The isolation and properties of a DNA-directed RNA polymerase from yeast mitochondria

331

Biochirnica et Biophysica Acta, 442 ( 1 9 7 6 ) 3 3 1 - - 3 4 2 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s

BBA 9 8 6 7 5

THE ISOLATION AND PROPERTIES OF A DNA-DIRECTED RNA POLYMERASE FROM YEAST MITOCHONDRIA

A L A N H. S C R A G G *

The National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA (U.K.) (Received M a r c h 1 2 t h , 1976)

Summary A method is described for the rapid isolation of yeast mitochondrial DNAdirected RNA polymerase. The enzyme obtained had a specific activity of 1.56 nmol UMP incorporated per mg protein in 20 min at 37°C, and is some 95% pure. This purified enzyme upon polyacrylamide gel electrophoresis consists of a single polypeptide of 68 000 mol. wt. However, the enzyme forms aggregates easily which are affected by ionic strength, an increase decreasing the apparent molecular weight of the aggregates. This property also explains the presence of two peaks of activity upon DEAE-cellulose chromatography. The purified enzyme is still sensitive to rifamycin and to a number of rifamycin derivatives. The enzyme's sensitivity to rifamycin and rifamycin derivatives was compared with Escherichia coli and yeast nuclear RNA polymerases.

Introduction

Isolated mitochondria are capable of independent RNA synthesis [1--4], using a DNA-directed RNA polymerase located within the mitochondrion [5]. As many of the mitochondrial components are bacterial in nature [6--8], it was suggested that the mitochondrial RNA polymerase would also be similar, and sensitive to rifamycin a bacterial RNA polymerase inhibitor [9,10]. However, both rifamycin sensitivity and resistance has been reported for isolated mitochondria and mitochondrial extracts. Isolated yeast mitochondria, and crude extracts from petite mitochondria have shown resistance to rifamycin [11,12]. In contrast, rifamycin sensitivity has been reported with isolated rat liver mitochondria [13], and crude extracts from both yeast [14] and rat liver mitochondria [15]. * Present address: Microbiological Research Establishment, Porton, Salisbury, Wiltshire, SP4 0JG, U.K.

332 Recently a number of mitochondrial RNA polymerases have been solubilized, purified and characterized, showing that these enzymes exhibit properties clearly different from those of their respective nuclear RNA polymerases, and those of bacteria. Unlike both the bacterial [16], and nuclear [17] RNA polymerases, consisting of high molecular weight complexes containir_g a number of different subunits, mitochondrial RNA polymerases appear to consist in the main of a single low molecular weight subunit. The molecular weights are 65 000 forNeurospora crassa [18], 64--68 000 for rat liver [19--21], 46 000 for Xenopus laevis [22] and 60--70 000 for the yeast mitochondrial RNA polymerase [23,24]. These isolated mitochondrial RNA polymerases although clearly different from the nuclear enzyme still exhibit both rifamycin resistance and sensitivity. The isolated mitochondrial RNA polymerases from N. crassa [18], rat liver [19,21] and yeast [14,23] are sensitive to riamycin, whereas those from X. laevis [22] and yeast [24--26] have been reported to be rifamycin resistant. Here the rapid isolation and characterizarion of a low molecular weight (68 000) yeast mitochondrial RNA polymerase, sensitive to rifamycin, is reported. Materials and Methods

Strains and growth conditions. Saccharomyces cerevisae (Strain 239 N.C.Y.C.) was grown to stationary phase in 2% peptone/l% yeast extract/l% glucose at 25°C. Preparation o f mitochondria. Mitochondria were prepared normally from 6 l of cells as described previously [27], but using only one wash before solubi]ization of RNA polymerase. Solubilization o f mitochondrial R N A polymerase. The isolated mitochondria were suspended in buffer B (0.01 M Tris. HC1, pH 7.8/0.002 M MgC12/0.5 mM EDTA/5 mM dithiothreitol/10% glycerol) and either sonicated for 1 min at maximum power (Sonniprobe, Dawe Instruments Ltd., Type 7530A), or incubated with digitonin (final concentration 1%) at 4°C for 30 min. The debris, and unbroken mitochindria were removed by centrifugation for 30 min at 15 000 X g. The supernatant is referred to as a crude extract. Purification o f mitochondrial R N A polymerase. The crude extract (20--30 ml) was applied to a DEAE-cellulose column (20 X 2.5 cm) previously equilibrated with buffer B. The column was washed with 100 ml buffer B, and enzyme activity eluted with a further 100 ml of buffer B containing 0.21 M KC1 and 2-ml fractions were collected. The fractions containing RNA polymerase activity were pooled and dialysed overnight at 4°C against buffer B. This dialysed fraction was either concentrated (X 5) and submitted to glycerol gradient centrifugation, or applied to a DNA-cellulose column. The glycerol gradients (5 ml) were 10--30% (v/v) made up in buffer B containing 0.05 M KC1. These were centrifuged at 4°C for 2 h at 169 000 X g, 0.28 ml fractions collected, and assayed for enzyme activity. The DNA-cellulose column was prepared using denatured calf thymus DNA as described by Alberts [28]. The dialysed enzyme (40 ml) was applied to the column (20 X 1 cm) previously equilibrated with buffer C {0.02 M Tris • HC1,

