Vol. 130, No. 3
JOURNAL OF BACTERIOLOGY, June 1977, P. 1387-1389 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Inhibition of Histoplasma capsulatum Ribonucleic Acid Polymerases by Homologous and Heterologous Ribonucleic Acid RAY McMILLIAN,* B. VIJAY KUMAR, GERALD MEDOFF, DAVID SCHLESSINGER, AND G. S. KOBAYASHI Divisions ofInfectious Diseases, Dermatology and Laboratory Medicine, Departments of Medicine and Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110 Received for publication 21 January 1977
The ribonucleic acid (RNA) polymerases from the yeast phase of Histoplasma capsulatum are differentially sensitive to RNA isolated from the yeast and mycelial phases of this fungus and from Escherichia coli. Low-molecular-weight RNA from H. capsulatum was the most effective inhibitor. The pathogenic dimorphic fungus Histoplasma capsulatum grows in a multicellular filamentous phase when incubated at 230C and in a yeast phase at 370C. Previous studies from this laboratory have shown that yeast-phase cells contain three species of ribonucleic acid (RNA) polymerases (2), whereas mycelia have one species of RNA polymerase of very low activity (1). In our attempts to purify the RNA polymerases of H. capsulatum, we have discovered that the yeast-phase RNA polymerases are differentially sensitive to different RNA classes from both phases of H. capsulatum and from Escherichia coli. Here we present the data from these findings. H. capsulatum (Down strain, mating-type [-] from our laboratory) was grown in the yeast phase at 370C and in the mycelial phase at 250C in glucose-yeast extract medium as previously described (2). E. coli B was grown at 250C in minimal salts medium fortified with 0.8% Casamino Acids (7). Cells were harvested by filtration, washed with distilled water, and processed immediately. RNA polymerases from the yeast phase of H. capsulatum were partially purified by phosphocellulose chromatography and assayed (2). One unit of RNA polymerase activity was defined as the amount of enzyme that catalyzed the incorporation of 1 pmol of [3H]uridine 5'-monophosphate into RNA in 60 min. The nomenclature of RNA polymerases from H. capsulatum was discussed in our previous publication (2) and is also used throughout this report. Inhibition of RNA synthesis was measured by adding 8 to 320 ng of RNA to the RNA polymerase reaction mixture and comparing the incorporation of [3H]uridine 5'triphosphate into RNA with the incorporation in the absence of exogenously added RNA.
The RNA was prepared from H. capsulatum by homogenizing yeast or mycelial cells with glass beads in a Braun homogenizer (3) and extracting the RNA from the lysate by hot sodium dodecyl sulfate-phenol (5). The procedure used to prepare RNA from E. coli B has been described (10). The RNA was fractionated on 10 to 30% sucrose gradients (10), and individual fractions corresponding to 26S, 18S, and 4 to 6S RNA from H. capsulatum and 23S, 16S, and 4 to 5S fromE. coli were pooled. There was no cross-contamination among these pooled components, as judged by polyacrylamide gel electrophoresis. Low-molecular-weight RNA (4 to 6S) was fractionated on 6.8% polyacrylamide gels (6). Electrophoresis was carried out for 2.5 h at 5 mA per gel. The effects of 26S, 18S, and 4 to 6S RNA from the yeast phase of H. capsulatum on yeastphase RNA polymerases and E. coli RNA polymerase are shown in Fig. 1. RNA polymerase PCIII was sensitive to low concentrations of all three classes of RNA but was most sensitive to 4 to 6S RNA (100% inhibition at 160 ng/125 ,ul of RNA). RNA polymerase PCI was not very sensitive to 26S and 18S RNA (30% inhibition at 320 ng/125 ,ul of RNA) but was as sensitive to the 4 to 6S RNA as PCIII. RNA polymerase PCII was not very sensitive to any of the three RNA classes (30% inhibition at 320 ng/125 ,ul of RNA). E. coli RNA polymerase was resistant to all of the concentrations of 26S and 18S RNA used in the assay, but it was sensitive to 4 to 6S RNA (85% inhibition at 320 ng/125 ,ul of RNA). The effects of 26S, 18S, and 4 to 6S RNA from the mycelial phase of H. capsulatum on yeastphase RNA polymerases and E. coli RNA polymerase were also examined. The pattern of
