Partial purification and characterization of DNA-dependent RNA polymerases I and II from cherry salmon (Oncorhynchus masou)

Comp. Biochem. Physiol. Vol. 74B, No. 4, pp. 719 to 723, 1983

0305-0491/83/04071%05503.00/0 © 1983 Pergamon Press Ltd

Printed in Great Britain

PARTIAL P U R I F I C A T I O N A N D C H A R A C T E R I Z A T I O N O F D N A - D E P E N D E N T R N A P O L Y M E R A S E S I A N D II F R O M CHERRY S A L M O N (ONCORHYNCHUS MASOU) CHIKAO NAKAYAMA, MINEO SANEYOSHI*, SHIGEHARU TAKIYA and MASAKI IWABUCHIJ" *Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan and tDepartment of Botany, Faculty of Science, Hokkaido University, Sapporo 060, Japan

(Received 23 Au(just 1982)

Abstract--1. DNA-dependent RNA polymerases I and II were purified approx 3900- and 13,000-fold, respectively, from sonicated nuclear extract of cherry salmon (Oncorhynchus masou) liver by DEAESephadex, heparin-Sepharose and DNA-cellulose column chromatography. 2. The purified RNA polymerases exhibited a requirement for four kinds of ribonucleoside 5'-triphosphates, an exogeneous template and divalent cation. 3. The activities of RNA polymerases I and II were inhibited by Actinomycin D (24/~g/ml) but not by Rifampicin (200 #g/ml). 4. RNA polymerase I preferred native DNA as template, while polymerase II preferred singlestranded DNA. 5. RNA polymerase II was inhibited by a low concentration of c~-amanitin (0.02 #g/ml). RNA polymerase I was also inhibited by the relatively high concentration of ~-amanitin (IC50 = 100pg/ml and IC70 = 750 ~g/ml). 6. RNA polymerases from cherry salmon exhibited a higher activity at low temperature than from rat liver.

INTRODUCTION RNA polymerases of eukaryotes are k n o w n to be multiple forms and their properties were extensively investigated in mammals, insects, protozoa, yeast and plants (Chambon, 1974; Roeder, 1976). These multiple forms of R N A polymerases can be classified into three types on the basis of their order of elution from D E A E - S e p h a d e x column (RNA polymerase I, II and III) or by the sensitivity to ~-amanitin (RNA polymerase A, B and C). R N A polymerases have been purified from vertebrates, a great part of study was concerned with m a m m a l i a n R N A polymerases. Gillam et al. (1979) recently reported the partial characterization of trout RNA polymerase. However, very little is known of the general properties of RNA polymerases from fishes. Cherry salmon (Oncorhynchus masou) is recognized as the most primitive species of pacific salmon (genus Oncorhynchus) and is found only on the Asian side of north-western Pacific Ocean. Generally, the salmon family (Salmonidae) inhabits relative cold environments and the habitable surroundings are quite different from those for m a m m a l s and other eukaryotes. Therefore, it is of interest to compare the properties of R N A polymerases of salmonid fishes with those of mammals. In this paper, we describe the purification procedure for R N A polymerases I and II of cherry salm o n liver and some of their properties. The results s h o w e d that the R N A polymerase I differed from that of other eukaryotes in c~-amanitin sensitivity and in having a significant activity at low temperature.

MATERIALS A N D M E T H O D S

Cherry salmon Mature cherry salmon of 3-yr old were killed and their livers removed and promptly frozen with solid carbon dioxide and kept at -80°C until use.

Chemicals [5-3H]UTP (19-23 Ci/mmole) was purchased from New England Nuclear Corporation. ATP, CTP, GTP and UTP were obtained from Yamasa Shoyu Co. Ltd. Salmon sperm DNA from P-L Biochemicals; Ribonuclease A, heparin, dithiothreitol (DTT), rifampicin, actinomycin D and bovine serum albumin (BSA) from Sigma; Ribonuclease T~ from Worthington Biochemical; c~-amanitin from Boehringer Mannheim; All other chemicals were of analytical grade. Heparin-Sepharose and native DNA~ellulose was prepared by the methods of Irverius (1971) and Litman (1968), respectively.

Protein determination Protein was determined according to Lowry et al. (195 l) with BSA as standard.