333 pH 7.4/1 mM EDTA/1 mM ~-mercaptoethanol/10% glycerol/100 pg/ml bovine serum albumin). The column was washed with buffer C until no further material adsorbing at 280 nm was eluted, the column was then further washed with buffer C containing 0.05 M NaCI, and finally the enzyme activity was eluted with C containing 2 M NaCI. In some cases after the first wash a gradient of 0--2 M NaCI was applied. In all cases 2.5-ml fractions were collected. Assay of RNA polymerase activity. The standard reaction mixture contained, per ml; 50 pmol Tris. HCI (pH 7.5 at room temperature), 20 #mol magnesium acetate, 1 pmol MnC12, 1 pmol each of GTP and CTP, 2 pmol ATP, 0.05 pmol UTP, 6 pCi [3H] UTP (10.5 Ci/mmol), 1 mg denatured calf thymus DNA and 0.1--0.4 ml enzyme. Standard assays (55 pl) were incubated for 20 min at 37°C, a 50 pl sample removed, and applied to a Whatman No. 1 filter paper disc (2.4 cm diameter). The discs were treated as described by Bollum [29]. Preparation of yeast nuclear and E. coli RNA polymerases. Yeast nuclear RNA polymerase II(B) was preapred as described by Dezelee and Sentenac [30] and ItNA polymerase I(A) by a modified method of Buhler et al. [31] (omitting the phosphocellulose batch step). Both preparations were used from the glycerol gradient step in these experiments. E. coli RNA polymerase was prepared from frozen E. coli MRE 600 cells and assayed according to Burgess [32]. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate (SDS) was performed as described be Weber and Osborn [33] and gel electrophoresis in the absence of SDS as described by Ornstein and Davis [34]. Materials. Unlabelled nucleoside triphosphates were purchased from Sigma or Boehringer, and [3H]UTP from the Itadiochemical Centre (Amersham, Bucks, U.K.). Ethidium bromide was obtained from Boots Pure Drug Co. Ltd., (Nottingham, U.K.), distamycin A from Sigma, and rifamycin SV from Calibiochem. The rifamycin derivatives were a kind gift from Dr. G. Lancini of Lepetit S.p.A. (Milano, Italy). The frozen E. coli MItE 600 cells were supplied by the Microbiological Itesearch Establishment, (Porton, Salisbury, U.K.). Results

Purification of the mitochondrial RNA polymerase One of the major problems in the purification of the yeast mitochondrial RNA polymerase has been its solubilization in sufficient quantities. A number of methods of solubilization have been tried including treatment with detergents such as Lubrol, Brij 58, Nonidet P40, Triton X-100 and high salt (0.5 M KC1). The only methods found to give consistent results were sonication in low salt buffer, or treatment with digitonin. It has been shown that the mitochondrial ItNA polymerase can be separated from contaminating nuclear RNA polymerase by DEAE-cellulose chromatography [ 23]. Using a KC1 gradient for elution, the mitochondrial enzyme elutes at 0.18--0.21 M KC1 as two peaks of activity, prior to the nuclear enzymes eluting at 0.3 and 0.37 M KC1. This can be simplified and made more rapid by use of a step elution (Fig. 1) which again gives two peaks of activity.

334

B.G.G. CoL

-o9o

1.0 1.5

87c

O.8 E

E

c

c~

1.0 ~ 3.5 Q.

~3( 0.5

2O

,

%

.

.

.

.

