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RNA polymerase from Bacillus amyloliquefaciens
280
BIOCHIMICAET BIOPHYSICAACTA
BBA 97336
RNA POLYMERASE FROM B A C I L L U S
AMYLOLIQUEFACIENS
J M HERMOSO, J AVILA, F JIMt~NEZ AND M S.~L~S Dcpa~tment of Molecula~ B~ology, Inst~t~ttr (; Mara~dn (C S i C ). VelSzq~tez I:44, Madrtd ~) (Received l~arch i6th, 1972)
SUMMARY Highly purified DNA-dependent RNA polymerase has been obtained from Bacdlus amylol,que/ac,ens cells. By electrophoresls in polyacrylamlde gels in the presence of sodium dodecyl sulfate three different bands, named ~'fl, a and ~ (molecular weight 15o ooo, 54 ooo and 42 ooo, respectively) were obtained. The molecular weight values of these subumts are very similar to those found for the Bacdlus subt~l~s RNA polymerase.
INTRODUCTION Recently the subunit composition of Bacillus subtdzs RNA polymerase has been described The enzyme consists of four different subumts, fl', t5, a and ~, with molecular weight values of 154 ooo (for fl' and/3), 56 ooo and 43 ooo, respectively x-4. Taking into account the unrelatedness of B subtll,s and Bac,llus amylol~que/ac,ens ~ and the fact that phage q~29 attacks both species of Bacillus 6, it was of Interest to study the subunlt composition of the RNA polymerase from B. amylol~que/aczcn~ in order to study further the modification that might take place m the RNA polymerase in both bacteria after q)29 infection. We report here the isolation of homogeneous B amvlol~que/ac~ens RNA polymerase and the study of its subunit composition which is similar to that of the B. subt~l~s enzyme MATERIALS AND METHODS E n z y m e purz]wahon and assay B amylol~que/ac,ens RNA polymerase was purified as already described for the purification of the B. subtil, s enzyme 1,2 The method consisted of breaking the cells with alumina, fractionation of the extracts in a two-phase polyethyleneglycoldextran sulfate system 7, precipitation with ammonium sulfate, DEAE-cellulose chromatography and ~b29 DNA-cellulose chromatography as described by Alberts et al s Protein was determined by the method of Lowry et al.9. The RNA polymerase activity was assayed as described previously using (/)29 DNA as template 1 Polyacrylannde gel electrophores~s 12 cm long gels containing 5 °'o (w/v) acrylamlde, o 15 % (w/v) N,N'-methyleneblsacrylamide and I °/o sodium dodecy] sulfate were prepared as described by B~och~m. B~ophys :tcta, 277 (1972) 2,So 283
B amylohque/aczens RNA POLYMERASE
281
Vlfiuela et al lO. The sample for electrophoresis was prepared as described 2. The gels were stained with a 0.25 o~ (w/v) solution of Coomassie brilliant blue in methanolacetic acid-water (5 : I : 5, v/v/v) for at least 6 h. The gels were destained by shaking in 7.5 o~ acetic acid Proteins of known molecular weight (fl-galactosidase, bovine serum albumin, ovalbumin and immunoglobulin G) were run in parallel gels to calculate the molecular weight values of the polypeptide chains of the RNA polymerase subumts as described by Shapiro et al. 11.
