ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 278, No. 2, May 1, pp. 475-477,199O
COMMUNICATION RNA Elongation by RNA Polymerase II Is Not Inhibited by A/-Ethylmaleimide or lodoacetamide Robert C. Krueger Department of Molecular College of Medicine,
Genetics, Biochemistry and Microbiology, University 231 Bethesda Avenue, Cincinnati, Ohio 45267-0524
Received October 23, 1989, and in revised form January
5, 1990
The elongation of RNA by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide. Three systems were studied: a soluble chromatin, purified RNA polymerase II with DNA, and a HeLa cell exCC) isso tract with an adenovirus 2 promoter sequence. Academic
Press,
of Cincinnati
Inc.
RNA polymerase II is reportedly a sulfhydryl enzyme and is inactivated by mercurials and alkylating agents (1, 2). This information derives from studies with purified enzyme tested by preincubation of enzyme and inhibitor. I show in this report that N-ethylmaleimide and iodoacetamide are ineffective as inhibitors of RNA elongation by RNA polymerase II. Three systems have been studied: a naturally occurring transcription complex, purified polymerase II with DNA, and a HeLa cell extract with a promoter sequence.
inhibited 92% by a-amanitin at 2 pg/ml and the specific activity was 10.8 nmol [“HJUTP incorporation/l0 min/mg protein. Adenovirus 2 major latepromotor transcription. This was performed by Mr. Randy Bechard in Professor Donal Luse’s laboratory in this department according to methods detailed in Ref. (8). The DNA template was a plasmid containing the adenovirus 2 major late promoter while the RNA polymerase II and transcription factors were provided by a HeLa cell nuclear extract. N-ethylmaleimide was added either before initiation or at a point where short transcripts (7-10 nucleotides) had been formed. The details are noted in the legend to Fig. 4.
RESULTS AND DISCUSSION The original observation demonstrating a lack of sensitivity to N-ethylmaleimide in transcription with a chromatin tem-
12000
METHODS l’ranscription complex-pol.ymerase II assay. The soluble chromatin isolated from rabbit thymus nuclei and studied in this laboratory (35) was found to contain polymerase II activity as assayed by incorporation of [“HIUTP or [“H]CTP when supplemented with MgS04 and nucleoside triphosphates. The soluble chromatin is a heterogeneous, nucleosomal type of complex consisting of 10 to 100 nucleosomes, a full complement of histones, and some nonhistone protein. The polymerase activity is designated II by virtue of a 96% inhibition with (Yamanitin at 2 pg/ml; it is in the elongation mode for it is insensitive to the rifamycin derivative AF/013 (6). A typical assay mixture in a final volume of 0.5 ml contained 32 mM K&SO,, 16 mM Tris, pH 7.6, 2 mM MgSO,, 0.1 mM ATP, GTP, and CTP, soluble chromatin (about 300 pg DNA). and 2 &i [3H]UTP (Amersham, 24 Ci/mmol). Tubes were set up in duplicate and after incubation at 37°C the reaction was quenched with trichloroacetic acid and the precipitate was collected on Whatman GF/C filters, washed, and counted. All incorporations were corrected for t = 0 counts. On average, the difference between duplicate determinations was 7%. Purification of RNA polymera.se II. A partially purified preparation of the enzyme was prepared from rabbit thymus tissue (PelFreeze, Rogers, AR) following, in general, the procedures described by Chambon and co-workers (7). This involved sonication in high salt, precipitation with ammonium sulfate, and adsorption to and elution from DEAE-Sephadex. The activity was measured in a Tris buffer at pH 7.9 using purified rabbit thymus DNA as a template. The activity was 0003-9861/90 $3.00 Copyright $:I 1990 hy All rights of reproduction
Academic Press, Inc. in any
form
reserved.
0
10
20
Minutes FIG. 1. The effect of N-ethylmaleimide on transcription with a chromatin template. The chromatin (357 fig DNA) in Tris bufIer, pH 7.6, was incubated at 21°C for 10 min with 1 mM N-ethylmaleimide and RNA synthesis was initiated by addition of MgSO,, nucleoside triphosphates, and [“HIUTP. (X) Control, (0) N-ethylmaleimide. 475
476
ROBERT
C. KRUEGER addition
of GTP
continuation
onto
the 12 mer in lanes
5-8.
