Deoxyribonuclease resistance of DNA-RNA polymerase complexes

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593

i G. INGRAM,Methods of Organic Elemental Micro Analysis, Reinhold, New York, 1962, p. 18o. 2 F. D. SNELL AND C. T. SNELL, Colorimetric Methods o/ Analysis, Vol. II, Vail Nostrand, Princeton, N. J., 3rd ed., 1949, p. 725 • 3 K. W. BRAMMER, Biochim. Biophys. Acta, 72 (1963) 217. 4 C. T. Y u AND e . C. ZAMECNIK, Biochim. Biophys. Acta, 76 (1963) 209. 5 R. W. HOLLEY, J. APGAR, G. A. EVERETT, J. T. MADISON, M. MARQUISEE, S. H. MERRILL, J. R. PENSWICK AND A. ZAMIR, Science, 147 (1965) 1462. 6 J. APGAR, R. W. HOLLE¥ AND S. H. I\~ERRILL, J. Biol. Chem., 237 (1962) 796.

7 8 9 io ii 12 13 14

J. R. PENSWICKAND R. W. HOLLEY, Proc. Natl. Acad. Sci. U.S., 53 (1965) 543. R, V. TOMLINSONAND G. M. TENER, J. Am. Chem. Soc., 84 (1962) 2644. J. APC,AR, G. A. EVERETTAND R. W. HOLLEY,J. Biol. Chem., 241 (1966) 12o6. J. A. CARBON,Biochem. Biophys. Res. Commun., 15 (I964) i. J. A. CARBON,Biochim. Biophys. Acta, 95 (1965) 55°. R. W. HOLLEY,J. T. MADISONANDA. ZAMIR,Biochem. Biophys. Res. Commun., 17 (1964) 389 . G. W. RusHIzK¥ AND H. n. SOBER, Biochem. Biophys. Res. Commun., 14 (1964) 276. J. T. MADISON,G. A. EVERETTAND H. I{. KUNG, J. Biol. Chem., 242 (1967) 1318.

Received September 28th, 1967 Biochim. Biophys. Acta, 149 (1967) 590-593

BBA 93280 Deoxyribonucleose resistonce of D N A - R N A polymerose complexes We have found, as have others l-a, t h a t during the in vitro R N A polymerase (nucleosidetriphosphate: R N A nucleotidyltransferase, EC 2.7.7.6) reaction a ternary complex is formed a m o n g the native D N A template, the R N A polymerase enzyme, and the product R N A (the D N A - R N A p o l y m e r a s e - R N A complex). A bin a r y D N A - R N A polymerase complex is formed in the absence of R N A synthesis 4. HAYASHIa treated the t e r n a r y complex with pancreatic ribonuclease (EC 2.7.7.16) and found t h a t only the most recently synthesized portion of the RNA, a 5o-nucleotide-long segment, was resistant to ribonuclease attack. He attributed this resistance to a hybridization between the R N A product and one strand of the D N A template. In this paper, we report on the resistance of part of the D N A in b o t h the binary and in the t e r n a r y complex to digestion b y pancreatic deoxyribonuclease (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5) 5. The reaction mixture for the synthesis of the t e r n a r y complex contained, in a total volume of 2.5 ml, 250/~moles of Tris-HC1 buffer (pH 7-5) ; 4 .0 #moles of spermidine trihydrochloride; 6.25 #moles of MnCI~; 2.o#moles each of ATP, GTP, U T P , and CTP; 500 #moles of KC1; 0.8 ml of a2P-labelled native Bacillus subtilis D N A (.42e0 m/z 12.o) containing a total of 37 500 counts/minute; and 5oo,ug of micrococcal R N A polymerase protein. I o u g of polymerase enzyme catalyzed the incorporation of 1,6.1o -6 mM of A T P into R N A in IO minutes (37 °). The nucleoside triphosphates were omitted for the synthesis of the binary D N A - R N A polymerase complex, and the R N A polymerase was omitted in the D N A control experiments. R N A polymerase Fraction VI was isolated from M . lysodeikticus as described b y NAKAMOTO et al. ~. The D N A was isolated from Bacillus subtilis according to the procedure of MARMUR7. The D N A was further purified b y equilibrium centrifugation t h r o u g h 0.0o5 M Tris0.0o5 M E D T A (pH 8.0) CsCI~ gradient (p = 1.7o6 g/ml) 7 and dialyzed against 2 X I-1 volumes of o.oi M NaC1 before use. The Bacillus subtilis cells were grown in I 1 of Biochim. Biophys. Acta, 149 (1967) 593-595

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Fig. I. Sephadex G-2oo c h r o m a t o g r a p h y of (A) the D N A - R N A p o l y m e r a s e - R N A complex, (B) th 9 D N A - R N A polymerase complex a n d (C) the D N A control, each after the action of deoxyribonuclease. The void volumes for A, B and C,are io, 15, and 15 ml, respectively. IO ml were allowed to pass t h r o u g h columns A a n d B before s t a r t i n g to collect fractions.

