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The effect of trypsin digestion and ionic strength on RNA polymerase of rat liver
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
The Effect of Trypsin
362-366
118,
Digestion
Polymerase A. E. PEGG Department
(1967)
and Ionic
Strength
on RNA
of Rat Liver AND
of Biochemistry,
A. I-ZORNER
University
Received
of Cambridge,
England
May 25, 1966
RNA polymerase of rat liver nuclei was assayed at low and at high ionic strength following incubation of the nuclei with trypsin. Digestion with trypsin, to remove histone, stimulated RNA polymerase activity when this was assayed at low ionic strength, but not when RNA synthesis had already been stimulated by the presence of a high salt concentration. Stimulation of RNA polymerase by high ionic strength increased the time during which RNA synthesis was observed. The optimal concen tration of divalent cations (Mg++ or Mn++), which are required for incorporation, was changed by the presence of a high salt concentration. After trypsin treatment, however, nuclei assayed at low ionic strength showed characteristics similar to those of nuclei assayed at high ionic strength, but the incorporation by such nuclei was less than that, of nuclei stimulated by high ionic strengt,h.
The presence of a high salt concentration in the assay medium stimulates the activity of RNA polymerase of various mammalian tissues when this is measured in crude preparations where endogenous DNA is the sole primer of the reaction (l-5). All such preparations contain histones which are known to inhibit RNA polymerase (668). Nucleohistone complexes are known to dissociate in solutions of high ionic strength (9, 10). Trypsin, which hydrolyzes bonds adjacent to a basic amino acid, can be used to digest histones which are rich in basic amino acids (11). We have studied the RNA polymerase activity of rat liver nuclei in solutions of low and high ionic strength after preincubation of the nuclei with trypsin. MATERIALS
AND
METHODS
Materi&. Trypsin was obtained from Worthington Biochemical Corporation, New Jersey, and was free of ribonuclease activity. Actinomycin D was a gift from Merck, Sharp and Dohme, Rahway, New Jersey. [3H]-CTP was obtained from Sigma Chemical Company. Preparation of nuclei. Female albino rats, 362
bred in t,he laboratory, were killed by decapitation and the livers were rapidly removed into icecold 0.5 M-sucrose containing 3 mM magnesium chloride. The livers were cut up finely with scissors, rinsed free of blood, weighed by displacement and homogenised in four volumes of the same medium. The homogenat,e was filtered through gauze and centrifuged at SOOgfor 5 minutes. The pellet produced was washed once by suspension in the sucrose-magnesium chloride medium and recentrifuged at 6OOg for 5 minutes. The pellet was then suspended in 2.2 M sucrose containing 3 rnrvr magnesium chloride and centrifuged at 22,000 rpm for 1 hour in the S.W.25 rotor of the Spinco model L Centrifuge. The purified nuclei produced by this procedure were suspended in 0.1 M KC1 adjusted to pH 7.9 with Tris-HCl. Assay of RNA polymerase. RNA polymerase activity was assayed in 0.5 ml total volume containing 50 pmoles Tris-HCl pH 7.9, 0.75 rmole MnC12, 2.5 pmoles mercaptoethanol, 20 Imoles KCl, 0.2 pmole each of ATP, GTP, and UTP, 0.04 pmole [‘HI-CTP, and nuclei containing approximately 0.3 mg of DNA. For assays at high ionic strength, 0.05 ml of saturated ammonium sulphate solution was added. Tubes were incubated at 37” for varying times and the reaction was stopped by the addition of 1 ml of ice-cold saturated sodium pyrophosphate solution. Bovine serum albumin (1 mg) was added as carrier fol-
TRYPSIN
DIGESTION
ANl)
RNil
363
POI,Yi\II+:RASl~
low-ed by 5 ml of ice-cold 5y0 perchloric acid. The precipitate was washed three times with cold 5y0 perchloric acid, twice with ethanol-ether [l: 1) and finally with ether. The dried precipitate was dissolved in hyamine and counted in a scintillation counter. Results are expressed in mpmoles of [“II]-CTP incorporated per milligram of DNA present. Incorporation was dependent on the presence of all four nucleoside triphosphates. l’rypsin treatment. Nuclei containing about 0.3 mg of DNA were preincubated for 15 minutes at 37” with varying amounts of trypsin in a total volume of 0.3 ml containing 50 bmoles Tris-HCl pH 7.9 and 20 pmoles KCl. RNA polymerase was then assayed as described during a furi,her incubation after the addition of 0.2 ml of a solution containing the other reagents. DNA was estimated by the method of Burtson
(12) Ii’igure 1 shows the time course of incorporation of [“HI-CTP into RN,4 by isolated rat-liver nuclei. When assayed at low ionic strength, incorporation was still increasing at 40 minutes. Figure 1 demonstrates that, when 13H]-CTP incorporationassayed at low ionic strength-had ceased,
FIG. 1. Time course of [3H]-CTP incorporation into RNA by rat-liver nuclei assayed at low ionic strength (A), assayed at low ionic strength when actinomycin (50 pg) was added after 15 minutes (A), assayed at low ionic strength when 0.05 ml of saturated ammonium sulphate was added after 15 minutes (a) and assayed at high ionic strength (0). Incorporation is in mpmoles of [aHI-CTP incorporated per mg of DNA present.