Four bands are seen in lane 1 because suboptimum amounts of CTP and UTP were used and these bands represent paused transcripts. (See Ref. (8) for details.) As shown in Fig. 4, addition of N-ethylmaleimide (lanes 3 and 4) to the enzyme-DNA complex prior to addition of nucleotides (ApC, UTP, CTP) completely inhibited the synthesis of RNA. The uninhibited reaction is limited to the first 10 nucleotides because GTP was not included. If, however, N-ethylmaleimide is added at a point where short transcripts (7-10 nucleotides) had been produced and GTP was included to allow further synthesis no inhibition of elongation was seen (compare lanes 7 and 8 with lane 5). While kinetics were not examined in the present study it is
8000
known
(Fig.
1, Ref. (8)) that
in this system
the top two bands
in lane 5 are not found by short term incubations (e.g., 1.5 min) and the presence of these bands in the reaction with NEM (lanes 7 and 8) represent synthesis roughly equivalent to that seen in lane 5 and this demonstrates that NEM is noninhibitory. In previous reports from another laboratory (g-11), methyl mercury and selenotrisulfide were shown to behave in a fashion similar to that observed here. With purified RNA polymerase II, these compounds were inhibitory when added before initiation while were without effect or stimulatory when added after
10
20
Minutes FIG. 2. The effect of iodoacetamide andp-hydroxymercuribenzoate on transcription with a chromatin template. The chromatin (311 pg DNA) in Tris buffer, pH 7.6, was incubated at 21°C for 30 min with either compound before addition of MgS04, nucleoside triphosphates, (A) and [“HIUTP. (X) Control, (0) 1 mM p-hydroxymercuribenzoate, 1 mM iodoacetamide. and (0) 2 mM iodoacetamide.
5 6000
.E E 2 E a 3
plate is shown in Fig. 1. An activation rather than an inhibition was observed. The activation was noticeable with as little as a 2-min preincubation with 1 mM N-ethylmaleimide and was at a maximum (twofold stimulation) at 30 min (data not shown). Figure 2 documents a similar activation with iodoacetamide
and, in addition, shows the classical inhibition of activity with a mercurial as described in the literature (1,2). The results with purified enzyme and DNA shown in Fig. 3 are particularly instructive. With N-ethylmaleimide added to the reaction components before enzyme, good inhibition is observed (0, Fig. 3). Furthermore, with a lo-min preincubation of enzyme and N-ethylmaleimide there is even more inhibition (90%) than noted here (data not shown). After 10 min of RNA synthesis, where presumably gation mode, IV-ethylmaleimide
the enzyme is mainly in the elonnow activates rather than in-
hibits (A, Fig. 3). It should be noted that Gissinger et al. (1) reported complete inhibition of RNA polymerase II by 2 X 10m4 M NEM. This differential effect of N-ethylmaleimide was also observed with another system containing a HeLa cell extract and the major late promoter of adenovirus 2. The sequence being transcribed is ACUCUCUUCCGCAU. With addition of ApC, and suboptimal quantitive UTP and CTP, we expect initiation
and elongation
up to the 10 mer in the first four lanes and with
Minutes FIG. 3. The effect of N-ethylmaleimide on transcription with purified RNA polymerase II. Rabbit thymus DNA (25 pg) was mixed at 21°C in a final volume of 0.43 ml containing the following: 63 mM Tris, pH 7.9, 12.5% glycerol, 5 mM MgS04, 1.3 mM MnC&, 2.1 mM 2-mercaptoethanol, 0.5 mM each ATP, GTP, and CTP, 0.05 mM UTP, and 2.2 &CL [3H]UTP. At t = -1 and t = 10 min, 0.05 ml 42 mM N-ethylmaleimide was added (final, net = 2.0 mM). Purified RNA polymerase II (20 ~1) was added at t = 0 to start the reaction and tubes were incubated at 37°C and precipitated with TCA as indicated. (X) control, (0) Nethylmaleimide at t = -1 min, (A) N-ethylmaleimide at 10 min.
