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Biochimica et Biophysica Acta, 475 (1977) 193--196 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 91441

INHIBITION OF DNA-ENZYME BINDING BY AN R N A POLYMERASE I N H I B I T O R FROM T4 PHAGE-INFECTED E S C H E R I C H I A COLI

AUDREY STEVENS

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.S.A.) (Received November 29th, 1976)

Summary T w o different methods have been used to study the effect of an R N A polymerase inhibitor on DNA-enzyme binding. They show that the inhibitor, isolated from T4 phage-infected Escherichia coli, prevents binding of R N A polymerase to T4 and T7 DNA.

Previous studies [ 1,2] showed than an inhibitor o f R N A polymerase can be isolated from T4 p h a g e i n f e c t e d gscherichia coli and that it copurifies with the small polymerase binding protein of 10 000 daltons. The inhibitor catalyzed a salt-promoted inhibition of R N A synthesis when T4 D N A was used as a template. Little inhibition was found with calf t h y m u s D N A or poly(dA-dT) as templates. The inhibitor was shown to inhibit the formation of rifampicinresistant polymerase-T4 D N A complexes, suggesting an inhibitory role at the stage of enzyme-DNA binding or at the level of DNA-enzyme preinitiation complex formation. In this paper it is shown that the inhibitor also inhibits T7 D N A transcription, and that with both T4 and T7 D N A enzyme-DNA binding is inhibited. For these studies R N A polymerase core enzyme and sigma were prepared from uninfected E. coli as described previously [3]. The inhibitor was isolated from T4 phage-infected E. coli and was an agarose gel fraction obtained as described previously [2]. T4 and T7 D N A were isolated from T4D and T7 phages according to the procedure of Thomas and Abelson [4]. With T4 D N A as a template, the competing template m e t h o d of Mueller [ 5] was used to measure the effect of the inhibitor on binding of R N A polymerase to DNA. Fig. 1A shows the effect of the inhibitor on the overall reaction with T4 DNA. Fig. 1B shows a measurement of the a m o u n t of enzyme b o u n d with the same amounts of inhibitor. Binding of T4 D N A and enzyme is inhib-

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Fig. 1 E f f e c t o f t h e i n h i b i t o r o n t h e o v e r a l l r e a c t i o n w i t h T4 D N A ( A ) a n d o n t h e b i n d i n g o f e n z y m e t o T 4 D N A (B). T h e r e a c t i o n m i x t u r e s ( 0 . 2 m l ) i n ( A ) c o n t a i n e d [14C] A T P ( S c h w a r z / M a r m ; 0 . 2 5 m M , 1 5 0 0 c p m / n m o l ) , U T P , C T P , a n d G T P (P-L B i o c h e m i c a l s , I n c . ; 0 . 2 5 m M e a c h ) , Tris b u f f e r ( p H 7 . 8 , 2 0 m M ) , MgCI 2 ( 1 0 m M ) , 2 - m e r c a p t o e t h a n o l ( 1 0 m M ) , KCI ( 0 . 2 M), b o v i n e s e r u m a l b u m i n ( S c h w a r z / M a n n ; 1 0 0 / ~ g ) , T 4 D N A ( 1 0 ~ g ) , c o r e e n z y m e (1 ~ g ) a n d s i g m a ( 0 . 7 5 / ~ g ) . T h e i n h i b i t o r w a s a d d e d t o r e a c t i o n m i x t u r e s a t 0 ° C b e f o r e e n z y m e a d d i t i o n . I n c u b a t i o n w a s f o r 1 0 r a i n a t 37VC a n d d e t e r m i n a t i o n o f r a d i o a c t i v i t y i n c o r p o r a t e d i n t o R N A w a s c a r r i e d o u t a s p r e v i o u s l y d e s c r i b e d [ 6 ] . In (B), t h e a s s a y o f R N A p o l y m e r a s e b i n d i n g t o T 4 D N A w a s c a r r i e d o u t e s s e n t i a l l y as d e s c r i b e d b y M u e l l e r [ 5 ] . T h e r e a c t i o n m i x t u r e s ( 0 . 2 m l ) c o n t a i n e d Tris b u f f e r ( p H 7 . 8 , 2 0 r a M ) , M g C l 2 ( 1 0 m M ) , 2 - m e r c a p t o e t h a n o l ( 1 0 m M ) , b o v i n e s e r u m a l b u m i n ( 1 0 0 / ~ g ) , T 4 D N A ( 1 0 / ~ g ) , KCI ( 0 . 2 M), c o r e e n z y m e ( l ~ g ) a n d s i g m a ( 0 . 7 5 ~g). i n h i b i t o r w a s a d d e d as d e s c r i b e d a b o v e . I n c o n t r o l r e a c t i o n m i x t u r e s , T 4 D N A w a s o m i t t e d . A f t e r a 1 0 - r a i n i n c u b a t i o n p e r i o d a t 3 7 ° C , 3 . 9 / ~ g o f p o l y ( d A - d T ) (Miles L a b o r a t o r i e s ) , 5 0 n m o l o f [ t 4 C ] A T P (spec. a c t . , 4 0 0 0 c p m / n m o l ) , a n d 5 0 n m o l o f U T P w e r e a d d e d . T h e r e a c t i o n w a s c o n t i n u e d at 37°C for another 10 ~ and determination of radioactivity incorporated into acid-insoluble material w a s c a r r i e d o u t as d e s c r i b e d a b o v e . T h e r e a c t i v i t y o f p o l y ( d A - d T ) , as c o m p a r e d w i t h t h e c o n t r o l , measured the amount of unbound enzyme left in the reaction mixture.