1387
1388
J. BACTERIOL.
NOTES
RNA polymerase sensitivity to the mycelial RNAs was the same as that seen with the yeast RNAs, but the mycelial RNAs were 1.5 times more inhibitory (data not shown). Figure 2 shows the effects of 23S, 16S, and 4 to 5S RNA from E. coli on the yeast-phase RNA polymerases and E. coli RNA polymerase. The sensitivity of the yeast-phase RNA polymerases to E. coli 23S and 16S RNA was similar to that of H. capsulatum 26S and 188 RNA. The RNA polymerases were less sensitive to 4 to 5S RNA from E. coli (60, 35, and 30% inhibition at 320 ng/125 ,ul of RNA for PC0II, PCI, and E. coli RNA polymerase, respectively) than to E. coli 23S and 16S RNA and H. capsulatum 4 to 6S RNA. Yeast- and mycelial-phase 5.8S, 58, and 4S 10 o0
II
26S
30 _ D
RNAs purified from the 4 to 68 RNA fraction were as inhibitory as the total fraction of lowmolecular-weight RNA when tested against the yeast-phase RNA polymerases. RNA polymerases PCI and PCIII were very sensitive to the three species of low-molecular-weight RNA isolated from both phases of H. capsulatum, whereas RNA polymerase PCII was resistant (data not shown). The sensitivity of RNA polymerase PCIII was not altered by the addition of increasing amounts of RNA polymerase PCII to the reaction mixture, which implies that the resistance to RNA inhibition associated with polymerase PCI1 was specific and could not protect polymerase PCIII from the RNA inhibition (data not shown). Moreover, the addition of large
PC
!//
0
6
0-
2)
oA K
A 80
' Aco/,
160
240
320 0
80
160
240
320 0
IE
80
Histoplasma capsulatum yeast phase RNA, (ng/125il)
FIG. 1. Inhibition of RNA polymerases from the yeast phase of H. capsulatum and from E. coli by sucrose gradient fractionated yeast-phase RNAs. RNA polymerases PCI (1.8 U, 0), PCII (2.0 U, 0), and PCIn (2.2 U, 0) and E. coli RNA polymerase (3.0 U, A) were added to the standard assay mixture containing varying amounts of RNA. The amount of inhibition was determined as described in the text. l
l_ _
100 _ 23S
16S
4
C,
55
PC
80 -
o2
E
60 0
40
-
PC
a-
PC~~~~~~~~~~~~~~~~~~~~~~~P PC~~~~~~~~",
20
C~~
0
0
80
-A-A 160 240 320 0 80
160
240
320 0
160
240
320
Escherichia coli RNA, (ng/125 uil)
FIG. 2. Inhibition of RNA polymerases from the yeast phase of H. capsulatum and from E. coli by sucrose gradient fractionated E. coli RNAs. RNA polymerases PCI (1.8 U, 0), PCII (2.0 U, 0), and PCIII (2.2 U, 0), and E. coli RNA polymerase (3.0 U, A) were added to the standard assay mixture containing varying amounts of RNA. The amount of inhibition was determined as described in the text.
