49
Biochimica et Biophysica Acta, 432 (1976) 49--59 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98557
PURIFICATION AND PROPERTIES OF DNA-DEPENDENT RNA POLYMERASE FROM MYCOBACTERIUM TUBERCULOSIS H3~R~
R.M. HARSHEY and T. RAMAKRISHNAN
Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore 560 012 (India) (Received October 14th, 1975)
Summary RNA polymerase (nucleosidetriphosphate : RNA nucleotidyltransferase (DNA-dependent), EC 2.7.7.6) was purified approximately 200 fold from Mycobacterium tuberculosis H37R~ cells. The purified enzyme has a molecular weight of about 330 000--350 000 and is composed of four subunits. The subunits ~'fl and o have molecular weights different from those of Escherichia coil polymerase; the fourth, a subunit has a similar weight. The purified enzyme is a thousand-fold more sensitive to rifampicin, a potent antitubercular drug, than the E. coil RNA polymerase, probably because of the difference in the ~ subunits. This, with other data presented in this paper, indicate that the RNA polymerase of M. tuberculosis differs in its properties from that of E. coll. Introduction
The antibiotic rifampicin is a potent antitubercular agent. It also inhibits, to a smaller extent, the growth of Escherichia coli, and in this organism the target of action of the drug has been shown to be DNA-dependent RNA polymerase [ 1,2 ]. In view of the fact that the chemotherapy of tuberculosis using rifampicin is complicated by the emergence of drug-resistant strains of the bacillus, it was thought desirable to study the properties of DNA-dependent RNA polymerase in Mycobacterium tuberculosis, including the effect of other drugs on the enzyme. Such a study might also enable us to design other antitubercular agents on a rational basis. DNA-dependent RNA polymerase has been isolated and purified from several prokaryotic sources [3--5]. This enzyme, which transcribes the message carried by DNA, could have important implications in transcriptional regulation. The subunit structure of the enzyme and its modifications during sporulation [6] or following phage infection [7] amply support such a deduction. The study of this enzyme is important, therefore, not only for its therapeutic significance but also in understanding some of the regulatory mechanisms operative in this
50 organism. In this paper we report the isolation and purification of DNA-dependent RNA polymerase from M. tuberculosis H37Rv, and some of its properties. Materials and Methods
Organisms. M. tuberculosis H37Rv, originally obtained from the National Collection of Type Cultures (NCTC 7416), was grown on Sauton's synthetic liquid media [8] as a surface mat. Mycobacterium smegmatis (BM1) was obtained from Dr. R. Bonicke, Institute for Experimental Biology and Medicine, Borstel, G.F.R. Mycobacteriophage I3 was isolated in our laboratory [9]. Phage T4 was a gift from Dr. J.D. Padayatty, Department of Biochemistry, Indian Institute of Science, Bangalore, India. Materials. DEAE-cellulose, phosphocellulose, ATP, GTP, UTP and CTP were obtained from Sigma Chemical Co., St. Louis, Missouri, U.S.A. [3H] CTP, was from Schwarz-Mann, Orangeburg, N.Y., U.S.A. Ethambutol and pyrazinamide were gifts from Tuberculosis Chemotherapy Centre, Madras, India. 8-Hydroxyquinoline derivatives were gifts from Dr. Warren Levinson, San Francisco, Calif. [ 14C] Rifampicin-38 was a gift from Dr. Giancarlo Lancini, Milan, Italy. Buffers. The following buffers were prepared: Buffer G, 0.05 M Tris • HC1 pH 7.9/0.001 M MgC12/0.2 M KC1/0.1 mM dithiothreitol/0.1 mM EDTA/5% (v/v) glycerol; Buffer A, 0.01 M Tris • HC1 pH 7.9/0.001 M MgC12/0.1 mM dithiothreitol/5% glycerol; Buffer C, 0.05 M Tris • HC1 pH 7.9/0.1 mM EDTA/ 0.1 mM dithiothreitol/5% glycerol. Preparation o f DNA. M. tuberculosis and M. smegmatis DNA were prepared according to Marmur [10] and DNA from bacteriophages T4 and I3 according to Mandel et al. [11]. Assay o f R N A polymerase. The assay mixture contained in 0.25 ml, 0.02 M Tris • HC1 pH 7.9 at 25°C, 0.001 M magnesium acetate/0.1 mM EDTA pH 6.9/ 0.1 mM dithiothreitol/bovine serum albumin 20 pg/ml/0.4 mM UTP, GTP and ATP/0.1 mM [3H] CTP (1.1 • l 0 s d p m / m m o l ) and 10 pg of calf thymus DNA. The assay mixtures were incubated for 5 min at 37°C, chilled in ice and 0.1 ml spotted onto Whatman 3MM paper discs (1.5 × 1.5 cm). The discs were dried under an infrared lamp and dipped into 5% trichloroacetic acid. They were washed 3--4 times in 5% trichloroacetic acid, twice in 80% ethanol, once in ether, dried and counted in a Beckman LS-100 liquid scintillation spectrometer, in 10 ml of toluene-based scintillation fluid (4 g of PPO and 50 mg of POPOP per 1 of toluene). One activity unit of enzyme incorporates 1 nmol of CMP in 5 min of incubation at 37°C under the conditions described above. Suitable corrections were made for the efficiency of counting, which is 3% for 3H on paper, when expressing the enzyme activities. Polynucleotide phosphorylase incorporates ribonucleotides well under these conditions because of small amounts of contaminating ribonucleoside diphosphates. This incorporation can be inhibited completely by the presence of 0.4 mM potassium phosphate pH 7.5, in the assay mixture [12]. All of the assays reported here contain added phosphate. This addition has no effect upon polymerase activity. Rifampicin binding with R N A polymerase. We have found that purified
