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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 RNA POLYMERASE INHIBITOR FROM T4 PHAGE-INFECTED ESCHERICHIA COLI

AUDREY STEVENS

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.S.A.)

(Received November 29th, 1976)

Summary

Two different methods have been used to study the effect of an RNA polymerase inhibitor on DNA-enzyme binding. They show that the inhibitor, isolated from T4 phage-infected Escherichia coli, prevents binding of RNA polymerase to T4 and T7 DNA.

Previous studies [ 1,2] showed than an inhibitor o f RNA polymerase can be isolated from T4 phageinfec ted gscherichia coli and that it copurifies with the small polymerase binding protein of 10 000 daltons. The inhibitor catalyzed a salt-promoted inhibition of RNA synthesis when T4 DNA was used as a tem- plate. Little inhibition was found with calf thymus DNA or poly(dA-dT) as templates. The inhibitor was shown to inhibit the formation of rifampicin- resistant polymerase-T4 DNA 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 DNA transcription, and that with both T4 and T7 DNA enzyme-DNA binding is inhibited.

For these studies RNA 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 DNA were isolated from T4D and T7 phages according to the procedure of Thomas and Abelson [4] .

With T4 DNA as a template, the competing template method of Mueller [ 5] was used to measure the effect of the inhibitor on binding of RNA polyme- rase to DNA. Fig. 1A shows the effect of the inhibitor on the overall reaction with T4 DNA. Fig. 1B shows a measurement of the amount of enzyme bound with the same amounts of inhibitor. Binding of T4 DNA and enzyme is inhib-

194

0.5t A

--~ 0.4-o

Q2 ~ 0.3- Z_

• ~ 0 . 2

0,1-

o~

o.'3o o.~o o~3o o.~o INHIBITOR (~g)

-I00

80

v

60 130

40 z w

• 20

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 overa 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 ) in (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 ) , UTP, CTP, a n d G T P (P-L B i o c h e m i c a l s , Inc . ; 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 was 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 in 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 T4 D N A w a s c a r r i e d o u t e s sen t i a l l y as d e s c r i b e d b y Mue 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 ml ) c o n t a i n e d Tr is b u f f e r ( p H 7 .8 , 2 0 raM) , MgCl 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 (10 /~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 . In 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 [ t4C] A T P (spec . ac 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 was c o n t i n u e d a t 3 7 ° C f o r a n o t h e r 1 0 ~ 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 a c i d - i n s o l u b l e m a t e r i a l w a s c a r r i e d o u t as d e s c r i b e d above . 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 , m e a s u r e d t h e a m o u n t o f u n b o u n d e n z y m e l e f t in t h e r e a c t i o n m i x t u r e .

i GIO n I

0,05.

A

\ \ \\o

~ , ~ 3b 6b INHIBITOR (rig)

1 ~0

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 overa l l r e a c t i o n w i t h T7 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 ) in A w e r e as d e s c r i b e d f o r A in 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 T7 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 igma. In B, t h e a s say o f e n z y m e b i n d i n g t o T7 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 in 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 3H-labe led T 7 D N A ( p r e p a r e d 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 ] ; s p e c . a c t . , 8 4 0 0 c p m / ~ g ) , 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 w e r e used .

195

T A B L E I

E F F E C T O F S A L T O N T H E I N H I B I T I O N O F B I N D I N G W I T H T7 D N A

The 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 in 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 igma.

E x p e r i m e n t I n h i b i t o r [SH] D N A (ng) r e t a i n e d

(%)

1. Minus KCI

2. 0 .1 M KCI

N o n e 78 6 0 52

1 2 0 3 0

N o n e 6 5 6 0 2 5

1 2 0 5

ited to almost the same degree as is the overall reaction. It was found that the reaction with T7 DNA is also inhibited by the in-

hibitor. Its effect on the overall reaction is shown in Fig. 2A. With 3H-labeled T7 DNA, the nitrocellulose filter assay 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 DNA. The results are shown in Fig. 2B. Again, binding of the enzyme to DNA 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 DNA was promoted by salt. It could also be shown that the effect of the inhibitor on binding of enzyme to T7 DNA 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 DNA. Enzyme and 3H-labeled T7 DNA 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

E F F E C T O F T H E I N H I B I T O R A F T E R P R E I N C U B A T I O N O F E N Z Y M E A N D T 7 D N A

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 in Fig . 2B 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 in 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 . In 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 ra in , 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 .

E x p e r i m e n t I n h i b i t o r [SH] D N A (ng) r e t a i n e d

(%)

1. I n h i b i t o r a d d e d N o n e 6 5 b e f o r e enzyme 6 0 1 8 a t 0°C

2. Inhib i tor added None 61 a f t e r p r e i n c u b a t i n g 6 0 51 e n z y m e a n d D N A f o r 1 0 r a in a t 3 7 ° C

1 9 6

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 repres- sors which exert their effect by binding to the DNA. In the case of this inhib- itor, there is no evidence for DNA binding. The extent of inhibition does not vary with the DNA concentrat ion and the inhibitor does not bind to T4 DNA- cellulose columns.

This investigation was supported by the Energy Research and Develop- ment Administration under contract with the Union Carbide Corporation.

References

1 Stevens, A. and Rhoton, J.C. (1975) Biochemistry 14, 5074--5079 2 Stevens, A. (1976) in RNA Polymerase (Losick, R. and Chamberlin, M., eds.), pp. 617--627, Cold

Spring Harbor Laboratory, New York 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 York 5 Mueller, K. (1971) MoL Gen. Genet. 111 ,273 - - 296 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, 157--185