
126
IHARA
AND
KAWAKAMI
cubated with DNA in the absence of AF/ 013 (Fig. 2a). In contrast, Fig. 2b shows that, when enzyme II is preincubated with DNA at 37°C in the absence of AF/013, RNA synthesis takes place even in the presence of AF/013 after the addition of substrates, and that RNA synthesis is depressed by AF/013 when preincubation is done at 0°C or in the presence of AF/013. These results are consistent with previous findings that AF/013 blocks the reaction of form II RNA polymerase at a step(s) preceding initiation but not at the initiation or elongation steps in transcription (10, 15). Additionally, our results support a recent concept that there exists a certain temperature-dependent step in the preinitiation process of transcription by form II but not by form I enzyme (10). Stimulation Fraction
of Activity
of Enzyme
II
hibitor effect on RNA synthesis. As described below, we have explored the presence of a factor capable of stimulating the preinitiation process of RNA synthesis by enzyme II, using an assay procedure (Assay II, see Materials and Methods) composed of a preincubation of the enzyme and DNA, incubation with ribonucleotides, and determination of UMP incorporation. A suitable amount of rifamycin AF/013 was added to the incubation mixture to prevent progression of the preinitiation process during the incubation. Figure 3 shows the effect of different
by
DE-O
The influence of a flow-through fraction (DE-O) of a DEAE-cellulose column on various RNA polymerase activities is shown in Table I. Fraction DE-O seemsto exert stimulatory effect on RNA synthesis, mainly when enzyme II is assayed using native DNA as template. This is in agreement with several recent reports on factors obtained from different eucaryotic sources (l-4, 16-18). The change in UMP incorporation thus observed, however, corresponds to the summation of overall influences of individual factors present in fraction DE-O, which have stimulatory or in-
0
Enzyme*
OF FRACTION
Template
DNA
OF
BIOCHEMISTRY
Stimulatory
AND
BIOPHYSICS
Biology
123-130
(1977)
Effect of a Factor Extracted from Mouse Myeloma on Preinitiation Process of RNA Synthesis SETSUNOSUKE
Molecular
183,
Laboratory,
IHARA
School
of Medicine, Kanagawa, Received
MASAYA
AND
Kitasato Japan
September
Nuclei
KAWAKAMI
University,
I, Asamizodai,
Sagamihara,
3, 1976
A flow-through fraction (fraction DE-O) obtained by diethylaminoethyl-cellulose chromatography of an extract from nuclei of mouse myeloma cells, MOPC 31C, was found to stimulate the activity of a homologous RNA polymerase (enzyme II) when native DNA was used as template. A remarkable enhancement of RNA synthesis resulted from preincubation of enzyme II and DNA with the fraction DE-O at 37” followed by incubation with substrate ribonucleotides and rifamycin AFI013. The effect of the preincubation was time- and temperature-dependent. The rate of RNA synthesis was reduced greatly if any one of the enzyme, DNA, and the fraction was omitted from the reaction mixture during preincubation. RNA synthesis was prevented completely by adding 0.3 M ammonium sulfate at any time during preincubation even if fraction DE-O was present. These results indicate that a factor in fraction DE-O participates in a step(s) of the preinitiation process in transcription by enzyme II.