51 RNA polymerase binds strongly to glass fibre filters which allowed us to develop a simple, sensitive assay for rifampicin binding to the enzyme. 8000 cpm of [14C] rifampicin (spec. act. = 2.3 • 107 cpm//~mol) were incubated with 50 pg of purified enzyme in 0.02 M Tris • HC1 buffer pH 7.9, containing 0:001 M magnesium acetate/0.1 mM EDTA pH 6.9/0.1 mM dithiothreitol at 37°C for 20 min. The reaction mixture was then passed through a glass fibre filter which was washed with 20 ml of the same buffer and after drying, counted for radioactivity. After a 20-min incubation of the reaction mixture with [14C]rifampicin, a five-fold excess of unlabelled rifampicin was added and, at various time intervals, suitable aliquots were drawn and the a m o u n t of radioactivity retained on the filters determined as described above. Protein determination. Protein was measured by the procedure of Lowry et al. [13], using crystalline bovine serum albumin as a standard. Column chromatography. DEAE-celiulose was stirred with 5 vol. of 0.5 M HC1 for 20--30 min, filtered and rinsed with distilled water until the rinse was about pH 4. The cake was stirred with 5 vol. of 0.5 M NaOH for 20--30 min and rinsed with water until the pH was about neutral. The cake was washed repeatedly in buffer A. The column was packed at 25°C and equilibrated with several column volumes of buffer A at 4°C. Phosphocellulose was washed in the same way except that the base wash preceded the acid wash. The column was equilibrated with buffer C containing 0.1 M KC1. Sephadex G-200 was mixed with 2 vol. of buffer A at room temperature and allowed to settle. The supernatant was discarded, another 2 vol. of buffer added and the slurry poured into a column, which was equilibrated at 4°C with buffer A. Polyacrylamide gel electrophoresis. Polyacrylamide gels, pH 8.7, were prepared and run according to the general methods of Ornstein [14] and Davis [15]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run according to the m e t h o d of Weber and Osborn [16]. The gels contained 5% polyacrylamide. They were stained with 2% Coomassie brilliant blue in methanol/ acetic acid/water (5 : 1 : 5) for 2--10 h and destained with 7.5% acetic acid/5% methanol. Results
The purification procedure followed was mainly as described by Burgess [12], with some modifications. Unless otherwise indicated, all operations were carried o u t at 4°(3. The purification procedure is described below, while the results of a typical preparation from 20 g of cells are summarised in Table I. Preparation of extracts. Cells were harvested on the eighth day of growth. 20 g of cells were suspended in 30 ml of buffer G and subjected to sonic oscillation in a R a y t h e o n 9 KH2 sonic oscillator, for 20 min. The suspension was centrifuged in a Sorvall RC-2B refrigerated centrifuge for 30 min at 20 000 X g. The supernatant (referred to hereafter as S-20), was centrifuged in a Beckman model L2-50 ultracentrifuge at 100 000 X g for 90 min. The supernatant (S-100) was further processed. Ammonium sulfate fractionation. Solid a m m o n i u m sulfate was added to S-100 slowly with stirring to give a solution 30% saturated with a m m o n i u m sulfate. The pH was prevented from dropping below 7 by the addition of
52 TABLE I S U M M A R Y O F M. T U B E R C U L O S I S
H37R v RNA POLYMERASE
PURIFICATION
D e t a i l s o f t h e s t e p s are d e s c r i b e d in t h e t e x t . Step
Total protein (mg)
Total activity X 10 -4 ( p m o l CMP incorporated/5 min)
Spec. act. ( p m o l C M P incorporated/rag protein/5 rain)
% Yield
1. C r u d e ( S - 2 0 ) f r a c t i o n 2. S - 1 0 0 f r a c t i o n 3. 3 0 - - 5 0 % A m m o n i u m s u l p h a t e f r a c t i o n 4. P o o l e d D E A E - c e l l u l o s e p e a k 5. 3 0 - - 5 0 % A m o n i u m s u l p h a t e f r a c t i o n 6. P o o l e d g l y c e r o l g r a d i e n t p e a k 7. P o o l e d S e p h a d e x G - 2 0 0 p e a k
910 627 264 40 10 1.5 1.4
20 10.4 16.0 10.4 3.6 6.0 5.6
220 166 606 2600 3600 40000 40000
100 52 80 52 18 30 28
0.05 ml of 1 M NaOH per 10 g of a m m o n i u m sulfate. The solution was stirred for 15 min and the precipitate removed by centrifugation at 10 000 × g for 20 min in a Sorvall centrifuge. The supernatant was brought to 50% saturation with a m m o n i u m sulfate and the precipitate collected by centrifugation. All the RNA polymerase activity came down with the precipitate. It was dissolved in a small volume of buffer A and dialysed overnight against buffer A. DEAE-cellulose chromatography. The dialysed 30--50% a m m o n i u m sulfate fraction was applied to a DEAE-cellulose column (20 ml column volume per 20 g of cells). The column was washed with 10 ml of buffer A and then 80 ml of buffer A + 0.24 M KC1. The polymerase was eluted with 60 ml of buffer A + 0.3 M KC1 (Fig. la). The fractions containing the bulk of the activity were pooled. Second ammonium sulfate fractionation. As in the first a m m o n i u m sulfate fractionation, the concentration of a m m o n i u m sulfate was brought to 30% by adding solid a m m o n i u m sulfate. The stringy precipitate was centrifuged and T A B L E II REQUIREMENTS
OF RNA POLYMERASE
O F M. T U B E R C U L O S I S
H37R v
T h e c o m p l e t e a s s a y s y s t e m is as d e s c r i b e d u n d e r M e t h o d s , e x c e p t t h a t i n s t e a d o f c a l f t h y m u s D N A , m y c o b a c t e r i a l D N A w a s u s e d as t e m p l a t e . T h e e n z y m e c o n c e n t r a t i o n p e r a s s a y w a s 25 ~g. Additions
Activity (pmol of labelled nucleoside m o n o p h o s p h a t e incorporated/5 min)
Complete --DNA +DNAase +RNAase --UTP --ATP --CTP * --GTP
800 0 20 15 100 260 40 141
* I n t h i s case t h e l a b e l l e d n u c l e o s i d e t r i p h o s p h a t e u s e d w a s U T P .