INC”EiTION
2Tqh4E h%Sl
40
Fro. 2. Time course of incorporation of [“HICTP by isolated nuclei assayed at low ionic skength after pre-incubation for 15 minutes with the amount of trypsin shown (in pg).
resumpt’ion could be induced by the addition of ammonium sulphate. The new rate curve was approximately paralle1 to that of the curve obtained when the assay was carried out in the presence of ammonium sulphate from the start of incubat,ion. When actinomycin was added after the completion of incorporation, at low ionic strength, no increase was observed in t,he usual small loss of label from RWA. It seems likely, thcreforc, that the plateau observed does not result from a balance between breakdown and synthesis of RNA. Figure 2 shows the time course of incorporat,ion of [3H]-CTP into RNA of nuclei which had previously been incubated for 15 minutes with trypsin at the concentrat,ion indicated and subsequently assayed at low ionic strength. Incorporation was stimulated by the pre-incubation mit,h trypsin and can continue for longer periods than with nuclei not treated with trypsin. The pattern of incorporation is similar to that seer, with nuclei assayed at, high ionic strength without trypsin treatment. Maximal stimulation was obtained with approximately 10 pg of trypsin per tube. When greater amounts of trypsin were used a similar curve was obtained but less incorporation was observed. Probably some
364
PEGG
AND
trypsin digestion of the enzyme had occurred. Figure 3 shows similar results with nuclei pre-incubated with trypsin and assayed at high ionic strength. When low concentra-
FIG. 3. Time course of incorporation of [3H]CTP by isolated nuclei assayed at high ionic strength after pre-incubation for 15 minutes with the amount of trypsin shown (in pg).
KORNER
tions of trypsin were used, little change in incorporation was seen ; when higher amounts of trypsin were employed, there was a marked inhibition of incorporation. Even after incubation with 100 pg trypsin, a stimulation of activity by high ionic strength was noted (compare Figs. 2 and 3). Estimation of the acid-soluble protein (13) remaining after treatment with 100 pg of trypsin revealed that at most only 10 % of the histone was still present. RNA polymerase requires either manganese or magnesium ions for activity. Figure 4 shows the effect of manganese ion concentration on RNA synthesis and Fig. 5 the effect of magnesium ions. Each figure shows results obtained with nuclei assayed at low ionic strength, at high ionic strength and at low ionic strength after pre-treatment with trypsin. The optimal concentration of Mn++ or Mg++ is altered by the ionic strength of the medium as previously reported (4). The activation curves for trypsin-treated nuclei, although assayed at low ionic strength, were similar to those obtained with nuclei assayed at high ionic strength although the amount of incorporation obtained was less.
0
k
FIG. 4. Effect of Mn++ concentration on incorporation of [3H]-CTP into RNA assayed at low ionic strength (O), assayed at high ionic strength (0), and assayed at low ionic strength after pre-incubation with 50 pg trypsin (a). Incorporation is in mrmoles of [3H]-CTP incorporated per mg of DNA present in 15 minutes. The values for assay at high ionic strength have been halved to reduce the scale of the figure.