RESULTS AND DISCUSSION
Table I shows a summary of the results of the purification of B. amylohaue]ac~ens RNA polymerase. The final yield in the purification process is approximately 3 ° °/o and the specific activity is similar to that obtained for B. subtihs RNA polymerase 2. TABLE
I
SUMMARY OF
B amylolzq,tefac2ens RIN'A POLWMERASE PURIFICATION
T h e d a t a g i v e n r e f e r t o t h e p u r i f i c a t i o n o f 26 g o f B amylohquefac,ens, s t r a t a H Activity units are expressed as nmoles of UMP mcorporated m io man under the assay condltmns described prev i o u s l y I u s m g q929 D N A a s t e m p l a t e
Extract Ammomum sulfate DEAE-cellulose DNA-cellulose
Total prote,n (mg)
Total actzvzty (un*ts)
820 41 i9 I
189o 184o I I 18 587
o z 5 2
Spec~t~cactw,ty (unzts/mg) 2 3
44 5 57.3 49 °
Yzeld (%) ioo 97 62 31
The purified enzyme was sublected to gel electrophoresis in the presence of sodium dodecyl sulfate to study its subunit composition. As shown in Fig IA, there are three protein bands which we named fl'fl, a and a in order of increasing electrophoretic moblhty, which move to the same position as the bands obtained with the B. subtilis enzyme (Fig IB). From the densitometry tracings the enzyme is calculated to be more than 95 % pure The molecular weight values of the three bands, calculated according to the method of Shapiro et al. xl, were approx. 15o ooo, 54 ooo and 42 ooo, respectively (Fig. 2). These values (mean of eight determinations) closely agree with those obtained for the B. subtd~s enzyme 1-4. The molar ratio of the subunits, calculated from the densitometry tracings, was 2 2 : 0.5 : 2 for fl'fl, a and e, respectively, which agrees with the structure fl'flo~2 for the core enzyme. The fact that the molar ratio of the a subunit is less than one probably means that some enzyme molecules have lost their ~ subunlt and contain only the fl', fl and ~ subunits. The presented results indicate that the molecular weights of the subumts of B. suht,hs and B. amylolique/aciens RNA polymerases are similar and differ from the Escherichm col, enzyme in the molecular weight of the a subunit. Bzochzm. Bzophys. Acta, 2 7 7 (1972) 2 8 0 - 2 8 3
2~2
J .Xl HERMO.~() dt a/
p'p
/
r
b2o,I '
i
,
r
8 ....... A
B ~
l
02
04
06
08
i
R E L.ATIV~ MO~IL.[TY
Fig i Electrophoresls on p o l y a c r y l a l m d e gels of B amylohquefaczens and B. subtd~s R N A polymerases 12-cm gels containing 5 % acrylamxde and I °/o s o d m m dodecyl sulfate were p r e p a r e d and stained as already descmbed ~ P r o t e i n migration was t o w a r d s the anode, s h o w n at the b o t t o m Electrophoresls was carried o u t at a c o n s t a n t voltage of 2 5 V / c m for 16 h (A) A p p r o x 4 ° llg ot I3 amylohquefaczens R N A p o l y m e r a s e (B) A p p r o x 4 ° p g of B. subtd~s R N A polymerase Fag 2 Entimation of the molecular weights of the s u b u m t s of B amylohquefac~ens R N A polymerase Purified RNA p o l y m e r a s e w a s dissociated a n d subjected to electrophorems as described in Fig i Protem~ of k n o w n molecular w e i g h t (~-galactomdase, mol w t 13 o ooo la, bovine s e r u m albumin, mol wt 08 ooo **, o v a l b u m m , tool w t 43 oool4, l m m u n o g l o b u h n G, tool w t 5 ° ooo and 23 5oo for the h e a v y and light chains, respectively 15) were sublected to electrophoresm in a parallel gel and their m o b l h t l e s relative to t h e tracking dye determined The moblhtles of the s u b u m t s of R N A polymerase are m d m a t e d b y arrows
ACKNOWLEDGEMENTS Thls mvestlgation has been aided by a grant from the Jane Coffin Ch]Ids Memorial Fund for Medmal Research. J M. H. has been the recipient of a fellowshlp from Antlbi6t]cos, S. A. J.A. Is a fellow of Fondo Nacxonal para la Formac16n de Personal Investigador. We are grateful to Dr E. Vifiuela for stlmulatmg discussions and to Mr ]esfis Lopez for the growth of the cells. Bwchtm B~ophys Acta, 277 (I972) 28o 283
B. amylolique[ac,ens RNA POLYMERASE
283
REFERENCES 1 2 3 4 5 6 7 8