VOL. 130, 1977
NOTES
1389
amounts (500 ,ug/125 /.l) of bovine serum albu- Kobayashi, manuscript in preparation). min to the reaction mixture did not affect the This work was supported by Public Health Service sensitivity of RNA polymerase PCIII to 4S grants AI 10622 and Al 06213 from the National Institute of RNA, which indicates that the resistance of Allergy and Infectious Diseases; training grants AI 00459 of Allergy and RNA polymerase PCII to RNA inhibition was and AI 07015 from the National Institute Infectious Diseases, and AM 05611 from the National Instinot secondary to the presence of large amounts tute of Arthritis, Metabolism and Digestive Diseases; and of nonspecific protein (data not shown). by grants from the John A. Hartford Foundation and the The inhibition of bacterial RNA polymerase Research Corporation (Brown-Hazen Fund). R. A. Mcby RNA has been described previously (4, 8, Millian was supported by Public Health Service fellowship from the National Institute of Allergy 11), but there have been only two reports on the 5 F 22 AI 01132-02 Diseases. inhibition of other eucaryotic RNA polymer- and Infectious LITERATURE CITED ases by RNA (9, 12). They have shown that RNA binds to the enzyme and that the inhibi- 1. Boguslawski, G., G. Medoff, D. Schlessinger, and G. Kobayashi. 1975. Histin, an RNA polymerase inhibtion can be reversed by the addition of increasitor isolated from Histoplasma capsulatum. Biochem. ing amounts of DNA. Our studies systematically Biophys. Res. Commun. 64:625-632. looked at the effects of three classes of RNA 2. Boguslawski, G., D. Schlessinger, G. Medoff, and G. from H. capsulatum and E. coli on the RNA Kobayashi. 1974. Ribonucleic acid polymerases of the yeast phase of Histoplasma capsulatum. J. Bacteriol. polymerases from these organisms and found 118:480-485. that RNA polymerase PCIII. was sensitive and 3. Cheung, S-S. C., G. S. Kobayashi, D. Schiessinger, and was inhibited by all of the RNAs tested; RNA G. Medoff. 1974. RNA metabolism during morphopolymerase PCI and E. coli RNA polymerase genesis in Histoplasma capsulatum. J. Gen. Microbiol. 82:301-307. were only sensitive to low-molecular-weight C. F., R. I. Gumport, and S. B. Weiss. 1965. RNA; and RNA polymerase PCII was not sensi- 4. Fox, enzymatic synthesis of ribonucleic acid. V. The The tive to any of the classes of RNA. This differeninteraction of ribonucleic acid polymerase with nutial sensitivity of the enzymes to the different cleic acids. J. Biol. Chem. 240:2101-2109. RNAs indicates that the inhibition is not non- 5. Kirby, K. S. 1965. Isolation and characterization of ribosomal ribonucleic acid. Biochem. J. 96:266-269. specific. In addition, a further experiment U. E. 1967. The fractionation of high molecuwhich supports the notion that the inhibition of 6. Loening, lar weight ribonucleic acid by polyacrylamide gel RNA polymerases by low-molecular RNA is electrophoresis. Biochem. J. 102:251-257. specific for certain classes of RNA is that the 7. Nikolaev, N., L. Silengo, and D. Schlessinger. 1973. A role for ribonuclease III in processing of ribosomal RNA polymerases were not inhibited by polyribonucleic acid and messenger ribonucleic acid preadenylic acid or polycytidylic acid (B. V. Kucursors in Escherichia coli. J. Biol. Chem. 248:7967mar, R. McMillian, G. Medoff, D. Schlessinger, 7969. and G. S. Kobayashi, manuscript in prepara- 8. Richardson, J. P. 1966. The binding of RNA polymerase to DNA. J. Mol. Biol. 21:83-114. tion). R., H. Goto, K. Arima, and Y. Sasaki. 1974. It is not clear whether RNA inhibition of 9. Sasaki, Effect of polyribonucleotides on eukaryotic DNA-deRNA polymerases has any role in intact H. pendent RNA polymerases. Biochim. Biophys. Acta 366:435-442. capsulatum. However, it may help to explain D., M. Ono, N. Nikolaev, and L. Silengo. why extracts of the mycelial phase of the orga- 10. Schlessinger, of 30S preribosomal ribonucleic 1974. Accumulation nism show only a single RNA polymerase peak acid in an Escherichia coli mutant treated with chlorof low activity (1), because the single RNA amphenicol. Biochemistry 13:4268-4271. polymerase peak of low activity can be con- 11. So, A. G., E. W. Davie, R. Epstein, and A. Tissieres. 1967. Effects of cations on DNA-dependent RNA poverted to several RNA polymerases with higher Proc. Natl. Acad. Sci. U.S.A. 58:1739-1746. lymerase. are purified 12. Stewart, L. E., activity when the enzyme extracts and R. C. Krueger. 1976. Nuclear riboof contaminating RNA (R. McMillian, B. V. nucleoproteins as inhibitors of mammalian RNA polymerase. Biochim. Biophys. Acta 425:322-333. Kumar, G. Medoff, D. Schlessinger, and G. S.