FIG. 5. Effect of Mg++ concentration on incorporation of [3H]-CTP into RNA assayed at low ionic strength (a), assayed at high ionic strength (0), and assayed at low ionic strength after preincubation with 10~ typsin (A). Incorporation is in mrmoles of [“HI-CTP incorporated per mg of DNA present in 15 minutes.
nism other t,han that seen w&h the purified enzyme. One explanation of t’he rise in RKA It is possible that the high salt acts by polymerase a&i&y of mammalian prepamodifying the act)ivity of t’rypsin still rations mhcn the assay is carried out at present during assay. However, trypsin high ionic skength is that, the salt uncovers inhibitor, added at the same time as the more of the DNA primer by removal of [aH]-CTP, did not modify t’he stimulation basic proteins (14). by added salt so this explanation seems Our results offer confirmation of this unlikely. sugg&ion, for treatment of nuclei with The high salt may act by inhibit,ing t,rypsin, which is kno\\rn to degrade histones, ribonwlease activity. Indeed the RNA stimulated RSA polymerase nctivit,y in a formed at low ionic strength is of low molecumanner similar to that seen in the presence lar weight (16, 17) so this possibility is a of high salt concentrations. Our finding is real one. Although it was observed (Fig. 1) supported by the observation (11) t’hat that few counts were lost from labeled incorporation of adcnine, erotic acid, and RNA after inhibition of RNIZ synthesis, phosphate into RNA was enhanced by keatit is possible that t’he RNA may have alment of thymus nuclei with t’rypsin. Furtherready been degraded to a ribonucleasemore, as would be expected, we noted that resistant piece, trypsin t’reat’ment, did. not stimulate RN&4 The values obtained for the optimal conpolymerase activity when t,his had already centrat,ion of divalent cations agree reabeen stimulated by the presence of high salt concentrations, suggesting that bot,h sonably well with those previously reported m&hods of stimulation are acting in the by Widnell and Tata (4), although these workers used less ammonium sulphate same way, by removing basic proteins t,o than in our experiments and the nuclei in uncovw more primer DNA. our experiments, unlike in t,heir experiments, The results of Chambon et al. (14) show had been preincubat’ed for 15 minutes. The that, purified RNA polymerase, free of inhibit’ion of RKA polymerase activity endogenous D?yTA, is inhibited by high produced by excess manganese or magnesium concentrations of salt, but inhibition is ions is more marked when the enzyme is greatly reduced if the enzyme is first aassayed at low ionic strength. The solulowed to form a complex wit’h added DKA bility of nucleohist,one is reduced if excess and has begun to synthesize RSA. Our finding that RNA polymerase activity was manganese or magnesium is present (IS). It is possible, therefore, t)hat the change in st,ill obGned even after incubation of nuclei the values obt)ainetl for the optimal concenwith 100 pg of trypsin shows that RX&4 t’rabion of divalent cation, after addition of polymerasc is very resist,atit to trypsin ammonium sulphat’e or after trypsin t,reatdigestion. It is possible that the enzyme is ment, is due t’o the greater solubility of the protected by being present as a compkx enzynicPDNA complex after the removal of