INHIBITION
OF RNA
POLYMERASE
477
II
example the resistance of the elongating enzyme to sarkosyl or high salt (18, 19). The slight activation with the alkylating agents may be of interest in relation to possible control mechanisms for RNA synthesis. For instance, is there a relationship between this activation and the stimulation of elongation observed with protein factors (20, al)? It may also be of interest to determine if these alkylating agents can modify enzyme activity that has been inhibited at so-called “pause-sites” that have been described in eukaryotic systems (22,23). ACKNOWLEDGMENTS Thanks are due to Dr. T. Wieland, Heidelberg, for providing the (Yamanitin and to Dr. W. J. Hudak, Merrell-Dow Research Institute, Cincinnati, Ohio, for a sample of rifamycin AF/013. Discussion with Donal Luse regarding the polymerase preparation and transcription reactions is much appreciated as is the technical assistance of Mr. Randy Bechard.
REFERENCES 1. Gissinger,
F., Kedinger,
C., and Chambon,
P. (1974) Biochimie
56,319-333. 2. Chambon, P. (1974) in The Enzymes (Boyer P., Ed.), Vol. X, pp. 261-331, Academic Press, San Diego. ACUCUCUUCCGCAU 1
5
10
* Order of addition of reagents to the preinitiation
complex.
on transcription from the FIG. 4. The effect of N-ethylmaleimide adenovirus 2 major late promoter. Full description of the procedure is given in Ref. (8). In summary, the plasmid containing the promoter sequence (ACUCUCUUCCGCAU; transcript of the anticoding strand) was incubated in a total volume of 0.93 ml with HeLa extract in Tris buffer, pH 7.4, containing 79 mM KC1 and 8.4 mM MgCi, for 20 min at 25°C. The preinitiation complex was isolated from this mixture by chromatography on Rio-Gel A 1.5. Various additions were made to the complex as indicated above. Incubations with reagents A and B were at 25°C for 5 min, with
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 278, No. 2, May 1, pp. 475-477,199O
COMMUNICATION RNA Elongation by RNA Polymerase II Is Not Inhibited by A/-Ethylmaleimide or lodoacetamide Robert C. Krueger Department of Molecular College of Medicine,
Genetics, Biochemistry and Microbiology, University 231 Bethesda Avenue, Cincinnati, Ohio 45267-0524
Received October 23, 1989, and in revised form January
5, 1990
The elongation of RNA by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide. Three systems were studied: a soluble chromatin, purified RNA polymerase II with DNA, and a HeLa cell exCC) isso tract with an adenovirus 2 promoter sequence. Academic
Press,
of Cincinnati
Inc.
RNA polymerase II is reportedly a sulfhydryl enzyme and is inactivated by mercurials and alkylating agents (1, 2). This information derives from studies with purified enzyme tested by preincubation of enzyme and inhibitor. I show in this report that N-ethylmaleimide and iodoacetamide are ineffective as inhibitors of RNA elongation by RNA polymerase II. Three systems have been studied: a naturally occurring transcription complex, purified polymerase II with DNA, and a HeLa cell extract with a promoter sequence.
inhibited 92% by a-amanitin at 2 pg/ml and the specific activity was 10.8 nmol [“HJUTP incorporation/l0 min/mg protein. Adenovirus 2 major latepromotor transcription. This was performed by Mr. Randy Bechard in Professor Donal Luse’s laboratory in this department according to methods detailed in Ref. (8). The DNA template was a plasmid containing the adenovirus 2 major late promoter while the RNA polymerase II and transcription factors were provided by a HeLa cell nuclear extract. N-ethylmaleimide was added either before initiation or at a point where short transcripts (7-10 nucleotides) had been formed. The details are noted in the legend to Fig. 4.