53
2o
i
1.5
L
30 ~ ~c
0.6 0
1.C 20 0.5
10
4o
2o Fraction (a)
,0 number
~m
0 5o
o.t,
i-
I I
0.2 , 0 ,~ bottom
~ =~ 4o
t .o Fraction
310 number
,,o
top
(b)
Fig. 1. D E A E - c e l l u l o s e c h r o m a t o g r a p h y and g l y c e r o l gradient c e n t r i f u g a t i o n o f R N A p o l y m e r a s e f r o m M. tuberculosis H 3 7 R v. ( a ) T h e 3 0 - - 5 0 % a m m o n i u m s u l p h a t e f r a c t i o n w a s a p p l i e d to t h e D E A E - c e l l u l o s e c o l u m n ( 2 0 m l c o l u m n v o l u m e for e v e r y 20 g cells). E l u t i o n o f the e n z y m e is d e s c r i b e d in the t e x t . (b) T h e p r o t e i n o b t a i n e d after the s e c o n d a m m o n i u m s u l p h a t e f r a c t i o n a t i o n (see t e x t ) w a s s u b j e c t e d t o g l y c e r o l gradient c e n t r i f u g a t i o n as d e s c r i b e d in the t e x t .
discarded. More ammonium sulfate was added to the supernatant to bring the saturation to 50%. The pellet obtained on centrifugation contained the RNA polymerase activity. Glycerol gradient centrifugation. The pellet was dissolved in 3 ml of buffer A and dialysed against 100 vol. of buffer A twice, for 2 h each. 1.5 ml of the dialysed solution were layered onto a 5--20% (v/v) glycerol gradient in buffer A without glycerol in a 30 ml tube. The gradients were centrifuged at 24 000 rev./min in an SW 25.1 rotor for 24 h at 4°C. 1 ml fractions were collected after puncturing the bottom of the tube with a syringe needle. The peak fractions were pooled (Fig. lb). Sephadex G-200 chromatography. The glycerol gradient fraction ( < 5 ml) was applied to a Sephadex G-200 column (45 × 2.5 cm) equilibrated with buffer A. It was eluted with the same buffer. The tubes containing the polymerase activity were pooled and stored. Protein purity. Two methods were used to determine the purity of this enzyme. The first was by polyacrylamide gel electrophoresis (see Methods). No
Plate 1. O u c h t e r l o n e y d o u b l e agar-gel d i f f u s i o n Pattern o b t a i n e d w i t h t h e purified R N A p o l y m e r a s e f r o m M. t u b e r c u l o s i s H 3 7 R w A n t i b o d i e s w e r e raised in rabbits against t h e c r u d e e n z y m e . T h e a n t i s e r u m w a s r e a c t e d against t h e purified e n z y m e . Well 1 c o n t a i n s t h e a n t i s e r u m , w h i l e w e l l s 2 and 3 c o n t a i n t h e purified enzyme.
54
I
=,' 0"6
o-
a. A
+1 @dye ~dye Ca) (b)
0.4
.J O
E c O o0 cd
"~
,.i,-
0.2
..-JJ 0
4
8
12
16
20
FRACTION
24
28
32
4O
36
NUMBER
F i g . 2. P h o s p h o c e l l u l o s e c h r o m a t o g r a p h y o f R N A p o l y m e r a s e f r o m M. t u b e r c u l o s i s H 3 7 R ~. A p o r t i o n o f the enzyme was applied to a phosphocellulose column (10 × 1.5 cm), equilibrated with buffer C containing 0 . 1 M KC1. F o r d e t a i l s o f e l u t i o n see t e x t . I n s e t : e l e c t r o p h o r e t i c p a t t e r n o f p u r i f i e d p o l y m e r a s e (a) h o l o e n z y m e a n d ( b ) c o r e e n z y m e f r o m M. t u b e r c u l o s i s H 3 7 R v o n s o d i u m d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e gels. P r e p a r a t i o n o f gels a n d e l e c t r o p h o r e s i 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 u n d e r M e t h o d s .
detectable impurity was observed. Purity was also determined b y raising antibodies in rabbits against the partially purified enzyme. The serum collected after 6 weeks, when reacted against the purified enzyme by the double agar-gel diffusion technique [ 1 7 ] , gave only one precipitin band showing that the enzyme preparation was homogeneous (Plate 1). Phosphocellulose chromatography. A portion of the purified enzyme was applied to a phosphocellulose column (10 × 1.5 cm) equilibrated with buffer C + 0.1 M KC1. The column was washed with 2 column volumes of buffer C + 0.25 M KC1. The polymerase was then eluted with buffer C + 0.35 M KC1. (Fig. 2). 800 M g ++
Nln ++
600 s~ ~ > 400
I I I I I I /
~
//
P
/ 200
I I
/
/ r 0
//
/ i
I
[
1
2
3
CONCN.
{raM)
4
0
I I I
I
I
1
2
3
CONCN.
&
{raM)
F i g . 3. E f f e c t o f M g 2+ a n d M n 2+ o n t h e R N A p o l y m e r a s e a c t i v i t y o f M. t u b e r c u l o s i s H 3 7 R ~. R N A p o l y m e r a s e 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 u n d e r m e t h o d s . 2 5 Mg o f t h e e n z y m e p r o t e i n w e r e u s e d p e r assay. E n z y m e a c t i v i t i e s are e x p r e s s e d as p m o l C M P i n c o r p o r a t e d / 5 m i n .