'

'

'

Fraction number

20

'

'

I

0

,

,

,

iO Froction number

,

1

I0

20

Fig. 1. D E A E - c e l I u l o s c c h r o m a t o g r a p h y o f c r u d e m i t o c h o n d r i a l e x t r a c t . T h e e x t r a c t ( 2 0 - - 3 0 m l ) w a s a p p l i e d t o a D E A E - c e l l u l o s e c o l u m n ( 2 0 × 2 . 5 c m ) e q u i l i b r a t e d w i t h b u f f e r B a n d e l u t e d as d e s c r i b e d u n d e r M e t h o d s . F r a c t i o n s o f 2 m l w e r e c o l l e c t e d a n d a s s a y e d f o r R N A p o l y m e r a s e a c t i v i t y as d e s c r i b e d under Methods. - - , A b s o r b a n c e a t 2 8 0 rim; e o, [ 3 H I U M P i n c o r p o r a t i o n . Fig. 2. G l y c e r o l g r a d i e n t c e n t r i f u g a t i o n o f m i t o c h o n d r i a i R N A p o l y m e r a s c . T h e D E A E - c e U u l o s e p e a k s w e r e p o o l e d , d i a l y s c d a g a i n s t b u f f e r B a n d c o n c e n t r a t e d (X 5). T h e c o n c e n t r a t e d e n z y m e w a s a p p l i e d t o a 5-ml, 1 0 - - 3 0 % (v/v) g l y c e r o l g r a d i e n t in b u f f e r B c o n t a i n i n g 0 . 0 5 M KC1. C e n t r i f u g a t i o n w a s c a r r i e d o u t a t 1 6 9 0 0 0 X g f o r 2 h a t 4 ° C in a S p i n c o SW 5 0 L r o t o r . F r a c t i o n s o f 0 . 2 8 m l w e r e c o l l e c t e d a n d a s s a y e d f p r R N A p o l y m e r a s e a c t i v i t y as d e s c r i b e d u n d e r M e t h o d s . o o, A b s o r b a n c e a t 2 8 0 n m ; • ~, [ 3 H I U M P i n c o r p o r a t i o n . Parallel g r a d i e n t s w e r e r u n c o n t a i n i n g b o v i n e g a m m a g l o b u l i n ( B . G . G . , tool. w t . 1 5 0 0 0 0 ) a n d c o U a g e n a s e (Col. t o o l . w t . 1 0 9 0 0 0 ) .

Further purification was achieved by either glycerol gradient centrifugation, or by DNA-cellulose chromatography. Fig. 2 shows the combined peaks from the DEAE-cellulose column subjected to glycerol gradient centrifugation. The major peak of activity ran somewhat faster than the protein profile with an apparent molecular weight of 250--300,000, when compared with protein markers run under similar conditions. Minor peaks can be detected, particularly if the centrifugation time was extended. When the enzyme was applied to a denature calf thymus DNA-cellulose column, prepared according to Alberts and Herrick [28], it was eluted with the 2 M NaC1 step. However, this particular preparation method often yielded a preparation containing some 3--4 bands when analysed by polyacrylamide gel electrophoresis, and as such was not used extensively. As can be seen from Fig. 3 the peak of activity from the glycerol gradient centrifugation yields a single band when analysed by polyacrylamide gel electrophoresis in the absence of SDS. This glycerol gradient preparation when analysed by SDS gel electrophoresis gave again a single band, and when compared with a number of known marker proteins this band had a molecular weight of 68 000 (Fig. 4). It has been reported previously [23] that the mitochondrial RNA polymerase had a molecular weight of 59--63 000, this difference is perhaps due to the use of different gel system. A typical preparation is summarized in Table I. The final value for specific activity was 1.56 nmol [3H] UMP incorporated/mg protein /20 min. This represents some 0.7% of the orginal mitochondrial protein but the increase in spe-

335

1.0

DYE

I

0.5

Migrotion

---,--

Fig. 3. E l e c t r o p h o r e s i s o f the m i t o c h o n d r i a i e n z y m e a t v a r i o u s s t a g e s of its p u r i f i c a t i o n . C r e f e r s t o t h e c r u d e e x t r a c t ; D r e f e r s t o t h e D E A E - c e l l u l o s e c o l u m n p e a k s p o o l e d and diaiysed against b u f f e r B: G r e f e r s t o t h e g l y c e r o l gradient f r a c t i o n 1 2 (Fig. 2). E l e c t r o p h o r e s i s in t h e a b s e n c e o f S D S w a s c a r r i e d o u t as described u n d e r M e t h o d s and gels w e r e s t a i n e d w i t h a m i d o s c h w a r t z .

cific activity cannot be accurately measured until the lysate supernatant, as the whole mitochindria and curde lysate contain a considerable proportion of nuclear R N A polymerase [14]. As such this represents an 18-fold increase in specific activity, a somewhat low figure. However, as stated previously [23], the mitochondrial R N A polymerase is particularly unstable, almost all activity being lost at either --20 or 4°C after 2--3 days even in the presence of 30% glycerol, and 5 mM dithiothreitol or bovine serum albumin. This would therefore give greatly reduced specific activities in the more purified prepatations. Mitochondrial extracts contain in particular a large amount of ribonuclease activity. The presence of ribonuclease activity in the various stages of preparation was measured by hydrolysis of 3H-labelled E. coli t R N A (Fig. 5). Clearly there is considreable ribonuclease activity in the crude mitochondrial extract, measured under the conditions of the R N A polymerase assay. This was r e d u c e d somewhat in the DEAE-cellulose preparations, and was absent in the glycerol gradient preparations. In addition, no DNA polymerase activity was found in the glycerol gradient preparations.