with DIVA and newly synthesized RY\;A. histories. Our results suggest that high ionic strength ACKNOWLEDGMENTS may st#imulate RNA polymerase activity This work was supported by grants from The in other whys as well as by uncovering Medical Research Council and The British Empire more DNA. It’ was noted that, when very Cancer Campaign. We are grateful to the Scientific little histone remained in the preparation Research Council for a Studentship for one of after treat,ment with trypsin, which had the authors (A.E.P.), and to Professor F. G. inhibited enzyme activity, t’he addition of Young for his encouragement. salt still caused an increase in RK\‘A polyREFERENCES merase activity. Purified RKA polymerase 1. GOLDBEIZG, I. 13. Hiochim. Biophys. Ac(a 51, is stimulated slightly by a small increase in 201, (1901). the ionic skength (1;) but, our result was 2. H.~NCOCK, R. I,., DELIS, R. F., SH.\V, M., only obtained when a very high salt conAND WILLIAMS-Asri~.\N, H. G., Riochenl. centrabion was added, suggesting a mechaLi3ophy.s Acta 65, 257 (1962). I>ISCIJSSION
366
PEGG
AND
3. GORSKI, J., J. Biol. Chem. 239, 889 (1964). 4. WIDNELL, C. C. AXD TATA, J. R., Biochim. Biophys. Acta 87, 531 (1964). 5. PEGG, A. E., AND KORR'ER, A., A’ature 206, 904 (1965). 6. HUANG, R. C., AND BONNER, J., Proc. Xall. Acad Sci. U.S. 48, 1216 (1962). 7. BARR, G. C. AND BUTLER, J. A. V. LVature 199, 1170 (1963). 8. HINDLEY, J. B&hem Biophys. Res. Commun. 12, 175 (1963). 9. HINDLEY, J., 6th Intern. Congr. Biochem., New York, Abstr I-82 (1964). 10. AKINRIMISI, E. O., BONNER, J., AND Ts’o, P. O., J. MOL. Biol. 11, 128 (1965). 11. ALLFREY, V.G., LITTAN, V. C., AND MIRSKY,
KORNER
12. 13. 14.
15. 16. 17. 18.
A. E., Proc. Natl. Acad. Sci. U.S. 49, 414 i1963). BUIZTON, K., Biochem. J. 62, 315 (1965). MIRSKY, A. E. AND RIS, H. J. Genl. Physiol. 31, 1 (1947). CHAMBON, P., RAMUZ, M. AND DOLY, J., Niochem. Biophys. Res. Commun. 21, 156 (1965). WILLIAMS-ASHMAX, H. G., J. Cell Camp. Physiol. 66 Suppl. 1, 111 (1965). WIDNELL, C. C., Biochem. J. 96, 42P (1965). PEGG, A. E. .~ND KORNER, A. (unpublished observations). BONNER, J. AND HUANG, R. C., Biochem. Biophys. Res Commun. 22, 211 (1966).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
The Effect of Trypsin
362-366
118,
Digestion
Polymerase A. E. PEGG Department
(1967)
and Ionic
Strength
on RNA
of Rat Liver AND
of Biochemistry,
A. I-ZORNER
University
Received
of Cambridge,
England
May 25, 1966
RNA polymerase of rat liver nuclei was assayed at low and at high ionic strength following incubation of the nuclei with trypsin. Digestion with trypsin, to remove histone, stimulated RNA polymerase activity when this was assayed at low ionic strength, but not when RNA synthesis had already been stimulated by the presence of a high salt concentration. Stimulation of RNA polymerase by high ionic strength increased the time during which RNA synthesis was observed. The optimal concen tration of divalent cations (Mg++ or Mn++), which are required for incorporation, was changed by the presence of a high salt concentration. After trypsin treatment, however, nuclei assayed at low ionic strength showed characteristics similar to those of nuclei assayed at high ionic strength, but the incorporation by such nuclei was less than that, of nuclei stimulated by high ionic strengt,h.