Assay of RNA polymerase activity The RNA polymerase activity was assayed as reported by Roeder (1974). The reaction mixture contained 50 mM Tris-HC1 (pH 7.9), 1 mM MnC12, 60 mM ammonium sulfate for polymerase I or 110 mM for polymerase II, 0.1 mM EDTA, 4 mM DTT, 200/~g/ml native salmon sperm DNA, 500,ug/ml BSA, 10% glycerol, 10pM [3H]UTP (2~Ci/ nmole), 0.5mM each of ATP, CTP and GTP in final volume of 51/ft. Each enzyme reaction was initiated with the addition of 10#1 of enzyme solution and incubation was carried out for 10 min at 25°C. Then the mixture was chilled and transfered to DEAE-cellulose paper discs (Whatman DE-81). The discs were washed with 5% 719

CHIKAO NAKAYAMAet al.

720

RESULTS

g ,0 /"

~0

Purification of RNA polymerases from cherry salmon liver nuclei

10,z 0.4

Jo e 0

10

20 30 fraction number

/gO

Fig. I. DEAE Sephadex column chromatography of nuclear RNA polymerases from cherry salmon (Oncorhynchus masou) liver. The column was washed with 200 ml of buffer B containing 0.05 M ammonium sulfate and eluted with 250 ml of a linear gradient of 0.05-0.5 M ammonium sulfate in buffer B at a flow rate of 30 ml/hr. Fractions of 5 ml were collected and assayed in the absence (© ©) and presence (.O) of e-amanitin. Absorbance at 280 nm (. . . . . ). Ammonium sulfate concentration ( ). Na2PO4 and dried and remaining radioactivity was measured in a Packard Tri-Carb liquid scintillation counter with toluene scintillator.

Isolation of nucleifrom cherry salmon liver Isolation of nuclei from cherry salmon liver was carried out by the method of Widnell & Tata (1964) with slight modifications. All of the procedures were performed at (~5°C unless otherwise noted. Cherry salmon liver (250 g) was minced in 750 ml of 0.34 M sucrose, 5 mM MgC12, 0.5~o Triton × 100 and the mixture was homogenized using Teflon pestle homogenizer (600 rev/min, 4 stroke). The homogenate was filtered through four layers of gauze and filtrate was centrifuged at 800 g for 5 min. The pellet was washed with 250 ml of 0.34 M sucrose containing 5 mM MgCI 2 by pipetting and centrifugated (800g, for 5 rain). The nuclear pellet was washed again and immediately used for the next step or stored at -20°C.

+

RNA polymerases from isolated nuclei were solubilized by the m e t h o d of Roeder & Rutter (1969) with a slight modification as follows. Isolated nuclei (from 250g of salmon liver) were suspended in buffer A [ 5 0 m M Tris-HCl, pH7.9, 5 m M MgCl2, 1 0 m M fl-mercaptoethanol (fl-ME), 0.1 m M EDTA, and 25~o glycerol] and sonicated in the presence of 0.5 M amm o n i u m sulfate until the viscosity of the solution was disappeared (usually, it is necessary to continue the sonication 12 times for 15 at 30 sec intervals under 180W in Tomy Seiko, UR-200P). The solution was centrifuged at 26,000 g for 40 min. To the supernatant (termed nuclear extract) was added polyethylene glycol[-(g)/nuclear extract ( m l ) = 0.3]. The supernatant was diluted to 0.05 M a m m o n i u m sulfate with buffer B (buffer A minus MgCI2) and it was mixed with D E A E - S e p h a d e x (dry weight, 8 g) which is previously equilibrated in a buffer B containing 0.05 M amm o n i u m sulfate. The suspension was stirred gently for 4 0 m i n and then poured into a column (1.6 x 21.5 cm). The column was washed with buffer B containing 0.05 M a m m o n i u m sulfate a n d eluted with a 0.054).5M linear a m m o n i u m sulfate gradient in buffer B. A typical elution profile of R N A polymerase activity on D E A E - S e p h a d e x column c h r o m a t o g r a p h y is shown in Fig. 1. The R N A polymerase activities were separated into three distinct peaks a n d the first peak ( D E I ) was resistant to 1 #g/ml of a-amanitin, while the second a n d third peaks were sensitive to the same concentration of ~-amanitin (Fig. 1). Therefore, we tentatively assigned the activity of the first peak to R N A polymerase I and those of the second and third peaks to R N A polymerase IIa and IIb, respectively, as reported for R N A polymerases of a m p h i b i a n (Roeder, 1974) and calf thymus (Kedinger & C h a m b o n , 1972). The polymerase activities in the second and third peaks were c o m b i n e d and termed the D E II fraction. The D E ! and D E II fractions were separately applied

o

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o

0.2

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00o

10 Fraction number

15

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Fraction number

Fig. 2. (A) Heparin-Sepharose column chromatography of RNA polymerase I and II (BB). The DE I and DE II fractions were applied to heparin-Sepharose (0.6 × 4cm) as described in the text. The column was washed with 20 ml of buffer B containing 0.2 M ammonium sulfate and then eluted with 20 ml of buffer B containing 0.5 M ammonium sulfate at a rate of l0 ml/hr. Fractions of 2-3 ml were collected and assayed as described in Methods. Activity of RNA polymerase ( ). Absorbance at 280 nm (. . . . . ).