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Fig. 2 E f f e c t o f t h e i n h i b i t o r o n t h e o v e r a l l r e a c t i o n w i t h T 7 D N A ( A ) a n d o n t h e b i n d i n g o f e n z y m e t o T7 D N A (B). T h e r e a c t i o n m i x t u r e s ( 0 . 2 m l ) i n A w e r e as d e s c r i b e d f o r A i n Fig. 1 e x c e p t t h a t t h e y c o n t a i n e d 0 . 1 M KC1, 5 # g o f T 7 D N A , 0 . 2 # g o f c o r e e n z y m e , a n d 0 . 3 # g o f s i g m a . I n B, t h e a s s a y o f e n z y m e b i n d i n g t o T 7 D N A w a s c a r r i e d o u t as d e s c r i b e d b y H i n k l e a n d C h a m b e r i i n [ 8 ] , e x c e p t t h a t 0 . 1 M KCI r e p l a c e d t h e NaCI i n t h e r e a c t i o n m i x t u r e s ( 0 . 1 m l ) . 2 # g o f 3 H - l a b e l e d T 7 D N A ( p r e p a r e d as described by Hinkle and Chamberiin [8] ; spec. act., 8400 cpm/~g), 0.1 #g of core enzyme and 0.15 #g of sigma were used.

195

TABLE I EFFECT OF SALT ON THE INHIBITION OF BINDING WITH T7 DNA T h e r e a c t i o n m i x t u r e s w e r e as d e s c r i b e d i n Fig. 2 B w i t h 0 . 1 # g o f c o r e e n z y m e a n d 0 . 1 5 # g o f s i g m a .

Experiment

Inhibitor (ng)

[SH] D N A retained (%)

1. M i n u s KCI

None 60 120

78 52 30

2. 0 . 1 M KCI

None 60 120

65 25 5

ited to almost the same degree as is the overall reaction. It was found that the reaction with T7 D N A is also inhibited by the inhibitor. Its effect on the overall reaction is shown in Fig. 2A. With 3H-labeled T7 D N A , the nitrocellulosefilterassay of Jones and Berg [7] as modified by Hinkle and Chamberlin [8] was used to test the effect of the inhibitor on the binding of enzyme to D N A . The results are shown in Fig. 2B. Again, binding of the enzyme to D N A is inhibited to almost the same degree as the overall reaction. The extent of inhibition did not increase with time past the usual 10-min incubation period. It was shown previously [1,2] that the inhibition of the reaction with T4 D N A was promoted by salt.It could also be shown that the effect of the inhibitor on binding of enzyme to T7 D N A was greater in the presence of 0.1 M KCI than in its absence. The results are shown in Table I. In all the experiments described here, the inhibitor was added to the reaction mixture at 0 ° C before the addition of enzyme. It was of interest to see if inhibition of binding was observed after a preincubation of enzyme and D N A . E n z y m e and 3H-labeled T7 D N A were incubated for I0 rain at 37°C; the inhibitor was then added and the reaction mixture was left at 37°C for another

T A B L E II EFFECT OF THE INHIBITOR AFTER PREINCUBATION

OF ENZYME AND T7 DNA

T h e r e a c t i o n m i x t u r e s w e r e as d e s c r i b e d i n Fig. 2 B w i t h 0 . 1 p g o f c o r e e n z y m e a n d 0 . 1 5 p g o f a i ~ n a . T h e m i x t u r e i n E x p . 1 w a s i n c u b a t e d f o r 2 0 m i n a t S 7 ° C . I n E x p . 2 t h e i n h i b i t o r w a s a d d e d a t 1 0 r a i n , and the i n c u b a t i o n w a s c o n t i n u e d f o r a n o t h e r 1 0 r a i n a t 3 7 ° C . Experiment

Inhibitor (ng)

[SH] D N A retained (%)

1. I n h i b i t o r a d d e d b e f o r e enzyme a t 0°C

None 60

65 18

2. I n h i b i t o r added after preincubating enzyme and DNA for 10 rain at 37°C

None 60

61 51

196

10 min. As the results in Table II show, there is only a small inhibition of binding in this case. Inhibition of RNA polymerase-DNA binding has been found with repressors which exert their effect by binding to the DNA. In the case of this inhibitor, there is no evidence for DNA binding. The extent o f inhibition does n o t vary with the DNA concentration and the inhibitor does n o t bind to T4 DNAcellulose columns. This investigation was supported by the Energy Research and Developm e n t Administration under contract with the Union Carbide Corporation. References 1 Stevens, A. and R h o t o n , J.C. (1975) Biochemistry 14, 5074--5079 2 Stevens, A. (1976) in R N A Polymerase (Losick, R. and Chamberlin, M., eds.), pp. 617--627, Cold Spring Harbor L a b o r a t o r y , New Y o r k 3 Stevens, A. (1974) Biochemistry 13, 493--503 4 Thomas, Jr., C.A. and Abelson, J. (1966) in Procedures in Nucleic Acid Research (Cantoni. G.L. and Davies, D.R., eds.), pp. 553--561, Harper and Row, New Y o r k 5 Mueller, K. (1971) MoL Gen. Genet. 1 1 1 , 2 7 3 - - 2 9 6 6 Stevens, A. and Henry, J. (1964) J. Biol. Chem. 239, 196--203 7 Jones, O.W. and Berg, P. (1966) J. Mol. Biol. 22, 199--209 8 I-Iinkle, D.C. and Chamberlin, M.J. (1972) J. Mol. Biol. 70, 1 5 7 - - 1 8 5

Inhibition of DNA-enzyme binding by an RNA polymerase inhibitor from T4 phage-infected Escherichia coli.

193 Biochimica et Biophysica Acta, 475 (1977) 193--196 © Elsevier/North-Holland Biomedical Press BBA Report BBA 91441 INHIBITION OF DNA-ENZYME BIND...
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