The presence of a high salt concentration in the assay medium stimulates the activity of RNA polymerase of various mammalian tissues when this is measured in crude preparations where endogenous DNA is the sole primer of the reaction (l-5). All such preparations contain histones which are known to inhibit RNA polymerase (668). Nucleohistone complexes are known to dissociate in solutions of high ionic strength (9, 10). Trypsin, which hydrolyzes bonds adjacent to a basic amino acid, can be used to digest histones which are rich in basic amino acids (11). We have studied the RNA polymerase activity of rat liver nuclei in solutions of low and high ionic strength after preincubation of the nuclei with trypsin. MATERIALS
AND
METHODS
Materi&. Trypsin was obtained from Worthington Biochemical Corporation, New Jersey, and was free of ribonuclease activity. Actinomycin D was a gift from Merck, Sharp and Dohme, Rahway, New Jersey. [3H]-CTP was obtained from Sigma Chemical Company. Preparation of nuclei. Female albino rats, 362
bred in t,he laboratory, were killed by decapitation and the livers were rapidly removed into icecold 0.5 M-sucrose containing 3 mM magnesium chloride. The livers were cut up finely with scissors, rinsed free of blood, weighed by displacement and homogenised in four volumes of the same medium. The homogenat,e was filtered through gauze and centrifuged at SOOgfor 5 minutes. The pellet produced was washed once by suspension in the sucrose-magnesium chloride medium and recentrifuged at 6OOg for 5 minutes. The pellet was then suspended in 2.2 M sucrose containing 3 rnrvr magnesium chloride and centrifuged at 22,000 rpm for 1 hour in the S.W.25 rotor of the Spinco model L Centrifuge. The purified nuclei produced by this procedure were suspended in 0.1 M KC1 adjusted to pH 7.9 with Tris-HCl. Assay of RNA polymerase. RNA polymerase activity was assayed in 0.5 ml total volume containing 50 pmoles Tris-HCl pH 7.9, 0.75 rmole MnC12, 2.5 pmoles mercaptoethanol, 20 Imoles KCl, 0.2 pmole each of ATP, GTP, and UTP, 0.04 pmole [‘HI-CTP, and nuclei containing approximately 0.3 mg of DNA. For assays at high ionic strength, 0.05 ml of saturated ammonium sulphate solution was added. Tubes were incubated at 37” for varying times and the reaction was stopped by the addition of 1 ml of ice-cold saturated sodium pyrophosphate solution. Bovine serum albumin (1 mg) was added as carrier fol-
TRYPSIN
DIGESTION
ANl)
RNil
363
POI,Yi\II+:RASl~
low-ed by 5 ml of ice-cold 5y0 perchloric acid. The precipitate was washed three times with cold 5y0 perchloric acid, twice with ethanol-ether [l: 1) and finally with ether. The dried precipitate was dissolved in hyamine and counted in a scintillation counter. Results are expressed in mpmoles of [“II]-CTP incorporated per milligram of DNA present. Incorporation was dependent on the presence of all four nucleoside triphosphates. l’rypsin treatment. Nuclei containing about 0.3 mg of DNA were preincubated for 15 minutes at 37” with varying amounts of trypsin in a total volume of 0.3 ml containing 50 bmoles Tris-HCl pH 7.9 and 20 pmoles KCl. RNA polymerase was then assayed as described during a furi,her incubation after the addition of 0.2 ml of a solution containing the other reagents. DNA was estimated by the method of Burtson
(12) Ii’igure 1 shows the time course of incorporation of [“HI-CTP into RN,4 by isolated rat-liver nuclei. When assayed at low ionic strength, incorporation was still increasing at 40 minutes. Figure 1 demonstrates that, when 13H]-CTP incorporationassayed at low ionic strength-had ceased,
FIG. 1. Time course of [3H]-CTP incorporation into RNA by rat-liver nuclei assayed at low ionic strength (A), assayed at low ionic strength when actinomycin (50 pg) was added after 15 minutes (A), assayed at low ionic strength when 0.05 ml of saturated ammonium sulphate was added after 15 minutes (a) and assayed at high ionic strength (0). Incorporation is in mpmoles of [aHI-CTP incorporated per mg of DNA present.
INC”EiTION
2Tqh4E h%Sl
40
Fro. 2. Time course of incorporation of [“HICTP by isolated nuclei assayed at low ionic skength after pre-incubation for 15 minutes with the amount of trypsin shown (in pg).