55 TABLE III E F F E C T O F R I F A M P I C I N ON E. C O L I A N D M. T U B E R C U L O S I S H37R v R N A P O L Y M E R A S E S T h e assay m i x t u r e for the E. coli e n z y m e c o n t a i n e d in 0 . 2 5 m l 0 . 0 4 M Tris - HCI p H 7 . 9 / 0 . 0 1 M MgC12/ 0.1 m M E D T A / 0 . 1 m M d i t h i o t h r e i t o l / 0 . 1 5 M K C I / 0 . 4 m M UTP, G T P a n d A T P / 0 . 1 m M [ 3 H ] C T P (1.1 • 108 d p m / m m o l ) a n d 20 # g o f c a l f t h y m u s D N A . T h e details o f assay o f the m y c o b a c t e r i a l e n z y m e axe described u n d e r M e t h o d s . T h e e n z y m e c o n c e n t r a t i o n per assay w a s 10 #g. Results axe e x p r e s s e d as p e r c e n t a g e activities o f t h e c o n t r o l . Rifampicin concentration (M)
A c t i v i t y (% o f c o n t r o l ) E. coli e n z y m e
10 -9
100
10 -8
98
--
10 -7
60
--
10 -6
40
--
10 -s
5
--
M. tuberculosis e n z y m e
33
Molecular weight determination and characterization of the subunits of RNA polymerase. The enzyme was layered on a glycerol gradient 10--25% (v/v) along with standard marker proteins (catalase, mol. wt. 244 000; pyruvate kinase, mol. wt. 237 000; alcohol dehydrogenase, mol. wt. 148 000;/3-galactosidase, mol. wt 130 000), and spun at 24 000 rev./min for 24 h in an SW-25.1 rotor. The distances of migration of protein markers were plotted against their log molecular weights. The molecular weight of the polymerase falls in the range of 330 000--350 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [16] was used for determining the molecular weights of the subunits by comparing with standard proteins (~-galactosidase, bovine serum albumin, catalase, pepsin, alcohol dehydrogenase, 7-chymotrypsin and lysozyme). The enzyme splits into 4 bands having mol. wts. 96 000, 86 000, 60 000 and 44 000
~I00 ~~.,....~.....~ .~ 6
o; =~
0
i
0 2
i
I
I
I
5
10
20
30
Time Fig. 4. Stability polymerase;
(rain)
of the [14C]rifampicin • RNA polymerase complex: o o, M. t u b e r c u l o s i s R N A z~, E. coli R N A p o l y m e r a s e . For e x p e r i m e n t a l details s e e M e t h o d s .
56
respectively (Fig. 2, inset a). In the phosphocellulose enzyme the band corresponding to 60 000 mol. wt. was absent (Fig. 2, inset b). Properties. Some of the properties of the enzyme are summarised in Table II. The enzyme requires a DNA template and all four ribonucleoside triphosphates for activity. The product is labile to ribonuclease and alkali treatment and resistant to DNAase treatment, showing that it is RNA. Divalent cation requirement. Mg2÷ is absolutely essential for enzyme activity but can be replaced by Mn 2÷ to a large extent (Fig. 3). Maximal activity was obtained at 2 mM concentration of either Mg2÷ or Mn 2÷. Beyond these concentrations both had an inhibitory effect on enzyme activity. Effect of rifampicin. 10 pg of the Mycobacterium RNA polymerase is completely inhibited at l f f 8 M rifampicin, whereas the same a m o u n t of E. coli enzyme, purified according to the procedure of Burgess [12], showed lower sensitivity to the drug, being completely inhibited only at 10 -5 M rifampicin (Table III). The inhibition by rifampicin could be overcome by increasing the enzyme concentration, showing that the enzyme is the target of drug action. Stability of the RNA polymerase . rifampicin complex. Binding studies with [14C] rifampicin show that the drug binds to the M. tuberculosis RNA polymerase with a higher affinity than to the E. coli RNA polymerase (Fig. 4). On incubation of the M. tuberculosis RNA polymerase • [14C] rifampicin complex with non-radioactive drug (see Methods), the label bound to the enzyme is released slowly, and at 30 min, 60% of the label is still bound to the enzyme. Under identical conditions, the radioactivity associated with the E. coli RNA polymerase is removed rapidly and at 30 min the enzyme retains only 20% of the original radioactivity. Effect of other antitubercular drugs. None of the antitubercular drugs tested had any effect on enzyme activity, although some (8-nitro-8-hydroxyquinoline;
T A B L E IV E F F E C T OF A N T I T U B E R C U L A R TUBERCULOSIS H37R v
AND
ANTIVIRAL
AGENTS
ON R N A P O L Y M E R A S E
O F M.
E n z y m e assays w e r e d o n e as d e s c r i b e d in the text w i t h a protein c o n c e n t r a t i o n of 25/~g p e r t u b e . Results are e x p r e s s e d as the percentage activities of t h e c o n t r o l .