Difco nutrient broth containing 1-2 mC of neutralized Hs'~2P0~. The polymerase reactions were run for 5 minutes at o7 ° before adding o.I m l o f deoxyribonuclease solution to the polymerase reaction m i x t u r e . This deoxyribonuclease solution conBiochim. Biophys. Acta, 149 (x967) 593-595

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tained I mg of Worthington's pancreatic deoxyribonuclease I dissolved in I ml of 0.5 M Tris-o.25 M MgSO~ (pH 7-5). The degradation with pancreatic deoxyribonuclease was allowed to proceed for 2 hours at 37 °. The digested mixture was then applied to Sephadex G-2oo columns. The columns were eluted with a o.oi M Tris buffer (pH 7.5). Figure I shows that part of the DNA, both in the ternary DNA-RNA polymerase-RNA complex and in the binary DNA-RNA polymerase complex, is resistant to deoxyribonuclease. In Fig. Ia 1.5 % (1125 out of 75 ooo counts) of the [~2P]DNA in the ternary complex is resistant to deoxyribonuclease. In Fig. Ib 2 % (71o out of 75 ooo counts) of the DNA in the binary complex is resistant to deoxyribonuclease. Both these values must be corrected for the o.5 % resistance (220 out of 48 ooo counts) found for the -"~P-labelledBacillus subtilis DNA control (Fig. IC). The deoxyribonuclease-resistant [3~P]DNA is eluted with the material possessing a molecular weight greater than 2oo ooo. However, when the isolated deoxyribonucleaseresistant DNA from the ternary complex is subiected to the action of pronase and then re-run on a Sephadex G-2oo column, all but 1 % of the 32p label is released from this high molecular weight peak. These studies show that the RNA polymerase in the binary and in the ternary complex surrounds a portion of the DNA in such a manner as to make it inaccessible to deoxyribonuclease attack. The deoxyribonuclease resistance is greater in the ternar~ complex than in the corresponding binary complex. The deoxyribonucleaseresistant material is released from the polymerase, and perhaps also from the RNA product, in the ternary complex by the proteolytic enzyme pronase. BLATTNER AND TI-IOMAS8 have also recently found that part of the binary T5 DNA-RNA polymerase (E. coli) complex is resistant to both pancreatic deoxyribonuclease and venom phosphodiesterase attack. We are presently taking advantage of the deoxyribonuclease resistance of the DNA in these polymerase complexes to determine the structure of the binding site on the DNA template for the RNA polymerase enzyme, and to determine whether or not the polymerase enzyme moves alovg the DNA template during transcription. This work was supported by a Brown-Hazen Research Grant from the Research Corporation,

Department o/ Biological Sciences, Purdue University, La/ayette, Ind. (U.S.A.)

ROBERT L.

NOVAK*

H.BREMER AND M. W. KONRAD, Proc. Natl. Acad. Sci. U.S., 51 (1964) 8Ol. P. BERG AND M. CHAMBERLIN, J. Mol. Biol., 8 (1964) 297. M. HAYASHI, Proc. Natl. Aead. Sci. U.S., 54 (1965) 1736. C. F. F o x , R. I. GUMPORT AND S. B. WEISS, J. Biol. Chem., 240 (I965) 21Ol. P a r t of t h i s w o r k w a s p r e s e n t e d a t t h e I 5 4 t h A m e r . C h e m . Soc. Meeting: R. L. NOVAK,Abstr, z54 Amer. Chem. Soc. Meeting, 1967, 46 C. 6 T. ~N~AKAMOTO,C. F. FOX AND S. g . WEISS~ J. Biol. Chem., 239 (1964) 167. 7 J. MARMUR,in S. P. COLOWICK AND N. O. KAPLAN,Methods in Enzymology, Vol. 6, A c a d e m i c Press, N e w York, 1963, p. 726. 8 V. R. BLATTNER AND C. A. THOMAS,Abstr. 7th intern. Congr. Biockem;, Tokyo, 1967, Vol. 4, T h e Science Council of J a p a n , T o k y o , p. 656.

I 2 3 4 5

Received September Ilth, 1967 * P r e s e n t a d d r e s s : D e p a r t m e n t of C h e m i s t r y , H a r v a r d U n i v e r s i t y , ' C a m b r i d g e , Mass. (UIS.A.)

Biochim. Biophys. Acta, i 4 9 ( i 9 6 7 ) 593-595