Properties o f the mitochondrial RNA polymerase The enzyme has an apparent molecular weight of 250--300 000 u p o n glycerol gradient centrifugation, but upon SDS gel electrophoresis appears to

336

5.0 4.8

"•enose

E4.6

~ ~

4.4

E c

chymotryp-.0 2 t ry~ps~n °gin~O~en 0,1

0

2

4

6

8

Distance from origin

10

O

Fig. 4. S D S gel e l e c t r o p h o r e s i s of the purified m i t o c h o n d r i a l e n z y m e . T h e p e a k f r o m the glycerol gradient centrifugation (Fig. 2) w a s subjected to S D S gel e l e c t r o p h o r e s i s as described by Weber and Osborn [ 3 3 ] . T h e gel was stained with a m i d o s c h w a r t z , and after destaining s c a n n e d with a Unicam gel scanner at 6 7 5 n m . Parallel gels were run w i t h m a r k e r p r o t e i n treated in the same manner; coLlagenasc, m o l . wt. 1 0 9 0 0 0 ; B S A , b o v i n e s e r u m a l b u m i n 6 4 0 0 0 ; c h y m o t r y p s i n o g e n , 2 5 700; trypsin inhibitor, 21 5 0 0 .

consist of a single polypeptide of 68 000 mol. wt. Thus it would appear to form aggregates easily. Such aggregation has been observed with the mitochondrial RNA polymerases from N. crassa [18], yeast [24] and X. laevis [22] where the ionic strength of buffers used has a considerable effect. Fig. 6 shows the effect of ionic strength (KC1) upon the sedimentation of the mitochondrial RNA polymerase through glycerol gradients. At 0.05 M KCI (as in Fig. 2) a single peak was obtained, which decreased in its apparent molecular weight as the KC1 concentration was increased. The slight shoulder obtained at 0.1 M KC1 (Fig. 6) suggested the reason for the two peaks obtained on the DEAE-cellulose columns (Fig. 1), may be due again to ionic strength. Fig. 7 shows the two peaks of activity obtained by DEAE-cellulose chromatography analysed by polyacrylamide gel electrophoreTABLE I P U R I F I C A T I O N OF M I T O C H O N D R I A L R N A P O L Y M E R A S E FROM Y E A S T

Steps

Whole mitochondria Lysate S u p e r n a t a n t (crude extract) DEAE-cellulose peak G l y c e r o l gradient p e a k

Specific activity * protein (mg)

(units/rag)

total enzyme units

Yield (%)

266 250 148 33.6 1.85

0.62 0.40 0.085 0.228 1.56

162 100 12.6 7.6 2.9

100 62.5 7.8 4.7 1.76

* A c t i v i t y unit, 1 n m o l UMP i n c o r p o r a t e d per m g p r o t e i n in 2 0 m i n at 3 7 ° C .

337

1,,

5O

L],

o

4C

o

E_ o_ 3C :E

>_ o

r~ ~_j2c

"1C

0

30

60

go 120 Time ( min )