resumpt’ion could be induced by the addition of ammonium sulphate. The new rate curve was approximately paralle1 to that of the curve obtained when the assay was carried out in the presence of ammonium sulphate from the start of incubat,ion. When actinomycin was added after the completion of incorporation, at low ionic strength, no increase was observed in t,he usual small loss of label from RWA. It seems likely, thcreforc, that the plateau observed does not result from a balance between breakdown and synthesis of RNA. Figure 2 shows the time course of incorporat,ion of [3H]-CTP into RNA of nuclei which had previously been incubated for 15 minutes with trypsin at the concentrat,ion indicated and subsequently assayed at low ionic strength. Incorporation was stimulated by the pre-incubation mit,h trypsin and can continue for longer periods than with nuclei not treated with trypsin. The pattern of incorporation is similar to that seer, with nuclei assayed at, high ionic strength without trypsin treatment. Maximal stimulation was obtained with approximately 10 pg of trypsin per tube. When greater amounts of trypsin were used a similar curve was obtained but less incorporation was observed. Probably some
364
PEGG
AND
trypsin digestion of the enzyme had occurred. Figure 3 shows similar results with nuclei pre-incubated with trypsin and assayed at high ionic strength. When low concentra-
FIG. 3. Time course of incorporation of [3H]CTP by isolated nuclei assayed at high ionic strength after pre-incubation for 15 minutes with the amount of trypsin shown (in pg).
KORNER
tions of trypsin were used, little change in incorporation was seen ; when higher amounts of trypsin were employed, there was a marked inhibition of incorporation. Even after incubation with 100 pg trypsin, a stimulation of activity by high ionic strength was noted (compare Figs. 2 and 3). Estimation of the acid-soluble protein (13) remaining after treatment with 100 pg of trypsin revealed that at most only 10 % of the histone was still present. RNA polymerase requires either manganese or magnesium ions for activity. Figure 4 shows the effect of manganese ion concentration on RNA synthesis and Fig. 5 the effect of magnesium ions. Each figure shows results obtained with nuclei assayed at low ionic strength, at high ionic strength and at low ionic strength after pre-treatment with trypsin. The optimal concentration of Mn++ or Mg++ is altered by the ionic strength of the medium as previously reported (4). The activation curves for trypsin-treated nuclei, although assayed at low ionic strength, were similar to those obtained with nuclei assayed at high ionic strength although the amount of incorporation obtained was less.
0
k
FIG. 4. Effect of Mn++ concentration on incorporation of [3H]-CTP into RNA assayed at low ionic strength (O), assayed at high ionic strength (0), and assayed at low ionic strength after pre-incubation with 50 pg trypsin (a). Incorporation is in mrmoles of [3H]-CTP incorporated per mg of DNA present in 15 minutes. The values for assay at high ionic strength have been halved to reduce the scale of the figure.
FIG. 5. Effect of Mg++ concentration on incorporation of [3H]-CTP into RNA assayed at low ionic strength (a), assayed at high ionic strength (0), and assayed at low ionic strength after preincubation with 10~ typsin (A). Incorporation is in mrmoles of [“HI-CTP incorporated per mg of DNA present in 15 minutes.
nism other t,han that seen w&h the purified enzyme. One explanation of t’he rise in RKA It is possible that the high salt acts by polymerase a&i&y of mammalian prepamodifying the act)ivity of t’rypsin still rations mhcn the assay is carried out at present during assay. However, trypsin high ionic skength is that, the salt uncovers inhibitor, added at the same time as the more of the DNA primer by removal of [aH]-CTP, did not modify t’he stimulation basic proteins (14). by added salt so this explanation seems Our results offer confirmation of this unlikely. sugg&ion, for treatment of nuclei with The high salt may act by inhibit,ing t,rypsin, which is kno\\rn to degrade histones, ribonwlease activity. Indeed the RNA stimulated RSA polymerase nctivit,y in a formed at low ionic strength is of low molecumanner similar to that seen in the presence lar weight (16, 17) so