J. Avfla, J M Hermoso, E Vlfiuela and M Salas, Nature, 226 (197o) 1244 J Avda, J M Hermoso, E. Vlfluela and M Salas, E u r J B~ochem , 21 (1971 ) 526 R Loslck, R G Shorensteln and A L Sonenshem, Nature, 227 (197 o) 91o. J C. C 1V~ala, P Kerlan and J Szulmajster, F E B S Left, 13 (1971 ) 269. VV E Welker and L L Campbell, J. Bacter~ol., 94 (1967) 1124 B E Redly and J. Splzlzen, J Bacterzol, 89 (1965) 782. C Babmet, Btochem B~ophys Res C o m m u n , 26 (1967) 639. 13 M Alberts, F J. Amodlo, M Jenkins, E D G u t m a n and F L Ferrls, Cold Sprzng Harbor Syrup Quant I?~ol., 33 (1968) 2899 0 H Lowry, N J Rosebrough, A L Farr and R J Randall, J Btol Chem , 193 (1951) 265 io E Vlfiuela, I D Algranatt and S. Ochoa, Eur J Bzochem , i (1967) 3i i A L Shapiro, E Vifiuela and J. v 1Vfalzel, Jr, Bzochem Brophys Res Commun , 28 (i967) 815 12 A Ullmann, M E Goldberg, D P e r r m and J Monod, Blochemzstry, 7 (1968) 261. 13 C Tanford, K K a w a h a r a and S Lapanle, J .4m Chem Soc, 89 (1967) 72914 F J Castelhno and R Barker, Bzochem~stry, 7 (1968) 2207 15 U Rutmhauser, 13 A C u m m m g h a m , C /3ennett, W H Konlgsberg and G M Edelman, Proc Nail Acad Scz U S , 61 (1968) 1414 B~och,m B*ophys Acta, 277 (1972) 280 283
BIOCHIMICAET BIOPHYSICAACTA
BBA 97336
RNA POLYMERASE FROM B A C I L L U S
AMYLOLIQUEFACIENS
J M HERMOSO, J AVILA, F JIMt~NEZ AND M S.~L~S Dcpa~tment of Molecula~ B~ology, Inst~t~ttr (; Mara~dn (C S i C ). VelSzq~tez I:44, Madrtd ~) (Received l~arch i6th, 1972)
SUMMARY Highly purified DNA-dependent RNA polymerase has been obtained from Bacdlus amylol,que/ac,ens cells. By electrophoresls in polyacrylamlde gels in the presence of sodium dodecyl sulfate three different bands, named ~'fl, a and ~ (molecular weight 15o ooo, 54 ooo and 42 ooo, respectively) were obtained. The molecular weight values of these subumts are very similar to those found for the Bacdlus subt~l~s RNA polymerase.
INTRODUCTION Recently the subunit composition of Bacillus subtdzs RNA polymerase has been described The enzyme consists of four different subumts, fl', t5, a and ~, with molecular weight values of 154 ooo (for fl' and/3), 56 ooo and 43 ooo, respectively x-4. Taking into account the unrelatedness of B subtll,s and Bac,llus amylol~que/ac,ens ~ and the fact that phage q~29 attacks both species of Bacillus 6, it was of Interest to study the subunlt composition of the RNA polymerase from B. amylol~que/aczcn~ in order to study further the modification that might take place m the RNA polymerase in both bacteria after q)29 infection. We report here the isolation of homogeneous B amvlol~que/ac~ens RNA polymerase and the study of its subunit composition which is similar to that of the B. subt~l~s enzyme MATERIALS AND METHODS E n z y m e purz]wahon and assay B amylol~que/ac,ens RNA polymerase was purified as already described for the purification of the B. subtil, s enzyme 1,2 The method consisted of breaking the cells with alumina, fractionation of the extracts in a two-phase polyethyleneglycoldextran sulfate system 7, precipitation with ammonium sulfate, DEAE-cellulose chromatography and ~b29 DNA-cellulose chromatography as described by Alberts et al s Protein was determined by the method of Lowry et al.9. The RNA polymerase activity was assayed as described previously using (/)29 DNA as template 1 Polyacrylannde gel electrophores~s 12 cm long gels containing 5 °'o (w/v) acrylamlde, o 15 % (w/v) N,N'-methyleneblsacrylamide and I °/o sodium dodecy] sulfate were prepared as described by B~och~m. B~ophys :tcta, 277 (1972) 2,So 283
B amylohque/aczens RNA POLYMERASE
281
Vlfiuela et al lO. The sample for electrophoresis was prepared as described 2. The gels were stained with a 0.25 o~ (w/v) solution of Coomassie brilliant blue in methanolacetic acid-water (5 : I : 5, v/v/v) for at least 6 h. The gels were destained by shaking in 7.5 o~ acetic acid Proteins of known molecular weight (fl-galactosidase, bovine serum albumin, ovalbumin and immunoglobulin G) were run in parallel gels to calculate the molecular weight values of the polypeptide chains of the RNA polymerase subumts as described by Shapiro et al. 11.