150

180

il JJ

0o Fr~ction number

Fig. 5. E s t i m a t i o n o f r i b o n u c l e a s e a c t i v i t y in various m i t o c h o n d r i a l p r e p a r a t i o n s . V a r i o u s p r e p a r a t i o n s w e r e a d d e d t o an R N A p o l y m e z a s e m i x t u r e m i n u s label b u t c o n t a i n i n g 0 . 4 5 A 2 6 0 n m units o f 3H-labelled E. coli t R N A ( 7 5 0 0 0 c p m ) and i n c u b a t e d at 3 7 ° C . 50-#I a l l q u o t s w e r e r e m o v e d at intervals and trichl o r o a c e t i c a c i d - i n s o l u b l e r a d i o a c t i v i t y m e a s u r e d as d e s c r i b e d in M e t h o d s . ~ ,7, 2 7 # g crude e x t r a c t ; o o, 1 4 # g D E A E - c e l l u l o s e p e a k If; ~ ~, 7 . 2 # g g l y c e r o l gradient p r e p a r a t i o n ; • A minus enzyme; ¢ -', 2 5 # g m l p a n c r e a t i c r i b o n u c l e a s e . Fig. 6. G l y c e r o l gradient c e n t r l f u g a t i o n o f m i t o c h o n d r l a l R N A p o l y m e r a s e at various KCI c o n c e n t r a t i o n s . T h e D E A E - c e l l u l o s e e n z y m e w a s c o n c e n t r a t e d and d i a l y s e d against b u f f e r B. 2 0 0 #i o f t h e c o n c e n t r a t e d e n z y m e w a s a p p l i e d t o three 5-rni, 1 0 - - 3 0 % (v/v) g l y c e r o l gradients p r e p a r e d in b u f f e r B c o n t a i n i n g t h r e e d i f f e r e n t KCI c o n c e n t r a t i o n s . T h e s e w e r e c e n t r i f u g e d at 1 6 9 0 0 0 X g f o r 1 . 6 h a t 4 ° C i n a S p i n c o SW 5 0 L r o t o r . F r a c t i o n s o f 1 6 d r o p s ( 0 . 2 9 m l ) w e r e c o l l e c t e d , and a s s a y e d for R N A p o l y m e r a s e a c t i v i t y as described under Methods. o c , 0 . 5 M KCI; • • , 0 . I M KCI; A ~ , 0 . 3 M KCI.

sis. The patterns obtained are very different with major bands running in different positions. However, upon pooling the two peaks, and dialysis against buffer containing 0.05 M KC1, the gel pattern obtained was similar to that of peak 2, with no indication of the major fast-running band found in peak 1. Both peaks 1 and 2 when analysed by SDS gel electrophoresis gave basically similar patterns (results not shown). Thus it would appear that the two peaks of activity obtained from DEAE-cellulose columns are due to the KC1 concentrations used and do not represent different enzymes. It has been shown previously that rifamycin SV has no effect, except at high concentrations, upon the yeast nuclear RNA polymerases [ 2 3 , 3 0 , 3 5 ] , whereas the mitochondrial enzyme has shown sensitivity [ 1 4 , 2 3 ] . The mitochondrial enzyme from X. laevis [22], and the yeast nuclear enzymes [30,35] have however shown sensitivity to some rifamycin derivatives. The effect of various rifamycin derivatives upon the yeast mitochondrial, nuclear and E. coli E N A polymerases, under normal conditions of assay, has been investigated. The resuslts are shown in Table II. As shown previously [9,23] rifamycin SV was only effective as an inhibitor upon the mitochondrial and E. coli R N A polymerases, the latter being far more affected. Eifamycin A F / 0 1 3 was the only effective inhibitor of the yeast nuclear polymerases I and II [30,35], results

338

T A B L E II INHIBITION OF VARIOUS RNA POLYMERASE ACTIVITIES BY RIFAMYCIN DERIVATIVES A s s a y s w e r e c a r r i e d o u t as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . R e s u l t s are e x p r e s s e d as p e r c e n t i n h i b i t i o n o f t h e a c t i v i t y m e a s u r e d in t h e s t a n d a r d a s s a y m i x t u r e . S o l u t i o n s o f i n h i b i t o r s w e r e m a d e u p f r e s h l y e a c h d a y , a n d a d d e d t o t h e e n z y m e at 0 ° C , 10 rain p r i o r t o s t a r t i n g t h e r e a c t i o n b y a d d i t i o n o f d e n a t u r e d c a l f t h y m u s D N A . I n c u b a t i o n w a s f o r 20 r a i n at 3 7 ° C . T h e 1 0 0 % a c t i v i t y f o r t h e v a r i o u s e n z y m e p r e p a r a t i o n s w a s (in n m o l U M P i n c o r p o r a t e d ) ; 0 . 0 3 f o r t h e m i t o c h o n d r i a l e n z y m e , 0 . 3 4 6 f o r t h e E. coli e n z y m e , 0 . 0 4 8 f o r t h e y e a s t n u c l e a r e n y z m e I, a n d 0 . 2 1 2 f o r t h e y e a s t n u c l e a r e n z y m e II. In e a c h case t h e i n c o r p o r a t i o n in c o n t r o l s , e i t h e r D N A or e n z y m e o m i t t e d w a s s u b s t r a c t e d f r o m t h e r e s u l t s ( 4 0 - - 6 0 c p m ) . Antibiotic

Concentration (~g/r~)