JOURNAL OF BACTERIOLOGY, June 1977, P. 1387-1389 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Inhibition of Histoplasma capsulatum Ribonucleic Acid Polymerases by Homologous and Heterologous Ribonucleic Acid RAY McMILLIAN,* B. VIJAY KUMAR, GERALD MEDOFF, DAVID SCHLESSINGER, AND G. S. KOBAYASHI Divisions ofInfectious Diseases, Dermatology and Laboratory Medicine, Departments of Medicine and Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110 Received for publication 21 January 1977
The ribonucleic acid (RNA) polymerases from the yeast phase of Histoplasma capsulatum are differentially sensitive to RNA isolated from the yeast and mycelial phases of this fungus and from Escherichia coli. Low-molecular-weight RNA from H. capsulatum was the most effective inhibitor. The pathogenic dimorphic fungus Histoplasma capsulatum grows in a multicellular filamentous phase when incubated at 230C and in a yeast phase at 370C. Previous studies from this laboratory have shown that yeast-phase cells contain three species of ribonucleic acid (RNA) polymerases (2), whereas mycelia have one species of RNA polymerase of very low activity (1). In our attempts to purify the RNA polymerases of H. capsulatum, we have discovered that the yeast-phase RNA polymerases are differentially sensitive to different RNA classes from both phases of H. capsulatum and from Escherichia coli. Here we present the data from these findings. H. capsulatum (Down strain, mating-type [-] from our laboratory) was grown in the yeast phase at 370C and in the mycelial phase at 250C in glucose-yeast extract medium as previously described (2). E. coli B was grown at 250C in minimal salts medium fortified with 0.8% Casamino Acids (7). Cells were harvested by filtration, washed with distilled water, and processed immediately. RNA polymerases from the yeast phase of H. capsulatum were partially purified by phosphocellulose chromatography and assayed (2). One unit of RNA polymerase activity was defined as the amount of enzyme that catalyzed the incorporation of 1 pmol of [3H]uridine 5'-monophosphate into RNA in 60 min. The nomenclature of RNA polymerases from H. capsulatum was discussed in our previous publication (2) and is also used throughout this report. Inhibition of RNA synthesis was measured by adding 8 to 320 ng of RNA to the RNA polymerase reaction mixture and comparing the incorporation of [3H]uridine 5'triphosphate into RNA with the incorporation in the absence of exogenously added RNA.
The RNA was prepared from H. capsulatum by homogenizing yeast or mycelial cells with glass beads in a Braun homogenizer (3) and extracting the RNA from the lysate by hot sodium dodecyl sulfate-phenol (5). The procedure used to prepare RNA from E. coli B has been described (10). The RNA was fractionated on 10 to 30% sucrose gradients (10), and individual fractions corresponding to 26S, 18S, and 4 to 6S RNA from H. capsulatum and 23S, 16S, and 4 to 5S fromE. coli were pooled. There was no cross-contamination among these pooled components, as judged by polyacrylamide gel electrophoresis. Low-molecular-weight RNA (4 to 6S) was fractionated on 6.8% polyacrylamide gels (6). Electrophoresis was carried out for 2.5 h at 5 mA per gel. The effects of 26S, 18S, and 4 to 6S RNA from the yeast phase of H. capsulatum on yeastphase RNA polymerases and E. coli RNA polymerase are shown in Fig. 1. RNA polymerase PCIII was sensitive to low concentrations of all three classes of RNA but was most sensitive to 4 to 6S RNA (100% inhibition at 160 ng/125 ,ul of RNA). RNA polymerase PCI was not very sensitive to 26S and 18S RNA (30% inhibition at 320 ng/125 ,ul of RNA) but was as sensitive to the 4 to 6S RNA as PCIII. RNA polymerase PCII was not very sensitive to any of the three RNA classes (30% inhibition at 320 ng/125 ,ul of RNA). E. coli RNA polymerase was resistant to all of the concentrations of 26S and 18S RNA used in the assay, but it was sensitive to 4 to 6S RNA (85% inhibition at 320 ng/125 ,ul of RNA). The effects of 26S, 18S, and 4 to 6S RNA from the mycelial phase of H. capsulatum on yeastphase RNA polymerases and E. coli RNA polymerase were also examined. The pattern of