There have been many reports indicating the presence of factors capable of stimulating eucaryotic RNA polymerase activity (l-9). These factors result in an increase in size or number of product RNA molecules in cell-free transcription, suggesting that they have an effect upon elongation rate (1, 5) or initiation frequency (4, 8, 9) of RNA synthesis. However, no evidence has been presented to answer the question of which step of transcription the factor acts upon directly. It is of interest to investigate the direct influence of the factor on individual steps in the transcription process. Recent extensive studies on eucaryotic transcription mechanisms (10, 11) have shed light on a process, called preinitiation, which involves a series of steps preceding the initiation step’ of transcription. It has been known that a rifamycin derivative, AF/013, blocks certain steps in the preinitiation process in which form II RNA polymerase participates, and that a DNA-enzyme complex formed at 37°C be’ The term initiation step is used in this paper to mean two reactions occurring successively, initiation complex formation and first phosphodiester bond formation. Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
fore addition of AF/013 is no longer dissociated by AF/013 (10). We applied such characteristics of AFl013 to the assay of the influence of stimulation factor on reactions in the preinitiation process. When preincubations of enzyme and DNA under different conditions, for example, in the presence and absence of the factor, are followed by incubations of these mixtures with the substrates and AFl013 under constant conditions, the difference in the amount of RNA synthesized should represent the difference in the rate of overall reaction in the preinitiation process, since nothing but preinitiation reactions take place during preincubation, while only chain growth proceeds during incubation. Using this method, we present evidence that there is a factor in mouse myeloma cells which enhances cell-free transcription by form II RNA polymerase of the same origin during a step(s) in the preinitiation process. MATERIALS
AND
METHODS
Biochemicals, buffers, and cells. chemicals were used in the present beled ribonucleoside triphosphates 123
The following studies: unla(Miles, Kan-
ISSN
0003-9861
124
IHARA
AND
kakee); 13HlUTP (New England Nuclear, Boston); calf thymus DNA (Sigma, Type I); cY-amanitin (Boehringer, Mannheim). Rifamycin AF/013, a gift from Dr. L. G. Silvestri (Gruppo Lepetit, Milano), was dissolved in dimethyl formamide giving a concentration of 12 mg/ml. An appropriate volume of this solution, or the same volume of dimethyl formamide for the control assay, was added to the reaction mixture for RNA synthesis. Buffer I contained 10 mM Tris-HCl (pH 7.9, 4”C), 5 mM MgCI,, 0.05 mg/ ml of phenylmethylsulfonyl fluoride (PMSF)*, and 0.5 mM dithiothreitol. Buffer II contained 20% (v/v) glycerol, 50 mM Tris-HCl (pH 7.9, 4”C), 1 rnM EDTA, 0.05 mg/ml of PMSF, and 0.5 mM dithiothreitol. The buffer with the double concentration (2x buffer II) was stored and diluted before use. In all cases, dithiothreitol was added immediately before use. Mouse myeloma, MOPC 31C (producing IgG,), was maintained in Balb/c mice by serial transfers. The excised tumors were stored frozen in liquid nitrogen until use. Isolation ofnuclei. All procedures described below were performed between 0 and 4°C unless specified. Ten grams each of thawed tissues was homogenized in 30 ml of 0.25 M sucrose in buffer I using a Teflonglass homogenizer (Dounce type, 15 strokes). The homogenate was filtered through two layers of cheesecloth and centrifuged for 10 min at 2100 rpm. Pooled pellet prepared from 30 g of tissue was resuspended by homogenization (live strokes) in 60 ml of 0.25 M sucrose in buffer I containing 0.1% Triton X100, filtered and centrifuged as above. The pellet was resuspended in 45 ml of the above medium and mixed with 90 ml of 2.2 M sucrose in buffer I. This suspension (45 ml each) was layered onto a lo-ml bed of 2.2 M sucrose in buffer I and centrifuged in an Hitachi RPS-25-2 rotor for 1 h at 22,000 rpm. Gelatinous pellets thus obtained were washed with a small volume of 0.34 M sucrose and used as the nuclear fraction. Extraction of RNA polymerases and stimulation factor. RNA polymerases were solubilized from the nuclear fraction by procedures modified from those of Schwartz et al. (12). The nuclear pellet prepared from 30 g of tissue was suspended in 15 ml of 0.34 M sucrose and mixed with equal volume of 2x buffer II. Ammonium sulfate (4 M, pH 7.6) was added into the suspension to give a final concentration of 0.3 M. Ten milliliters each of the viscous nuclear lysate was sonicated with a Tomy model UR-15OP sonitier (maximum power setting, for a total period of 5 min at intervals). The sheared lysate was centrifuged for 1 h at 50,000 rpm in an Hitachi RP-65 rotor. The supernatant (fraction Sl) was dialyzed overnight against 6.4 vol of buffer II, so that the ammonium 2 Abbreviation used: PMSF, phenylmethylsulfonyl fluoride; DEAE, diethylaminoethyl.