I n h i b i t i o n (%) Mitochondrial enzyme

E. coli enzyme

Yeast nuclear enzyme I

Yeast nuclear e n z y m e II

Rifamycin SV

0 10 20 50 100

0 31 79 88 95

0 81 83 83 84

0 6 10 10 13

0 0 0 0 34

AF/013

0 10 20 50 100

0 35 55 87 87

0 61 80 81 89

0 5 6 13 48

0 0 0 72 93

AF/05

0 10 20 50 100

0 33 61 83 87

0 77 77 79 81

0 0 0 0 0

0 0 0 0 18

AF/AP

0 10 20 50 100

0 10 11 17 20

0 85 85 87 87

0 0 0 0 0

0 0 0 0 0

AF/APBD

0 10 20 50 100

0 29 52 76 93

0 81 81 89 93

0 0 0 0 0

0 0 0 0 0

consistent with those found for mammalian RNA polymerases [36,37], with perhaps the exception of rifamycin AF/APBD. In contrast, both the mitochondrial and E. coil enzymes were sensitive to all derivatives, with perhaps the exception of rifamycin AF/AP which only gave some 20% inhibition at high concentrations with the mitochondrial enzyme. Further characteristics of the purified mitochondrial RNA polymerase are shown in Table III. The enzymes shows a marked preference for denatured calfthymus DNA as a template when compared with the native form. Native yeast mitochondrial DNA acts equally well as a template, with another small DNA,

339 TABLE

III

PROPERTIES

OF YEAST

MITOCHONDRIAL

RNA

POLYMERASE

Standard assays were those described under Methods, except that the D N A concentrations were 2/~g/ml in experiment I. T h e results are expressed as p m o l U M P incorporated/20 m i n / 3 7 ° C with the blank values subtracted. In experiment II the antibiotics were added to the template 5 rain prior to addition of enzyme. pmol UMP incorporated/ 20 min/37° C Expt. I Standard assay plus native calf thymus DNA plus T4 DNA plus native yeast mitochondrial

DNA

Expt. II Standard assay plus actinomycin D (5 pglPl) plus actinomycin D (20 pg//~l) plus distamycin A (5/~g/#al) plus distamycin A (20/~g//~l) plus ethidium b r o m i d e (5 pg//~l) plus ethidium b r o m i d e (26 #g//~l)

6.7 1.0 10.0 6.7

100 15 150 100

46.0 32.2 27.6 46.0 40.0 41.0 26.8

100 70 60 100 87 89 58

T4 showing an increased activity. Actinomycin D as expected inhibited the enzyme activity as did ethidium bromide. In contrast distamycin A gave little at similar concentrations.

TABLE IV SUMMARY

OF THE CHARACTERISTICS

Source

Yeast nuclear I (A) Y e a s t n u c l e a r II (B) Yeast mitochondrial 1.

2. 3. 4. N. crassa m i t o c h o n d r i a Rat liver mitochondria 1. 2. 3. X. laevis m i t o c h o n d r i a B, e m e r s o n i i m i t o c h o n drla W h e a t leaf polymerase

OF ISOLATED

Sensitivity

RNA POLYMERASES

Molecular weight

Specific activity *

Ref.

600 2000

31 30

a-Amanitin

Rifamycin

-+

---

large tool. wt. large mol. wt. 2000

--

-----

+ ---+

63--69 000 -67 0 0 0 high tool. wt. 63 000

---

+ +

65--70 000 66 000

--

- - **

46 000

---

+ +

-65 0 0 0

1.56 17.3 1--1.3 67.6 ---

26 24 39 18

8.63

19 21 20 22

23.32

41 38

0.322

--

* S p e c i f i c a c t i v i t y e x p r e s s e d as n m o l U M P i n c o r p o r a t e d / m g p r o t e i n / 2 0 r a i n / 3 0 - - - 3 7 ° C. * * A l t h o u g h i n s e n s i t i v e t o r i f a m y c i n t h i s e n z y m e is s e n s i t i v e t o o t h e r d e r i v a t i v e s . * * * S p e c i f i c a c t i v i t y e x p r e s s e d a s n m o l A M P i n c o r p o r a t e d a t 4 0 - - 4 8 ° C.

340

t T h ~'

B

C

Fig. 7. G e l e l e c t r o p h o r e s i s o f t h e D E A E - c e l l u l o s e c o l u m n f r a c t i o n s . E l e c t x o p h o r e s i s i n t h e a b s e n c e o f S D S w a s c a r r i e d o u t as d e s c r i b e d u n d e r M e t h o d s a n d gels w e r e s t a i n e d w i t h a m i d o s c h w a r t z . A r e f e r s t o p e a k I f r o m t h e D E A E - c e l l u l o s e c o l u m n (Fig. 1); B r e f e r s t o p e a k II f r o m t h e D E A E - c e l l u l o s e c o l u m n (Fig. 1); C r e f e r s t o P e a k s I a n d II p o o l e d , a n d d i a l y s e d a g a i n s t b u f f e r B c o n t a i n i n g 0 . 0 5 M KCI.