Agent
Activity
(% of control) A n t i t u b e r c u l a r drugs
S t r e p t o m y c i n ( 1 0 -3 M) Capreomycin ( 1 0 -3 M) K a n a m y c i n ( 1 0 -3 M) E t h a m b u t o l ( 1 0 -3 M) P y r a z i n a m i d e ( 1 0 -3 M)
I00 I00 I00 160 I00
Antivixal a g e n t s (also antitubereular) C o p p e r i s o n l c o t i n i c acid h y d r a z i d e ( 1 6 raM) 8 - a m i n o - 8 - h y d r o x y q u i n o l i n e (4 rnM) 8 o n i t r o - 8 - h y d r o x y q u i n o l i n e (4 raM) 5,7 d i c h l o r o - 8 - h y d r o x y q u i n o l i n e (4 mM) 5 - c h l o r o - 7 - i o d o - 8 - h y d r o x y q u i n o l l n e (4 m M )
100 100 175 145 155
57 5-chloro-7-iodo-8-hydroxyquinoline; 5,7
Biochimica et Biophysica Acta, 432 (1976) 49--59 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98557
PURIFICATION AND PROPERTIES OF DNA-DEPENDENT RNA POLYMERASE FROM MYCOBACTERIUM TUBERCULOSIS H3~R~
R.M. HARSHEY and T. RAMAKRISHNAN
Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore 560 012 (India) (Received October 14th, 1975)
Summary RNA polymerase (nucleosidetriphosphate : RNA nucleotidyltransferase (DNA-dependent), EC 2.7.7.6) was purified approximately 200 fold from Mycobacterium tuberculosis H37R~ cells. The purified enzyme has a molecular weight of about 330 000--350 000 and is composed of four subunits. The subunits ~'fl and o have molecular weights different from those of Escherichia coil polymerase; the fourth, a subunit has a similar weight. The purified enzyme is a thousand-fold more sensitive to rifampicin, a potent antitubercular drug, than the E. coil RNA polymerase, probably because of the difference in the ~ subunits. This, with other data presented in this paper, indicate that the RNA polymerase of M. tuberculosis differs in its properties from that of E. coll. Introduction
The antibiotic rifampicin is a potent antitubercular agent. It also inhibits, to a smaller extent, the growth of Escherichia coli, and in this organism the target of action of the drug has been shown to be DNA-dependent RNA polymerase [ 1,2 ]. In view of the fact that the chemotherapy of tuberculosis using rifampicin is complicated by the emergence of drug-resistant strains of the bacillus, it was thought desirable to study the properties of DNA-dependent RNA polymerase in Mycobacterium tuberculosis, including the effect of other drugs on the enzyme. Such a study might also enable us to design other antitubercular agents on a rational basis. DNA-dependent RNA polymerase has been isolated and purified from several prokaryotic sources [3--5]. This enzyme, which transcribes the message carried by DNA, could have important implications in transcriptional regulation. The subunit structure of the enzyme and its modifications during sporulation [6] or following phage infection [7] amply support such a deduction. The study of this enzyme is important, therefore, not only for its therapeutic significance but also in understanding some of the regulatory mechanisms operative in this
50 organism. In this paper we report the isolation and purification of DNA-dependent RNA polymerase from M. tuberculosis H37Rv, and some of its properties. Materials and Methods
Organisms. M. tuberculosis H37Rv, originally obtained from the National Collection of Type Cultures (NCTC 7416), was grown on Sauton's synthetic liquid media [8] as a surface mat. Mycobacterium smegmatis (BM1) was obtained from Dr. R. Bonicke, Institute for Experimental Biology and Medicine, Borstel, G.F.R. Mycobacteriophage I3 was isolated in our laboratory [9]. Phage T4 was a gift from Dr. J.D. Padayatty, Department of Biochemistry, Indian Institute of Science, Bangalore, India. Materials. DEAE-cellulose, phosphocellulose, ATP, GTP, UTP and CTP were obtained from Sigma Chemical Co., St. Louis, Missouri, U.S.A. [3H] CTP, was from Schwarz-Mann, Orangeburg, N.Y., U.S.A. Ethambutol and pyrazinamide were gifts from Tuberculosis Chemotherapy Centre, Madras, India. 8-Hydroxyquinoline derivatives were gifts from Dr. Warren Levinson, San Francisco, Calif. [ 14C] Rifampicin-38 was a gift from Dr. Giancarlo Lancini, Milan, Italy. Buffers. The following buffers were prepared: Buffer G, 0.05 M Tris • HC1 pH 7.9/0.001 M MgC12/0.2 M KC1/0.1 mM dithiothreitol/0.1 mM EDTA/5% (v/v) glycerol; Buffer A, 0.01 M Tris • HC1 pH 7.9/0.001 M MgC12/0.1 mM dithiothreitol/5% glycerol; Buffer C, 0.05 M Tris • HC1 pH 7.9/0.1 mM EDTA/ 0.1 mM dithiothreitol/5% glycerol. Preparation o f DNA. M. tuberculosis and M. smegmatis DNA were prepared according to Marmur [10] and DNA from bacteriophages T4 and I3 according to Mandel et al. [11]. Assay o f R N A polymerase. The assay mixture contained in 0.25 ml, 0.02 M Tris • HC1 pH 7.9 at 25°C, 0.001 M magnesium acetate/0.1 mM EDTA pH 6.9/ 0.1 mM dithiothreitol/bovine serum albumin 20 pg/ml/0.4 mM UTP, GTP and ATP/0.1 mM [3H] CTP (1.1 • l 0 s d p m / m m o l ) and 10 pg of calf thymus DNA. The assay mixtures were incubated for 5 min at 37°C, chilled in ice and 0.1 ml spotted onto Whatman 3MM paper discs (1.5 × 1.5 cm). The discs were dried under an infrared lamp and dipped into 5% trichloroacetic acid. They were washed 3--4 times in 5% trichloroacetic acid, twice in 80% ethanol, once in ether, dried and counted in a Beckman LS-100 liquid scintillation spectrometer, in 10 ml of toluene-based scintillation fluid (4 g of PPO and 50 mg of POPOP per 1 of toluene). One activity unit of enzyme incorporates 1 nmol of CMP in 5 min of incubation at 37°C under the conditions described above. Suitable corrections were made for the efficiency of counting, which is 3% for 3H on paper, when expressing the enzyme activities. Polynucleotide phosphorylase incorporates ribonucleotides well under these conditions because of small amounts of contaminating ribonucleoside diphosphates. This incorporation can be inhibited completely by the presence of 0.4 mM potassium phosphate pH 7.5, in the assay mixture [12]. All of the assays reported here contain added phosphate. This addition has no effect upon polymerase activity. Rifampicin binding with R N A polymerase. We have found that purified