RNA polymerases from cherry salmon

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~8

0

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number

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Fig. 3. Native DNA-cellulose column chromatography of RNA polymerase II. The dialysate of Hep II fraction was applied to a native DNA cellulose column (0.6 × 3.5 cm). The column was washed with 0.05 M ammonium sulfate in buffer C and eluted with 0.25 M ammonium sulfate in buffer C. Flow rate, 10ml/hr. Fraction size, 1 ml/tube, RNA polymerase activity ( ). Absorbance at 280nm ( .....

I.

to a column of h e p a r i n - S e p h a r o s e (0.6 x 4 c m ) pre-equilibrated with buffer B containing 0 . 2 M a m m o n i u m sulfate a n d then eluted with 0 . 5 M a m m o n i u m sulfate in buffer B. (Fig. 2).

721

The fractions containing polymerase activity were pooled termed Hep I a n d Hep II. The Hep II fraction was dialyzed against buffer C ( 5 0 m M Tris-HCl, pH 7.9, 0 . 1 m M EDTA, 1 0 m M fl-ME and 5 0 ~ glycerol) for 15 hr and then applied to a column of native DNA-cellulose previously equilibrated with 0.05 M a m m o n i u m sulfate in buffer C. The column was washed with buffer C containing 0.05 M a m m o n i u m sulfate and then eluted with 0.25 M a m m o n i u m sulfate in buffer C (Fig. 3). The fractions containing RNA polymerase activity were collected and termed D N A II. The extent of purification of R N A polymerase I and II are summarized in Table 1. R N A polymerases I and II were purified by 3900- and 13,300-fold, respectively, c o m p a r e d with the enzyme activity in the nuclear extract. This fraction was stable for several m o n t h s at - 8 0 ° C in the presence of 2 . 5 m g / m l of BSA in buffer C.

Requirement for RNA polymerase activity and characterization of reaction product Requirement for R N A polymerase activity was studied with H p I and D N A II fractions (Table 2). Both the enzymes required D N A as template, divalent cation, four kinds of ribonucleoside 5'-triphosphates. The R N A polymerase activity was inhibited in the presence of 2 4 p g / m l of actinomycin D. W h e n the reaction mixture was incubated with ribonuclease A

Table 1. Purification of RNA polymerases I and II from cherry salmon liver Total protein (mg)

Purification step Nuclear extract

(I) (II)

DEAE-Sephadex

(I) (II)

Heparin-Sepharose DNA~zellulose

1832

Total activity (units)

Specific activity (units/mg)

Fold

13.9 39.2

0.008 0.021

1 1

7.74 6.54

5.4 57.1

0.698 8.78

90 410

(I) (II)

0.2 l 0.50

6.2 26.5

29.52 53.00

3880 2480

(II)

0.18

51.4

285.50

13,340

Values were obtained in one representative experiment on 250g of cherry salmon liver. One unit = 1 nmol of UMP incorportated to RNA in 10 min at 25°C. In nuclear extracts, polymerase I activity was determined in the presence of ~-amanitin (1/~g/ml) and the polymerase II activity was estimated by subtracting polymerase I activity from the total activity.

Table 2. Requirements for RNA polymerase activity Condition Complete Complete CompleteComplete Complete Complete -

template DNA Mn 2+ Mn 2+ + Mg 2+ ATP, CTP, GTP DNA plus heat-denatured DNA

(~o) of Control synthesis RNA polymerase I RNA polymerase II 100 5 0 86

100 9 0 30

1

1

80

170

Assay conditions were as described in the Methods. Heat-denatured DNA was prepared by heating at 100° for 15 min and then cooled rapidly in an ice-bath.

CH[KAO NAKAYAMAet al.

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Fig. 4. The effect of Mn 2* and Mg 2+ on RNA polymerases I (A) and II (B). The enzyme activity were assayed under standard conditions except for the concentration of MnC12 ( ) and MgCI 2 (. . . . . ).

or Under the alkaline conditions (1 N NaOH), no radioactivity remained in the product. Therefore, the isolated enzymes must be D N A - d e p e n d e n t RNA polymerases. RNA polymerase I prefered native D N A to single-stranded D N A as template, while RNA polymerase II prefered single-stranded DNA. These properties were confirmed in other eukaryotic RNA polymerases I and II. In addition, salmon RNA polymerases were not inhibited even by a high concentration (200/~g/ml) of rifampicin, as reported in eukaryotic RNA polymerases.