RESULTS AND DISCUSSION The original observation demonstrating a lack of sensitivity to N-ethylmaleimide in transcription with a chromatin tem-
12000
METHODS l’ranscription complex-pol.ymerase II assay. The soluble chromatin isolated from rabbit thymus nuclei and studied in this laboratory (35) was found to contain polymerase II activity as assayed by incorporation of [“HIUTP or [“H]CTP when supplemented with MgS04 and nucleoside triphosphates. The soluble chromatin is a heterogeneous, nucleosomal type of complex consisting of 10 to 100 nucleosomes, a full complement of histones, and some nonhistone protein. The polymerase activity is designated II by virtue of a 96% inhibition with (Yamanitin at 2 pg/ml; it is in the elongation mode for it is insensitive to the rifamycin derivative AF/013 (6). A typical assay mixture in a final volume of 0.5 ml contained 32 mM K&SO,, 16 mM Tris, pH 7.6, 2 mM MgSO,, 0.1 mM ATP, GTP, and CTP, soluble chromatin (about 300 pg DNA). and 2 &i [3H]UTP (Amersham, 24 Ci/mmol). Tubes were set up in duplicate and after incubation at 37°C the reaction was quenched with trichloroacetic acid and the precipitate was collected on Whatman GF/C filters, washed, and counted. All incorporations were corrected for t = 0 counts. On average, the difference between duplicate determinations was 7%. Purification of RNA polymera.se II. A partially purified preparation of the enzyme was prepared from rabbit thymus tissue (PelFreeze, Rogers, AR) following, in general, the procedures described by Chambon and co-workers (7). This involved sonication in high salt, precipitation with ammonium sulfate, and adsorption to and elution from DEAE-Sephadex. The activity was measured in a Tris buffer at pH 7.9 using purified rabbit thymus DNA as a template. The activity was 0003-9861/90 $3.00 Copyright $:I 1990 hy All rights of reproduction
Academic Press, Inc. in any
form
reserved.
0
10
20
Minutes FIG. 1. The effect of N-ethylmaleimide on transcription with a chromatin template. The chromatin (357 fig DNA) in Tris bufIer, pH 7.6, was incubated at 21°C for 10 min with 1 mM N-ethylmaleimide and RNA synthesis was initiated by addition of MgSO,, nucleoside triphosphates, and [“HIUTP. (X) Control, (0) N-ethylmaleimide. 475
476
ROBERT
C. KRUEGER addition
of GTP
continuation
onto
the 12 mer in lanes
5-8.
Four bands are seen in lane 1 because suboptimum amounts of CTP and UTP were used and these bands represent paused transcripts. (See Ref. (8) for details.) As shown in Fig. 4, addition of N-ethylmaleimide (lanes 3 and 4) to the enzyme-DNA complex prior to addition of nucleotides (ApC, UTP, CTP) completely inhibited the synthesis of RNA. The uninhibited reaction is limited to the first 10 nucleotides because GTP was not included. If, however, N-ethylmaleimide is added at a point where short transcripts (7-10 nucleotides) had been produced and GTP was included to allow further synthesis no inhibition of elongation was seen (compare lanes 7 and 8 with lane 5). While kinetics were not examined in the present study it is
8000
known
(Fig.
1, Ref. (8)) that
in this system
the top two bands
in lane 5 are not found by short term incubations (e.g., 1.5 min) and the presence of these bands in the reaction with NEM (lanes 7 and 8) represent synthesis roughly equivalent to that seen in lane 5 and this demonstrates that NEM is noninhibitory. In previous reports from another laboratory (g-11), methyl mercury and selenotrisulfide were shown to behave in a fashion similar to that observed here. With purified RNA polymerase II, these compounds were inhibitory when added before initiation while were without effect or stimulatory when added after
10
20
Minutes FIG. 2. The effect of iodoacetamide andp-hydroxymercuribenzoate on transcription with a chromatin template. The chromatin (311 pg DNA) in Tris buffer, pH 7.6, was incubated at 21°C for 30 min with either compound before addition of MgS04, nucleoside triphosphates, (A) and [“HIUTP. (X) Control, (0) 1 mM p-hydroxymercuribenzoate, 1 mM iodoacetamide. and (0) 2 mM iodoacetamide.