51 RNA polymerase binds strongly to glass fibre filters which allowed us to develop a simple, sensitive assay for rifampicin binding to the enzyme. 8000 cpm of [14C] rifampicin (spec. act. = 2.3 • 107 cpm//~mol) were incubated with 50 pg of purified enzyme in 0.02 M Tris • HC1 buffer pH 7.9, containing 0:001 M magnesium acetate/0.1 mM EDTA pH 6.9/0.1 mM dithiothreitol at 37°C for 20 min. The reaction mixture was then passed through a glass fibre filter which was washed with 20 ml of the same buffer and after drying, counted for radioactivity. After a 20-min incubation of the reaction mixture with [14C]rifampicin, a five-fold excess of unlabelled rifampicin was added and, at various time intervals, suitable aliquots were drawn and the a m o u n t of radioactivity retained on the filters determined as described above. Protein determination. Protein was measured by the procedure of Lowry et al. [13], using crystalline bovine serum albumin as a standard. Column chromatography. DEAE-celiulose was stirred with 5 vol. of 0.5 M HC1 for 20--30 min, filtered and rinsed with distilled water until the rinse was about pH 4. The cake was stirred with 5 vol. of 0.5 M NaOH for 20--30 min and rinsed with water until the pH was about neutral. The cake was washed repeatedly in buffer A. The column was packed at 25°C and equilibrated with several column volumes of buffer A at 4°C. Phosphocellulose was washed in the same way except that the base wash preceded the acid wash. The column was equilibrated with buffer C containing 0.1 M KC1. Sephadex G-200 was mixed with 2 vol. of buffer A at room temperature and allowed to settle. The supernatant was discarded, another 2 vol. of buffer added and the slurry poured into a column, which was equilibrated at 4°C with buffer A. Polyacrylamide gel electrophoresis. Polyacrylamide gels, pH 8.7, were prepared and run according to the general methods of Ornstein [14] and Davis [15]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run according to the m e t h o d of Weber and Osborn [16]. The gels contained 5% polyacrylamide. They were stained with 2% Coomassie brilliant blue in methanol/ acetic acid/water (5 : 1 : 5) for 2--10 h and destained with 7.5% acetic acid/5% methanol. Results
The purification procedure followed was mainly as described by Burgess [12], with some modifications. Unless otherwise indicated, all operations were carried o u t at 4°(3. The purification procedure is described below, while the results of a typical preparation from 20 g of cells are summarised in Table I. Preparation of extracts. Cells were harvested on the eighth day of growth. 20 g of cells were suspended in 30 ml of buffer G and subjected to sonic oscillation in a R a y t h e o n 9 KH2 sonic oscillator, for 20 min. The suspension was centrifuged in a Sorvall RC-2B refrigerated centrifuge for 30 min at 20 000 X g. The supernatant (referred to hereafter as S-20), was centrifuged in a Beckman model L2-50 ultracentrifuge at 100 000 X g for 90 min. The supernatant (S-100) was further processed. Ammonium sulfate fractionation. Solid a m m o n i u m sulfate was added to S-100 slowly with stirring to give a solution 30% saturated with a m m o n i u m sulfate. The pH was prevented from dropping below 7 by the addition of
52 TABLE I S U M M A R Y O F M. T U B E R C U L O S I S
H37R v RNA POLYMERASE
PURIFICATION
D e t a i l s o f t h e s t e p s are d e s c r i b e d in t h e t e x t . Step
Total protein (mg)
Total activity X 10 -4 ( p m o l CMP incorporated/5 min)
Spec. act. ( p m o l C M P incorporated/rag protein/5 rain)
% Yield
1. C r u d e ( S - 2 0 ) f r a c t i o n 2. S - 1 0 0 f r a c t i o n 3. 3 0 - - 5 0 % A m m o n i u m s u l p h a t e f r a c t i o n 4. P o o l e d D E A E - c e l l u l o s e p e a k 5. 3 0 - - 5 0 % A m o n i u m s u l p h a t e f r a c t i o n 6. P o o l e d g l y c e r o l g r a d i e n t p e a k 7. P o o l e d S e p h a d e x G - 2 0 0 p e a k
910 627 264 40 10 1.5 1.4
20 10.4 16.0 10.4 3.6 6.0 5.6
220 166 606 2600 3600 40000 40000
100 52 80 52 18 30 28
0.05 ml of 1 M NaOH per 10 g of a m m o n i u m sulfate. The solution was stirred for 15 min and the precipitate removed by centrifugation at 10 000 × g for 20 min in a Sorvall centrifuge. The supernatant was brought to 50% saturation with a m m o n i u m sulfate and the precipitate collected by centrifugation. All the RNA polymerase activity came down with the precipitate. It was dissolved in a small volume of buffer A and dialysed overnight against buffer A. DEAE-cellulose chromatography. The dialysed 30--50% a m m o n i u m sulfate fraction was applied to a DEAE-cellulose column (20 ml column volume per 20 g of cells). The column was washed with 10 ml of buffer A and then 80 ml of buffer A + 0.24 M KC1. The polymerase was eluted with 60 ml of buffer A + 0.3 M KC1 (Fig. la). The fractions containing the bulk of the activity were pooled. Second ammonium sulfate fractionation. As in the first a m m o n i u m sulfate fractionation, the concentration of a m m o n i u m sulfate was brought to 30% by adding solid a m m o n i u m sulfate. The stringy precipitate was centrifuged and T A B L E II REQUIREMENTS
OF RNA POLYMERASE
O F M. T U B E R C U L O S I S
H37R v
T h e c o m p l e t e a s s a y s y s t e m is as d e s c r i b e d u n d e r M e t h o d s , e x c e p t t h a t i n s t e a d o f c a l f t h y m u s D N A , m y c o b a c t e r i a l D N A w a s u s e d as t e m p l a t e . T h e e n z y m e c o n c e n t r a t i o n p e r a s s a y w a s 25 ~g. Additions
Activity (pmol of labelled nucleoside m o n o p h o s p h a t e incorporated/5 min)
Complete --DNA +DNAase +RNAase --UTP --ATP --CTP * --GTP
800 0 20 15 100 260 40 141
* I n t h i s case t h e l a b e l l e d n u c l e o s i d e t r i p h o s p h a t e u s e d w a s U T P .