1387
1388
J. BACTERIOL.
NOTES
RNA polymerase sensitivity to the mycelial RNAs was the same as that seen with the yeast RNAs, but the mycelial RNAs were 1.5 times more inhibitory (data not shown). Figure 2 shows the effects of 23S, 16S, and 4 to 5S RNA from E. coli on the yeast-phase RNA polymerases and E. coli RNA polymerase. The sensitivity of the yeast-phase RNA polymerases to E. coli 23S and 16S RNA was similar to that of H. capsulatum 26S and 188 RNA. The RNA polymerases were less sensitive to 4 to 5S RNA from E. coli (60, 35, and 30% inhibition at 320 ng/125 ,ul of RNA for PC0II, PCI, and E. coli RNA polymerase, respectively) than to E. coli 23S and 16S RNA and H. capsulatum 4 to 6S RNA. Yeast- and mycelial-phase 5.8S, 58, and 4S 10 o0
II
26S
30 _ D
RNAs purified from the 4 to 68 RNA fraction were as inhibitory as the total fraction of lowmolecular-weight RNA when tested against the yeast-phase RNA polymerases. RNA polymerases PCI and PCIII were very sensitive to the three species of low-molecular-weight RNA isolated from both phases of H. capsulatum, whereas RNA polymerase PCII was resistant (data not shown). The sensitivity of RNA polymerase PCIII was not altered by the addition of increasing amounts of RNA polymerase PCII to the reaction mixture, which implies that the resistance to RNA inhibition associated with polymerase PCI1 was specific and could not protect polymerase PCIII from the RNA inhibition (data not shown). Moreover, the addition of large
PC
!//
0
6
0-
2)
oA K
A 80
' Aco/,
160
240
320 0
80
160
240
320 0
IE
80
Histoplasma capsulatum yeast phase RNA, (ng/125il)
FIG. 1. Inhibition of RNA polymerases from the yeast phase of H. capsulatum and from E. coli by sucrose gradient fractionated yeast-phase RNAs. RNA polymerases PCI (1.8 U, 0), PCII (2.0 U, 0), and PCIn (2.2 U, 0) and E. coli RNA polymerase (3.0 U, A) were added to the standard assay mixture containing varying amounts of RNA. The amount of inhibition was determined as described in the text. l
l_ _
100 _ 23S
16S
4
C,
55
PC
80 -
o2
E
60 0
40
-
PC
a-
PC~~~~~~~~~~~~~~~~~~~~~~~P PC~~~~~~~~",
20
C~~
0
0
80
-A-A 160 240 320 0 80
160
240
320 0
160
240
320
Escherichia coli RNA, (ng/125 uil)
FIG. 2. Inhibition of RNA polymerases from the yeast phase of H. capsulatum and from E. coli by sucrose gradient fractionated E. coli RNAs. RNA polymerases PCI (1.8 U, 0), PCII (2.0 U, 0), and PCIII (2.2 U, 0), and E. coli RNA polymerase (3.0 U, A) were added to the standard assay mixture containing varying amounts of RNA. The amount of inhibition was determined as described in the text.