5 6000
.E E 2 E a 3
plate is shown in Fig. 1. An activation rather than an inhibition was observed. The activation was noticeable with as little as a 2-min preincubation with 1 mM N-ethylmaleimide and was at a maximum (twofold stimulation) at 30 min (data not shown). Figure 2 documents a similar activation with iodoacetamide
and, in addition, shows the classical inhibition of activity with a mercurial as described in the literature (1,2). The results with purified enzyme and DNA shown in Fig. 3 are particularly instructive. With N-ethylmaleimide added to the reaction components before enzyme, good inhibition is observed (0, Fig. 3). Furthermore, with a lo-min preincubation of enzyme and N-ethylmaleimide there is even more inhibition (90%) than noted here (data not shown). After 10 min of RNA synthesis, where presumably gation mode, IV-ethylmaleimide
the enzyme is mainly in the elonnow activates rather than in-
hibits (A, Fig. 3). It should be noted that Gissinger et al. (1) reported complete inhibition of RNA polymerase II by 2 X 10m4 M NEM. This differential effect of N-ethylmaleimide was also observed with another system containing a HeLa cell extract and the major late promoter of adenovirus 2. The sequence being transcribed is ACUCUCUUCCGCAU. With addition of ApC, and suboptimal quantitive UTP and CTP, we expect initiation
and elongation
up to the 10 mer in the first four lanes and with
Minutes FIG. 3. The effect of N-ethylmaleimide on transcription with purified RNA polymerase II. Rabbit thymus DNA (25 pg) was mixed at 21°C in a final volume of 0.43 ml containing the following: 63 mM Tris, pH 7.9, 12.5% glycerol, 5 mM MgS04, 1.3 mM MnC&, 2.1 mM 2-mercaptoethanol, 0.5 mM each ATP, GTP, and CTP, 0.05 mM UTP, and 2.2 &CL [3H]UTP. At t = -1 and t = 10 min, 0.05 ml 42 mM N-ethylmaleimide was added (final, net = 2.0 mM). Purified RNA polymerase II (20 ~1) was added at t = 0 to start the reaction and tubes were incubated at 37°C and precipitated with TCA as indicated. (X) control, (0) Nethylmaleimide at t = -1 min, (A) N-ethylmaleimide at 10 min.
INHIBITION
OF RNA
POLYMERASE
477
II
example the resistance of the elongating enzyme to sarkosyl or high salt (18, 19). The slight activation with the alkylating agents may be of interest in relation to possible control mechanisms for RNA synthesis. For instance, is there a relationship between this activation and the stimulation of elongation observed with protein factors (20, al)? It may also be of interest to determine if these alkylating agents can modify enzyme activity that has been inhibited at so-called “pause-sites” that have been described in eukaryotic systems (22,23). ACKNOWLEDGMENTS Thanks are due to Dr. T. Wieland, Heidelberg, for providing the (Yamanitin and to Dr. W. J. Hudak, Merrell-Dow Research Institute, Cincinnati, Ohio, for a sample of rifamycin AF/013. Discussion with Donal Luse regarding the polymerase preparation and transcription reactions is much appreciated as is the technical assistance of Mr. Randy Bechard.
REFERENCES 1. Gissinger,
F., Kedinger,
C., and Chambon,
P. (1974) Biochimie
56,319-333. 2. Chambon, P. (1974) in The Enzymes (Boyer P., Ed.), Vol. X, pp. 261-331, Academic Press, San Diego. ACUCUCUUCCGCAU 1
5
10
* Order of addition of reagents to the preinitiation
complex.
on transcription from the FIG. 4. The effect of N-ethylmaleimide adenovirus 2 major late promoter. Full description of the procedure is given in Ref. (8). In summary, the plasmid containing the promoter sequence (ACUCUCUUCCGCAU; transcript of the anticoding strand) was incubated in a total volume of 0.93 ml with HeLa extract in Tris buffer, pH 7.4, containing 79 mM KC1 and 8.4 mM MgCi, for 20 min at 25°C. The preinitiation complex was isolated from this mixture by chromatography on Rio-Gel A 1.5. Various additions were made to the complex as indicated above. Incubations with reagents A and B were at 25°C for 5 min, with