53
2o
i
1.5
L
30 ~ ~c
0.6 0
1.C 20 0.5
10
4o
2o Fraction (a)
,0 number
~m
0 5o
o.t,
i-
I I
0.2 , 0 ,~ bottom
~ =~ 4o
t .o Fraction
310 number
,,o
top
(b)
Fig. 1. D E A E - c e l l u l o s e c h r o m a t o g r a p h y and g l y c e r o l gradient c e n t r i f u g a t i o n o f R N A p o l y m e r a s e f r o m M. tuberculosis H 3 7 R v. ( a ) T h e 3 0 - - 5 0 % a m m o n i u m s u l p h a t e f r a c t i o n w a s a p p l i e d to t h e D E A E - c e l l u l o s e c o l u m n ( 2 0 m l c o l u m n v o l u m e for e v e r y 20 g cells). E l u t i o n o f the e n z y m e is d e s c r i b e d in the t e x t . (b) T h e p r o t e i n o b t a i n e d after the s e c o n d a m m o n i u m s u l p h a t e f r a c t i o n a t i o n (see t e x t ) w a s s u b j e c t e d t o g l y c e r o l gradient c e n t r i f u g a t i o n as d e s c r i b e d in the t e x t .
discarded. More ammonium sulfate was added to the supernatant to bring the saturation to 50%. The pellet obtained on centrifugation contained the RNA polymerase activity. Glycerol gradient centrifugation. The pellet was dissolved in 3 ml of buffer A and dialysed against 100 vol. of buffer A twice, for 2 h each. 1.5 ml of the dialysed solution were layered onto a 5--20% (v/v) glycerol gradient in buffer A without glycerol in a 30 ml tube. The gradients were centrifuged at 24 000 rev./min in an SW 25.1 rotor for 24 h at 4°C. 1 ml fractions were collected after puncturing the bottom of the tube with a syringe needle. The peak fractions were pooled (Fig. lb). Sephadex G-200 chromatography. The glycerol gradient fraction ( < 5 ml) was applied to a Sephadex G-200 column (45 × 2.5 cm) equilibrated with buffer A. It was eluted with the same buffer. The tubes containing the polymerase activity were pooled and stored. Protein purity. Two methods were used to determine the purity of this enzyme. The first was by polyacrylamide gel electrophoresis (see Methods). No
Plate 1. O u c h t e r l o n e y d o u b l e agar-gel d i f f u s i o n Pattern o b t a i n e d w i t h t h e purified R N A p o l y m e r a s e f r o m M. t u b e r c u l o s i s H 3 7 R w A n t i b o d i e s w e r e raised in rabbits against t h e c r u d e e n z y m e . T h e a n t i s e r u m w a s r e a c t e d against t h e purified e n z y m e . Well 1 c o n t a i n s t h e a n t i s e r u m , w h i l e w e l l s 2 and 3 c o n t a i n t h e purified enzyme.
54
I
=,' 0"6
o-
a. A
+1 @dye ~dye Ca) (b)
0.4
.J O
E c O o0 cd
"~
,.i,-
0.2
..-JJ 0
4
8
12
16
20
FRACTION
24
28
32
4O
36
NUMBER
F i g . 2. P h o s p h o c e l l u l o s e c h r o m a t o g r a p h y o f R N A p o l y m e r a s e f r o m M. t u b e r c u l o s i s H 3 7 R ~. A p o r t i o n o f the enzyme was applied to a phosphocellulose column (10 × 1.5 cm), equilibrated with buffer C containing 0 . 1 M KC1. F o r d e t a i l s o f e l u t i o n see t e x t . I n s e t : e l e c t r o p h o r e t i c p a t t e r n o f p u r i f i e d p o l y m e r a s e (a) h o l o e n z y m e a n d ( b ) c o r e e n z y m e f r o m M. t u b e r c u l o s i s H 3 7 R v o n s o d i u m d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e gels. P r e p a r a t i o n o f gels a n d e l e c t r o p h o r e s i 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 u n d e r M e t h o d s .
detectable impurity was observed. Purity was also determined b y raising antibodies in rabbits against the partially purified enzyme. The serum collected after 6 weeks, when reacted against the purified enzyme by the double agar-gel diffusion technique [ 1 7 ] , gave only one precipitin band showing that the enzyme preparation was homogeneous (Plate 1). Phosphocellulose chromatography. A portion of the purified enzyme was applied to a phosphocellulose column (10 × 1.5 cm) equilibrated with buffer C + 0.1 M KC1. The column was washed with 2 column volumes of buffer C + 0.25 M KC1. The polymerase was then eluted with buffer C + 0.35 M KC1. (Fig. 2). 800 M g ++
Nln ++
600 s~ ~ > 400
I I I I I I /
~
//
P
/ 200
I I
/
/ r 0
//
/ i
I
[
1
2
3
CONCN.
{raM)
4
0
I I I
I
I
1
2
3
CONCN.
&
{raM)
F i g . 3. E f f e c t o f M g 2+ a n d M n 2+ o n t h e R N A p o l y m e r a s e a c t i v i t y o f M. t u b e r c u l o s i s H 3 7 R ~. R N A p o l y m e r a s e 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 u n d e r m e t h o d s . 2 5 Mg o f t h e e n z y m e p r o t e i n w e r e u s e d p e r assay. E n z y m e a c t i v i t i e s are e x p r e s s e d as p m o l C M P i n c o r p o r a t e d / 5 m i n .