KAWAKAMI sulfate concentration of the dialysate fell to 0.04 M. The sedimentable portion of the dialysate was removed by centrifugation as above. The supernatant (fraction S2) can be stored frozen at -70°C without loss of enzyme activity. Fraction 52 was chromatographed on a DEAEcellulose (Whatman DE-52) column, as shown in Fig. 1. The flow-through fraction (fraction DE-O), which had no RNA polymerase activity but exerted significant stimulatory effect on enzyme II, was stored at -7O”C, at which temperature the stimulatory activity was completely maintained at least for 1 year. Preparation of enzymes I and II. Two forms of RNA polymerase, enzymes I and II, were considerably purified from fractions DE-I and DE-II, respectively (see legend to Fig. 1). A detailed description of the purification procedures will be present elsewhere (S. Ihara and M. Kawakami, manuscript in preparation). In brief, enzyme I was further purified by carboxymethyl-Sephadex chromatography and phosphocellulose chromatography, and enzyme II by DEAE-cellulose rechromatography and two cycles of phosphocellulose chromatography. Both enzymes were dialyzed against 0.04 M ammonium sulfate in buffer II before assay. Enzyme activity was determined by the following procedures (Assay I). Incubation was performed at 37°C for 20 min in 60 ~1 of assay mixture containing
FIG. 1. DEAE-cellulose chromatography of fraction S2. Fraction S2 was applied to a DEAE-cellulose column (Whatman DE-52, 100 ml of bed/1000 mg of protein) equilibrated with 0.04 M ammonium sulfate in buffer II. After a wash with the same solution, adsorbed materials were eluted with a linear gradient of 0.04 to 0.4 M ammonium sulfate in buffer II. RNA polymerase activity in 15 ml each of fractions thus eluted was measured according to Assay I described under Materials and Methods. The reaction mixture contained (0- - -0) or did not contain (0-O) 10m6 M or-amanitan. Pairs of vertical lines 0, I, and II indicate the ranges of pooled fractions DE-O, DE-I, and DE-II, respectively. Concentrations of protein (-) and ammonium sulfate (- - - - -1 are shown.
FACTOR
FROM
MYELOMA
NUCLEI
2.4 pmol of Tris-HCl (pH 7.9, 4”C), 2.4 pmol of ammonium sulfate, 0.096 pmol of MnCl,, 0.024 pmol of EDTA, 0.24 pmol of dithiothreitol, 0.024 pmol each of ATP, GTP, and CTP, 0.0024 Fmol of 13H]UTP (260 mCi/mmol), 2.5 pg of heat-denatured calf thymus DNA, 20% (v/v) glycerol, and 10 ~1 of enzyme solution. After the incubation, an aliquot of the mixture was removed and applied to a Whatman GF/C disc. The disc was immediately placed in icecold 5% trichloroacetic acid in 0.01 M sodium pyrophosphate, and rinsed three times with ice-cold 2% trichloroacetic acid in 0.01 M sodium pyrophosphate and twice with ethanol. The radioactivity of the dried disc was counted in 5 ml of toluene-based scintillation fluid. One unit of activity represents incorporation of 1 nmol of UMP under these conditions. Specific activities (units per milligram of protein) of our enzyme I and II preparations are 10 and 320, respectively. Assay of stimulation factor (Assay II). Forty microliters of preincubation mixture contained 2 pmol of Tris-HCl (pH 7.9, 4”C), 1.6 pmol of ammonium sulfate, 0.08 pmol of MnCl,, 0.02 pmol of EDTA, 0.2 pmol of dithiothreitol, 1 pg of PMSF, 20% (v/v) glycerol, 10 pg of bovine serum albumin, 5 bg of native calf thymus DNA, and appropriate amounts of enzyme and factor, i.e., unless otherwise noted, 0.014 unit of enzyme I or 0.085 unit of enzyme II and 5 ~1 of fraction DE-O (4.25 pg of protein). After various periods of preincubation at 37 or o”C, incubation at 37°C for RNA synthesis was started by adding 20 ~1 of the solution containing 1 pmol of Tris-HCl (pH 7.9, 4”C), 0.8 pmol of ammonium sulfate, 0.04 pmol of MnCl,, 0.01 pmol of EDTA, 0.1 prnol of dithiothreitol, 20% (v/v) glycerol, 0.012 pmol each of ATP, GTP, and CTP, 0.0012 nmol of ?HlUTP (250 mCi/mmol), and 6 pg of rifamycin AFI 013 which gave a final concentration of 100 pg/ml. After a given period of the incubation, the samples were processed and the radioactivities were counted as described for Assay I. All data presented here are mean values in duplicate assays, corrected by subtracting a mean value in the control assay omitting the incubation. Other procedures. Escherichia coli RNA polymerase (holoenzyme) was purified from E. coli K12 by the method of Burgess (13), and the fraction obtained by the second glycerol sedimentation was used. Salt concentrations of dialysates and chromatographic eluates were determined by conductivity measurement using buffers with known salt concentration as standards. Protein was determined by the method of Lowry etal. (14). In order to remove interfering substances, the sample solution was mixed with trichloroacetic acid to a final concentration of 10% and left for 30 min at 0°C; the resulting precipitate was collected by centrifugation, washed with ice-cold 10% trichloroa-
STIMULATES
RNA
cetic acid and ice-cold orimetry.