El[fects of Mg 2+, M n 2+ and ammonium su!fate on R N A polymerase activity Figure 4 showed the effects of divalent cations such as Mg 2 +, M n 2 + and a m m o n i u m sulfate on the activities of RNA polymerases I and II. The optimal concentrations of Mg 2+ and M n 2+ for both enzyme activities were 5 and 1 mM, respectively. However, RNA polymerase I utilized both M g z+ and M n z+ to the almost same extent, while R N A polymerase II prefered M n 2+ rather to Mg 2+. The M n 2 + / M g 2+ ratio

0

, "3 10

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o n Pol. [I

r'.~ b. , -I 0 10 10

OL - a m a n i t i n ( pg/mL )

Fig. 6. Effects of ~-amanitin on RNA polymerases I and II. RNA polymerase activity was measured in the presence of c~-amanitin (10 3 to 7.5 x 102 #g/ml). RNA polymerase I from O. masou (O O), RNA polymerase I from rat liver (O O) RNA polymerase l] from O. masou ( A - - - G).

for the enzyme activity was estimated to be 1.2 for polymerse I and 3.3 for polymerase II. Figure 5 shows the effect of a m m o n i u m sulfate. The optimal salt concentration for polymerase I was lower than that for polymerase II (110 mM). These results were similar to those obtained with other eukaryotic RNA polymerases I and II.

~-Amanitin sensitivities Effect of c~-amanitin on R N A polymerases I and II was summarized in Fig. 6. R N A potymerase II from salmon liver exhibited the same behavior with :~-amanitin as other eukaryotic RNA polymerase II (ICso = 0.05/2g/ml) did. Interesting enough, the activity of R N A polymerase I of salmon liver was inhibited about 70% by 750#g/ml of c~-amanitin to which almost all of eukaryotic RNA polymerase I were resistant. Indeed, rat liver RNA polymerase I, which was prepared by essentially same procedure used for salmon liver, was not inhibited by ~-amanitin of the concentration used in our system.

Effect of temperature 100

>.

,~ 50

<

16o

260

(raM)

Fig. 5. Effects of ammonium sulfate concentration on RNA polymerases I and II. Activities of RNA polymerases were measured under the standard conditions except for the concentration of ammonium sulfate as shown in the figure. The open and closed circles represent RNA polymerases I and II, respectively.

The activities of salmon R N A polymerases I and II (Hep I and Hep II) were tested at various temperature and their temperature effect was compared with that of rat liver R N A polymerases (Fig. 7). In the case of salmon liver polymerase I, incubation at 20 and 15°C brought a b o u t 48 and 28% of enzyme activity at its optimal temperature. O n the other hand, in the case of rat liver RNA polymerase I only 25 and 15% of enzyme activity was observed at 20 and 15°C respectively. Thus, the activity of RNA polymerase I was retained to relatively high level at low temperature compared with that of rat liver RNA polymerase I. Similar p h e n o m e n o n was observed with polymerase II (Fig. 7). DISCUSSION

D N A - d e p e n d e n t RNA polymerases were partially purified from nuclear extract of cherry salmon (Oncorhynchus masou) liver. These polymerases belong to

RNA polymerases from cherry salmon

A

B

IO0

7v

8o

:_;Oo <4O d 20

20

o"

o" 10 °

20 °

30 °

40 °

50 °

Temperature ( C )

0

10"

20* 30* 40* 50' Temperature ( C )

Fig. 7. Effect of temperature on cherry salmon and rat RNA polymerases. Activities of RNA polymerases were measured in complete assay conditions at various temperature for 10 rain. The enzyme activity was compared with those at optimal temperature. O. masou (0 ¢) and rat