55 TABLE III E F F E C T O F R I F A M P I C I N ON E. C O L I A N D M. T U B E R C U L O S I S H37R v R N A P O L Y M E R A S E S T h e assay m i x t u r e for the E. coli e n z y m e c o n t a i n e d in 0 . 2 5 m l 0 . 0 4 M Tris - HCI p H 7 . 9 / 0 . 0 1 M MgC12/ 0.1 m M E D T A / 0 . 1 m M d i t h i o t h r e i t o l / 0 . 1 5 M K C I / 0 . 4 m M UTP, G T P a n d A T P / 0 . 1 m M [ 3 H ] C T P (1.1 • 108 d p m / m m o l ) a n d 20 # g o f c a l f t h y m u s D N A . T h e details o f assay o f the m y c o b a c t e r i a l e n z y m e axe described u n d e r M e t h o d s . T h e e n z y m e c o n c e n t r a t i o n per assay w a s 10 #g. Results axe e x p r e s s e d as p e r c e n t a g e activities o f t h e c o n t r o l . Rifampicin concentration (M)
A c t i v i t y (% o f c o n t r o l ) E. coli e n z y m e
10 -9
100
10 -8
98
--
10 -7
60
--
10 -6
40
--
10 -s
5
--
M. tuberculosis e n z y m e
33
Molecular weight determination and characterization of the subunits of RNA polymerase. The enzyme was layered on a glycerol gradient 10--25% (v/v) along with standard marker proteins (catalase, mol. wt. 244 000; pyruvate kinase, mol. wt. 237 000; alcohol dehydrogenase, mol. wt. 148 000;/3-galactosidase, mol. wt 130 000), and spun at 24 000 rev./min for 24 h in an SW-25.1 rotor. The distances of migration of protein markers were plotted against their log molecular weights. The molecular weight of the polymerase falls in the range of 330 000--350 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [16] was used for determining the molecular weights of the subunits by comparing with standard proteins (~-galactosidase, bovine serum albumin, catalase, pepsin, alcohol dehydrogenase, 7-chymotrypsin and lysozyme). The enzyme splits into 4 bands having mol. wts. 96 000, 86 000, 60 000 and 44 000
~I00 ~~.,....~.....~ .~ 6
o; =~
0
i
0 2
i
I
I
I
5
10
20
30
Time Fig. 4. Stability polymerase;
(rain)
of the [14C]rifampicin • RNA polymerase complex: o o, M. t u b e r c u l o s i s R N A z~, E. coli R N A p o l y m e r a s e . For e x p e r i m e n t a l details s e e M e t h o d s .
56
respectively (Fig. 2, inset a). In the phosphocellulose enzyme the band corresponding to 60 000 mol. wt. was absent (Fig. 2, inset b). Properties. Some of the properties of the enzyme are summarised in Table II. The enzyme requires a DNA template and all four ribonucleoside triphosphates for activity. The product is labile to ribonuclease and alkali treatment and resistant to DNAase treatment, showing that it is RNA. Divalent cation requirement. Mg2÷ is absolutely essential for enzyme activity but can be replaced by Mn 2÷ to a large extent (Fig. 3). Maximal activity was obtained at 2 mM concentration of either Mg2÷ or Mn 2÷. Beyond these concentrations both had an inhibitory effect on enzyme activity. Effect of rifampicin. 10 pg of the Mycobacterium RNA polymerase is completely inhibited at l f f 8 M rifampicin, whereas the same a m o u n t of E. coli enzyme, purified according to the procedure of Burgess [12], showed lower sensitivity to the drug, being completely inhibited only at 10 -5 M rifampicin (Table III). The inhibition by rifampicin could be overcome by increasing the enzyme concentration, showing that the enzyme is the target of drug action. Stability of the RNA polymerase . rifampicin complex. Binding studies with [14C] rifampicin show that the drug binds to the M. tuberculosis RNA polymerase with a higher affinity than to the E. coli RNA polymerase (Fig. 4). On incubation of the M. tuberculosis RNA polymerase • [14C] rifampicin complex with non-radioactive drug (see Methods), the label bound to the enzyme is released slowly, and at 30 min, 60% of the label is still bound to the enzyme. Under identical conditions, the radioactivity associated with the E. coli RNA polymerase is removed rapidly and at 30 min the enzyme retains only 20% of the original radioactivity. Effect of other antitubercular drugs. None of the antitubercular drugs tested had any effect on enzyme activity, although some (8-nitro-8-hydroxyquinoline;
T A B L E IV E F F E C T OF A N T I T U B E R C U L A R TUBERCULOSIS H37R v
AND
ANTIVIRAL
AGENTS
ON R N A P O L Y M E R A S E
O F M.
E n z y m e assays w e r e d o n e as d e s c r i b e d in the text w i t h a protein c o n c e n t r a t i o n of 25/~g p e r t u b e . Results are e x p r e s s e d as the percentage activities of t h e c o n t r o l .
Agent
Activity
(% of control) A n t i t u b e r c u l a r drugs
S t r e p t o m y c i n ( 1 0 -3 M) Capreomycin ( 1 0 -3 M) K a n a m y c i n ( 1 0 -3 M) E t h a m b u t o l ( 1 0 -3 M) P y r a z i n a m i d e ( 1 0 -3 M)
I00 I00 I00 160 I00
Antivixal a g e n t s (also antitubereular) C o p p e r i s o n l c o t i n i c acid h y d r a z i d e ( 1 6 raM) 8 - a m i n o - 8 - h y d r o x y q u i n o l i n e (4 rnM) 8 o n i t r o - 8 - h y d r o x y q u i n o l i n e (4 raM) 5,7 d i c h l o r o - 8 - h y d r o x y q u i n o l i n e (4 mM) 5 - c h l o r o - 7 - i o d o - 8 - h y d r o x y q u i n o l l n e (4 m M )
100 100 175 145 155
57 5-chloro-7-iodo-8-hydroxyquinoline; 5,7