VOL. 130, 1977
NOTES
1389
amounts (500 ,ug/125 /.l) of bovine serum albu- Kobayashi, manuscript in preparation). min to the reaction mixture did not affect the This work was supported by Public Health Service sensitivity of RNA polymerase PCIII to 4S grants AI 10622 and Al 06213 from the National Institute of RNA, which indicates that the resistance of Allergy and Infectious Diseases; training grants AI 00459 of Allergy and RNA polymerase PCII to RNA inhibition was and AI 07015 from the National Institute Infectious Diseases, and AM 05611 from the National Instinot secondary to the presence of large amounts tute of Arthritis, Metabolism and Digestive Diseases; and of nonspecific protein (data not shown). by grants from the John A. Hartford Foundation and the The inhibition of bacterial RNA polymerase Research Corporation (Brown-Hazen Fund). R. A. Mcby RNA has been described previously (4, 8, Millian was supported by Public Health Service fellowship from the National Institute of Allergy 11), but there have been only two reports on the 5 F 22 AI 01132-02 Diseases. inhibition of other eucaryotic RNA polymer- and Infectious LITERATURE CITED ases by RNA (9, 12). They have shown that RNA binds to the enzyme and that the inhibi- 1. Boguslawski, G., G. Medoff, D. Schlessinger, and G. Kobayashi. 1975. Histin, an RNA polymerase inhibtion can be reversed by the addition of increasitor isolated from Histoplasma capsulatum. Biochem. ing amounts of DNA. Our studies systematically Biophys. Res. Commun. 64:625-632. looked at the effects of three classes of RNA 2. Boguslawski, G., D. Schlessinger, G. Medoff, and G. from H. capsulatum and E. coli on the RNA Kobayashi. 1974. Ribonucleic acid polymerases of the yeast phase of Histoplasma capsulatum. J. Bacteriol. polymerases from these organisms and found 118:480-485. that RNA polymerase PCIII. was sensitive and 3. Cheung, S-S. C., G. S. Kobayashi, D. Schiessinger, and was inhibited by all of the RNAs tested; RNA G. Medoff. 1974. RNA metabolism during morphopolymerase PCI and E. coli RNA polymerase genesis in Histoplasma capsulatum. J. Gen. Microbiol. 82:301-307. were only sensitive to low-molecular-weight C. F., R. I. Gumport, and S. B. Weiss. 1965. RNA; and RNA polymerase PCII was not sensi- 4. Fox, enzymatic synthesis of ribonucleic acid. V. The The tive to any of the classes of RNA. This differeninteraction of ribonucleic acid polymerase with nutial sensitivity of the enzymes to the different cleic acids. J. Biol. Chem. 240:2101-2109. RNAs indicates that the inhibition is not non- 5. Kirby, K. S. 1965. Isolation and characterization of ribosomal ribonucleic acid. Biochem. J. 96:266-269. specific. In addition, a further experiment U. E. 1967. The fractionation of high molecuwhich supports the notion that the inhibition of 6. Loening, lar weight ribonucleic acid by polyacrylamide gel RNA polymerases by low-molecular RNA is electrophoresis. Biochem. J. 102:251-257. specific for certain classes of RNA is that the 7. Nikolaev, N., L. Silengo, and D. Schlessinger. 1973. A role for ribonuclease III in processing of ribosomal RNA polymerases were not inhibited by polyribonucleic acid and messenger ribonucleic acid preadenylic acid or polycytidylic acid (B. V. Kucursors in Escherichia coli. J. Biol. Chem. 248:7967mar, R. McMillian, G. Medoff, D. Schlessinger, 7969. and G. S. Kobayashi, manuscript in prepara- 8. Richardson, J. P. 1966. The binding of RNA polymerase to DNA. J. Mol. Biol. 21:83-114. tion). R., H. Goto, K. Arima, and Y. Sasaki. 1974. It is not clear whether RNA inhibition of 9. Sasaki, Effect of polyribonucleotides on eukaryotic DNA-deRNA polymerases has any role in intact H. pendent RNA polymerases. Biochim. Biophys. Acta 366:435-442. capsulatum. However, it may help to explain D., M. Ono, N. Nikolaev, and L. Silengo. why extracts of the mycelial phase of the orga- 10. Schlessinger, of 30S preribosomal ribonucleic 1974. Accumulation nism show only a single RNA polymerase peak acid in an Escherichia coli mutant treated with chlorof low activity (1), because the single RNA amphenicol. Biochemistry 13:4268-4271. polymerase peak of low activity can be con- 11. So, A. G., E. W. Davie, R. Epstein, and A. Tissieres. 1967. Effects of cations on DNA-dependent RNA poverted to several RNA polymerases with higher Proc. Natl. Acad. Sci. U.S.A. 58:1739-1746. lymerase. are purified 12. Stewart, L. E., activity when the enzyme extracts and R. C. Krueger. 1976. Nuclear riboof contaminating RNA (R. McMillian, B. V. nucleoproteins as inhibitors of mammalian RNA polymerase. Biochim. Biophys. Acta 425:322-333. Kumar, G. Medoff, D. Schlessinger, and G. S.