(o--o). type I and II enzymes on the basis of following criteria. (i) A characteristic elution pattern of the enzyme in D E A E Sephadex column chromatography; polymerase I was eluted at a lower concentration of ammonium sulfate than polymerase II. (ii) Difference in the template specificity: polymerase I prefered native D N A as template while polymerase II prefered single-stranded DNA. (iii) Difference in the Mn2+/Mg 2+ ratio for enzyme activity; polymerase II required much higher concentration of Mn 2÷ than Mg 2+, while polymerase I needed Mn 2+ and Mg 2÷ equally. (iv) Difference in the optimal concentration of ammonium sulfate for enzyme activity; the optimal concentration of ammonium sulfate for polymerase I activity was lower than that for polymerase II. Thus, the enzymatic properties of salmon RNA polymerases generally resemble those of the eukaryotic RNA polymerases which have been reported by many workers. However, polymerases I was quite unique in ~-amanitin sensitivity compared with polymerase I of other eukaryotes. It should especially be noted that RNA polymerase I of cherry salmon is sensitive to c~-amanitin, because it has not been reported yet that vertebrate RNA polymerase I is sensitive to the toxin. In yeast, however, RNA polymerase I has been shown to be sensitive to 300-600 #g/ml 7-amanitin (Sculz & Hall, 1976). In addition, Huet et al. (1975) have reported that yeast RNA polymerase I lacking two polypeptide chains compared to normal RNA polymerase I became more sensitive to c~-amanitin. This report implies that the sensitivity of RNA polymerase I to ct-amanitin is largely affected by the structure of the enzyme consisting of polypeptide subunits. The degree of the enzyme purification should not have influenced the c~-amanitin sensitivity, because rat liver RNA polymerase I was completely resistant to this toxin. Therefore, it is likely that the subunit structure of salmon polymerase I is different from that of other vertebrate polymerase I. Gillam et al. (1979) have recently reported that crude RNA polymerase I from rainbow trout (Salmon gairdneri) was resistant to ct-amanitin at lO0#g/ml. We also examined the ct-amanitin sensitivity of RNA polymerase I purified from rainbow trout liver. The c.~,P. 74/4B

[)

723

results showed that trout enzyme was completely resistant to ~-amanitin of 750 #g/ml (data not shown). So, it should be interesting to compare the subunit structure of R N A polymerases of salmon, trout yeast and mammals. Another interesting observation was the effect of temperature on the enzyme activity, As can be seen in Fig. 7, the activity of RNA polymerases was still retained to a significant extent at low temperature, compared with those of rat liver RNA polymerases. Gillam et al. (1979) reported that RNA polymerases from rainbow trout showed a significant activity at low temperature, although the data were not shown in their paper. The result may explain one of the reasons why cherry salmon can inhabit and adapt to cold water in northern environments. Note--This forms part of the requirements of Chikao Nakayama for a Ph.D degree from Hokkaido University (1981). Acknowled~lements--The authors are indebted to Mr Masaaki Uchiyama, Director of Mori Branch, Hokkaido Fish Hatchery and his staff for collecting cherry salmon liver in September, 1978-1980. We also thank to Mr. I. Sato, Mr. N. Yoshida and Mr. N. Imada for their maintaining cherry salmon in tank. Some of cherry salmon used in this study were collected in Shakotan river, Atsuta river and Furuu river under special collecting permit from Governor of Hokkaido. This work was supported in part by Grant-in-Aid for Scientific research from the Ministry of Education, Science and Culture of Japan to M.S. and M.I. REFERENCES

CHAMBON P. (1974) The Enzymes, 3rd edn, Vol. 10, pp. 261-331. Academic Press, New York. GILLAM S., ALHNE R., WYLE V., INGLESC. J. & SMITH M. (1979) RNA synthesis and RNA polymerase activities in germ cells of developing rainbow trout testis. Biochim. biophys. Acta 565, 275-292. HUET J., BUHLER J.-M., SENTENAGA., & FROMAGEOT P. (1975) Dissociation of two polypeptide chains from yeast RNA polymerase A. Proc. natn. Acad. Sci. U.S.A. 72, 3034-3038. IRERIUS P.-H. (1971) Coupling of glycosaminoglycans to agarose beads (Sepharose 4B). Biochem. J. 124, 677-683. KEOINGER C. & CHAMnONP. (1972) Animal DNA-dependent RNA polymerase. 3. Purification of calf thymus BI and BII enzymes. Eur. J. Biochem. 28, 283 290. LITMAN R. M. (1968) A deoxyribonucleic acid polymerase from M. luteus isolated on deoxyribonucleic acid cellulose. J. biol. Chem. 243, 6222-6233. LOWRY O. H., ROSEBROUGHN. J., FARR A. L., & RANDALL R. J. (1951)Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. ROEDER R. G. (1974) Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus larvis. Isolation and partial characterization. J. biol. Chem. 249, 241-248. ROEDER R. G. (1976) R N A polymerase, pp. 285-329. Cold Spring Harbor Lab. ROEDER R. G. & RUTTER W. J. (1969) Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature. Lond. 224. 234-237. SCHULZL. D. & HALL B. D. (1976) Transcription in yeast: ~-Amanitin sensitivity and other properties which dist inguish between RNA polymerase I and III. Proc. Nat. Acad. Sci, U.S.A. 73, 1029-1033. WIDNELL C. C. & TATA J. P. (1964) A procedure for the isolation of enzymically active rat-liver nuclei. Biochem. J. 92, 313-317.