125
SYNTHESIS acetone,
and subjected
to col-
RESULTS
Inhibition of RNA Polymerase Activity Rifamycin AFl013
by
We have confirmed in a preliminary experiment that AF/013 inhibits completely the activity of myeloma RNA polymerases, both of enzymes I and II, at concentrations above 50 pglml as long as the drug, DNA, and enzyme are added simultaneously to the reaction mixture for RNA synthesis. In order to investigate the effect of the presence of AFt013 on a course of RNA synthesis before or after a supposed formation of DNA-enzyme complex, AFI 013 was added to the reaction mixture giving a final concentration of 100 pg/ml at the beginning of preincubation of enzyme and DNA or just before the subsequent incubation of the enzyme and DNA with substrate ribonucleotides (Fig. 2). Evidently enzyme I is prevented by AF/013 from RNA synthesis even when it is prein-
[Ls
2 0 0
10 INCUBATION
20 TIME
(ml”)
FIG. 2. Inhibition of RNA polymerase activity by rifamycin AF/013. Enzyme I (a) or enzyme II (b) was preincubated with DNA for 10 min; then substrates (ATP, GTP, CTP, and 13HlUTP) were added and the incubation was performed at 37°C for the indicated period. AF/013 was added to the reaction mixture at the beginning of preincubation at 37°C (x1, at the beginning of incubation after preincubation at 37°C (U-0) or 0°C (a---a), or not added throughout preincubation at 37°C and incubation (0-O).
126
IHARA
AND
KAWAKAMI
cubated with DNA in the absence of AF/ 013 (Fig. 2a). In contrast, Fig. 2b shows that, when enzyme II is preincubated with DNA at 37°C in the absence of AF/013, RNA synthesis takes place even in the presence of AF/013 after the addition of substrates, and that RNA synthesis is depressed by AF/013 when preincubation is done at 0°C or in the presence of AF/013. These results are consistent with previous findings that AF/013 blocks the reaction of form II RNA polymerase at a step(s) preceding initiation but not at the initiation or elongation steps in transcription (10, 15). Additionally, our results support a recent concept that there exists a certain temperature-dependent step in the preinitiation process of transcription by form II but not by form I enzyme (10). Stimulation Fraction
of Activity
of Enzyme
II
hibitor effect on RNA synthesis. As described below, we have explored the presence of a factor capable of stimulating the preinitiation process of RNA synthesis by enzyme II, using an assay procedure (Assay II, see Materials and Methods) composed of a preincubation of the enzyme and DNA, incubation with ribonucleotides, and determination of UMP incorporation. A suitable amount of rifamycin AF/013 was added to the incubation mixture to prevent progression of the preinitiation process during the incubation. Figure 3 shows the effect of different
by
DE-O
The influence of a flow-through fraction (DE-O) of a DEAE-cellulose column on various RNA polymerase activities is shown in Table I. Fraction DE-O seemsto exert stimulatory effect on RNA synthesis, mainly when enzyme II is assayed using native DNA as template. This is in agreement with several recent reports on factors obtained from different eucaryotic sources (l-4, 16-18). The change in UMP incorporation thus observed, however, corresponds to the summation of overall influences of individual factors present in fraction DE-O, which have stimulatory or in-
0
Enzyme*
OF FRACTION
Template
DNA