Discussion A simple, rapid method for the isolation of yeast mitochondrial RNA polymerase has been developed. The mitochondrial enzyme was solublized by either sonication, or digitonin treatment, and separated from the contaminating nuclear enzymes by batch elution from DEAE-cellulose. The final purification was achieved by glycerol gradient centrifugation. This yielded an enzyme with a specific activity of 1.56 nmol UMP incorporated/mg protein/20 min/37°C. This is low whem compared with yeast nuclear polymerase 1 600 [2,31], polymerase II 2000 [30] and E. coli RNA polymerase [32] 906 nmol UMP incorporated/rag protein/20 min/37°C. This perhaps represents a minimum value as the enzyme is particularly unstable. However, the low specific activity appears to be quite a general property of mitochondrial and chloroplast RNA polymerases as can be seen from Table IV. The reason for this remains obscure. The final enzyme preparation has been shown by gel electrophoresis with or without SDS as being some 95% pure (Figs. 3 and 4). The enzyme appears to consist of a single small polypeptide of 68 000 mol.wt. This also appears to be a distinguishing feature of organelle RNA polymerases (Table IV) for similar-

341 sized single polypeptides have been found in yeast [24], N. crassa [18], X. laevis [22] rat liver [20,21] and wheat leaf chloroplasts [38]. The only exception has been the report of high molecular weight mitochondrial enzymes in yeast by Eccleshall and Criddle [39]. This is in contrast to the high molecular weight multicomponent complexes found in eukaryotic nuclei [17], and prokaryotes [16]. The only similar RNA polymerases found are those of bacteriophages T3 and T7 whose molecular weights are around 100 000 [40]. Although consisting of a single low molecular weight polypeptide, the yeast mitochondrial polymerase sediments as a high molecular weight complex (Fig. 2). The aggregation phenomenon has been observed with similar enzymes from yeast [24], N. crassa [18] and X. laevis [22] and is similarly affected by ionic strength; the higher the ionic strength the lower the apparent molecular weight. This has also been shown to be probably responsible for the occurrence of two peaks of activity upon DEAE-cellulose chromatography (Fig. 7). The enzyme as isolated here was sensitive to rifamycin SV an inhibitor of bacterial RNA polymerases [9], but not yeast nuclear enzymes [35] (Table II). However, the picture remains confusing, as although rifamycin sensitivity has been shown for the enzymes isolated from the mitochondria N. crassa [18], rat liver [19,21], Blastocladiella emersonii [41] and wheat leaf chloroplasts [38], resistance has been also reported for yeast [24,25] and X. laevis [22]. As the isolated enzymes are similar in many other properties the reason for this difference is obscure. Other rifamycin derivatives used all gave inhibition except rifamycin AF/AP which gave only 20% at high concentrations. Similar results, omitting rifamycin AF/AP, have been shown with the X. laevis enzyme [22] which was resistant to rifamycin SV. The E. coli RNA polymerase was sensitive to all derivatives used in contrast to the nuclear enzymes which were only affected by rifamycin AF/013 [30,35]. Further characteristics of the mitochondrial enzyme confirm previous work [24] with the enzyme showing a preference for denatured calf thymus DNA, and being capable of transcribing native DNA with a similar efficiency. Actinomycin D as expected inhibited enzyme activity, using denatured calf thymus as a template, as did ethidium bromide. Distamycin A however showed less inhibition. This could reflect their different binding activities, actinomycin D binding to d(G-C)-rich regions and distamycin A binding to d(A-T)-rich regions [42,43] affecting the mitochondrial RNA polymerases promoter site selection more or less. However, it is difficult to draw any conclusions from experiments done under limited conditions and with only one template.

Acknowledgements The author is grateful to Dr. T.S. Work, Dr. D.H. Williamson and Dr. D.Y. Thomas for their encouragement and useful discussions. I wish also to thank Mr. B. Trinnaman for his expert technical assistance.