this possibility is a of high salt concentrations. Our finding is real one. Although it was observed (Fig. 1) supported by the observation (11) t’hat that few counts were lost from labeled incorporation of adcnine, erotic acid, and RNA after inhibition of RNIZ synthesis, phosphate into RNA was enhanced by keatit is possible that t’he RNA may have alment of thymus nuclei with t’rypsin. Furtherready been degraded to a ribonucleasemore, as would be expected, we noted that resistant piece, trypsin t’reat’ment, did. not stimulate RN&4 The values obtained for the optimal conpolymerase activity when t,his had already centrat,ion of divalent cations agree reabeen stimulated by the presence of high salt concentrations, suggesting that bot,h sonably well with those previously reported m&hods of stimulation are acting in the by Widnell and Tata (4), although these workers used less ammonium sulphate same way, by removing basic proteins t,o than in our experiments and the nuclei in uncovw more primer DNA. our experiments, unlike in t,heir experiments, The results of Chambon et al. (14) show had been preincubat’ed for 15 minutes. The that, purified RNA polymerase, free of inhibit’ion of RKA polymerase activity endogenous D?yTA, is inhibited by high produced by excess manganese or magnesium concentrations of salt, but inhibition is ions is more marked when the enzyme is greatly reduced if the enzyme is first aassayed at low ionic strength. The solulowed to form a complex wit’h added DKA bility of nucleohist,one is reduced if excess and has begun to synthesize RSA. Our finding that RNA polymerase activity was manganese or magnesium is present (IS). It is possible, therefore, t)hat the change in st,ill obGned even after incubation of nuclei the values obt)ainetl for the optimal concenwith 100 pg of trypsin shows that RX&4 t’rabion of divalent cation, after addition of polymerasc is very resist,atit to trypsin ammonium sulphat’e or after trypsin t,reatdigestion. It is possible that the enzyme is ment, is due t’o the greater solubility of the protected by being present as a compkx enzynicPDNA complex after the removal of with DIVA and newly synthesized RY\;A. histories. Our results suggest that high ionic strength ACKNOWLEDGMENTS may st#imulate RNA polymerase activity This work was supported by grants from The in other whys as well as by uncovering Medical Research Council and The British Empire more DNA. It’ was noted that, when very Cancer Campaign. We are grateful to the Scientific little histone remained in the preparation Research Council for a Studentship for one of after treat,ment with trypsin, which had the authors (A.E.P.), and to Professor F. G. inhibited enzyme activity, t’he addition of Young for his encouragement. salt still caused an increase in RK\‘A polyREFERENCES merase activity. Purified RKA polymerase 1. GOLDBEIZG, I. 13. Hiochim. Biophys. Ac(a 51, is stimulated slightly by a small increase in 201, (1901). the ionic skength (1;) but, our result was 2. H.~NCOCK, R. I,., DELIS, R. F., SH.\V, M., only obtained when a very high salt conAND WILLIAMS-Asri~.\N, H. G., Riochenl. centrabion was added, suggesting a mechaLi3ophy.s Acta 65, 257 (1962). I>ISCIJSSION
366
PEGG
AND
3. GORSKI, J., J. Biol. Chem. 239, 889 (1964). 4. WIDNELL, C. C. AXD TATA, J. R., Biochim. Biophys. Acta 87, 531 (1964). 5. PEGG, A. E., AND KORR'ER, A., A’ature 206, 904 (1965). 6. HUANG, R. C., AND BONNER, J., Proc. Xall. Acad Sci. U.S. 48, 1216 (1962). 7. BARR, G. C. AND BUTLER, J. A. V. LVature 199, 1170 (1963). 8. HINDLEY, J. B&hem Biophys. Res. Commun. 12, 175 (1963). 9. HINDLEY, J., 6th Intern. Congr. Biochem., New York, Abstr I-82 (1964). 10. AKINRIMISI, E. O., BONNER, J., AND Ts’o, P. O., J. MOL. Biol. 11, 128 (1965). 11. ALLFREY, V.G., LITTAN, V. C., AND MIRSKY,
KORNER
12. 13. 14.
15. 16. 17. 18.
A. E., Proc. Natl. Acad. Sci. U.S. 49, 414 i1963). BUIZTON, K., Biochem. J. 62, 315 (1965). MIRSKY, A. E. AND RIS, H. J. Genl. Physiol. 31, 1 (1947). CHAMBON, P., RAMUZ, M. AND DOLY, J., Niochem. Biophys. Res. Commun. 21, 156 (1965). WILLIAMS-ASHMAX, H. G., J. Cell Camp. Physiol. 66 Suppl. 1, 111 (1965). WIDNELL, C. C., Biochem. J. 96, 42P (1965). PEGG, A. E. .~ND KORNER, A. (unpublished observations). BONNER, J. AND HUANG, R. C., Biochem. Biophys. Res Commun. 22, 211 (1966).