References 1 Luck, D.J.L. and Reich, E.P. (1964) Proc~ Natl. Acad. Sci. U.S. 52, 9 3 1 - - 9 3 8 2 Neubert, D. and Helge, H. (1965) Biochem. Biophys. Res. C o m m u n . 18, 600---605 3 Saccone, G., Gadaleta, M.N. and Quagliamello, E. (1969) Eur. J. Biochem. 10, 61--65

342 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

South, D.J. and Mahler, H.R. (1968) Nature 218, 1 2 2 6 - - 1 2 3 2 Saceone, C., Gadaleta, M.N. and Quagliariello, E. (1967) Biochim. Biophys. Acta. 138, 474--479 Kuntzel, H. (1969) FEBS Lett. 4, 140--142 Ciferri, O. and Parisi, B. (1970) Progr. Nucl. Acid Res. 10, 121--144 Borst, P. (1972) Annu. Rev. Biochem. 41, 333--376 Sippel, A.E. and Hartmann, G.R. (1970) Eur. J. Biochem. 16, 152--157 Buntz, E.K.F. and Buntz, F.A. (1970) Nature 226, 1 2 1 9 - - 1 2 2 2 Wintersberger, E. and Wintersberger, U. (1969) FEBS Letts. 6, 58--60 Yang, S. and Criddle, R.A. (1970) Biochemistry 9, 3 0 6 3 - - 3 0 7 2 Shmerling, Zh. G. (1969) Biochem. Biophys. Res. Commun. 37, 965---969 Scragg, A.H. (1971) Biochem. Biophys. Res. Commun. 45, 701--706 Wintersbcrger, E. (1970) Biochem. Biophys. Res. Commun. 40, 1179--1184 Losiek, R. (1972) Annu. Rev. Biochem. 4 1 , 4 0 9 - - 4 4 6 Chambon, P. (1975) Annu. Rev. Biochem. 44, 6 1 3 - ' 6 3 8 Kuntzel, H. and Schafer, K.P. (1971) Nat, New Biol. 231, 265--271 Reid, B.D. and Parsons, P. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2830--2834 Gallerani, R. and Saccone, C. (1974) in The Biogenesis of Mitoehondria (Kroon, A.M. and Saceone, C., eds.), pp. 59--69, Academic Press, New York Mukerjee, H. and Goldfeder, A. (1973) Biochemistry 12, 5096--5101 Wu, G.J. and David, I.B. (1972) Biochemistry 11, 3 5 8 9 - - 3 5 9 5 Scragg, A.H. (1974) in The Biogenesis of Mitochondria (Kroon, A.M. and Saccone, C., eds.), pp. 47-57, Academic Press, New York Rogall, G. and Wintersberger, E. (1974) FEBS Lett. 46, 333--336 Tsai, M.J., Michaelis, G. and Criddle, R.S. (1971) Proc. Natl. Acad. Sci. U.S. 68, 4 7 3 - 4 7 7 Wintersberger, E. (1972) Biochem. Biophys. Res. Commun. 48, 1287--1294 Thomas, D.Y. and Scragg, A.H. (1973) Eur. J. Biochem. 37, 585--568 Alberts, B. and Herrick, G. (1971) in Methods in Enzymol ogy (Colowick, S.P. and Kaplan, N.O., eds.), Vol. 21, pp. 198--217, Academic Press, New York Bollum, F.J. (1959) J. Biol. Chem. 234, 2 7 3 3 - - 2 7 3 4 Dezelee, S. and Sentenac, A. (1973) Eur. J. Biochem. 34, 41--52 Buhler, J.M., Sentenac, A. and Fromageot, P. (1974) J. Biol. Chem. 249, 5963--5970 Burgess, R.R. (1969) J. Biol. Chem. 244, 6 1 6 0 - - 6 1 6 7 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 Ornstein, L. and Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 1 2 1 , 3 2 1 - - 4 0 4 Adman, R., Schultz, L.D. and Hall, B.D. (1972) Proe. Natl. Acad. Sci. U.S. 69, 1 7 0 2 ~ 1 7 0 6 Juhasz, P.P., Benecke, B.J. and Siefart, K.H. (1972) FEBS Lett. 27, 30--34 Busiello, E., Girolamo, A.D., Girolamo, M.D., Fischer-Fantuzzi, L. and Vesco, C. (1973) Eur. J. Biochem. 35, 251--258 Polya, G.M. (1973) Arch. Biochem. Biophys. 155, 125--135 EccleshaU, T.R. and Criddle, R.S. (1974) in The Biogenesis of Mitochondria (Kroon, A.M. and Saccone, C., eds.), pp. 31--46, Academic Press, New York Beier, H. and Hausmann, R. (1974) Nature 251, 538--539 Horgen, P.A. and Griffin, D.H. (1971) Proc. Natl. Acad. Sci. U.S. 68, 338--341 Reich, E. and Goldberg, I.H. (1964) Progr. Nucl. Acid. Res. Mol. Biol. 3, 183--234 Puschendorf, B. and Grunicke, H. (1969) FEBS Lett. 4, 355--357