RESULTS AND DISCUSSION
Table I shows a summary of the results of the purification of B. amylohaue]ac~ens RNA polymerase. The final yield in the purification process is approximately 3 ° °/o and the specific activity is similar to that obtained for B. subtihs RNA polymerase 2. TABLE
I
SUMMARY OF
B amylolzq,tefac2ens RIN'A POLWMERASE PURIFICATION
T h e d a t a g i v e n r e f e r t o t h e p u r i f i c a t i o n o f 26 g o f B amylohquefac,ens, s t r a t a H Activity units are expressed as nmoles of UMP mcorporated m io man under the assay condltmns described prev i o u s l y I u s m g q929 D N A a s t e m p l a t e
Extract Ammomum sulfate DEAE-cellulose DNA-cellulose
Total prote,n (mg)
Total actzvzty (un*ts)
820 41 i9 I
189o 184o I I 18 587
o z 5 2
Spec~t~cactw,ty (unzts/mg) 2 3
44 5 57.3 49 °
Yzeld (%) ioo 97 62 31
The purified enzyme was sublected to gel electrophoresis in the presence of sodium dodecyl sulfate to study its subunit composition. As shown in Fig IA, there are three protein bands which we named fl'fl, a and a in order of increasing electrophoretic moblhty, which move to the same position as the bands obtained with the B. subtilis enzyme (Fig IB). From the densitometry tracings the enzyme is calculated to be more than 95 % pure The molecular weight values of the three bands, calculated according to the method of Shapiro et al. xl, were approx. 15o ooo, 54 ooo and 42 ooo, respectively (Fig. 2). These values (mean of eight determinations) closely agree with those obtained for the B. subtd~s enzyme 1-4. The molar ratio of the subunits, calculated from the densitometry tracings, was 2 2 : 0.5 : 2 for fl'fl, a and e, respectively, which agrees with the structure fl'flo~2 for the core enzyme. The fact that the molar ratio of the a subunit is less than one probably means that some enzyme molecules have lost their ~ subunlt and contain only the fl', fl and ~ subunits. The presented results indicate that the molecular weights of the subumts of B. suht,hs and B. amylolique/aciens RNA polymerases are similar and differ from the Escherichm col, enzyme in the molecular weight of the a subunit. Bzochzm. Bzophys. Acta, 2 7 7 (1972) 2 8 0 - 2 8 3
2~2
J .Xl HERMO.~() dt a/
p'p
/
r
b2o,I '
i
,
r
8 ....... A
B ~
l
02
04
06
08
i
R E L.ATIV~ MO~IL.[TY
Fig i Electrophoresls on p o l y a c r y l a l m d e gels of B amylohquefaczens and B. subtd~s R N A polymerases 12-cm gels containing 5 % acrylamxde and I °/o s o d m m dodecyl sulfate were p r e p a r e d and stained as already descmbed ~ P r o t e i n migration was t o w a r d s the anode, s h o w n at the b o t t o m Electrophoresls was carried o u t at a c o n s t a n t voltage of 2 5 V / c m for 16 h (A) A p p r o x 4 ° llg ot I3 amylohquefaczens R N A p o l y m e r a s e (B) A p p r o x 4 ° p g of B. subtd~s R N A polymerase Fag 2 Entimation of the molecular weights of the s u b u m t s of B amylohquefac~ens R N A polymerase Purified RNA p o l y m e r a s e w a s dissociated a n d subjected to electrophorems as described in Fig i Protem~ of k n o w n molecular w e i g h t (~-galactomdase, mol w t 13 o ooo la, bovine s e r u m albumin, mol wt 08 ooo **, o v a l b u m m , tool w t 43 oool4, l m m u n o g l o b u h n G, tool w t 5 ° ooo and 23 5oo for the h e a v y and light chains, respectively 15) were sublected to electrophoresm in a parallel gel and their m o b l h t l e s relative to t h e tracking dye determined The moblhtles of the s u b u m t s of R N A polymerase are m d m a t e d b y arrows
ACKNOWLEDGEMENTS Thls mvestlgation has been aided by a grant from the Jane Coffin Ch]Ids Memorial Fund for Medmal Research. J M. H. has been the recipient of a fellowshlp from Antlbi6t]cos, S. A. J.A. Is a fellow of Fondo Nacxonal para la Formac16n de Personal Investigador. We are grateful to Dr E. Vifiuela for stlmulatmg discussions and to Mr ]esfis Lopez for the growth of the cells. Bwchtm B~ophys Acta, 277 (I972) 28o 283
B. amylolique[ac,ens RNA POLYMERASE
283
REFERENCES 1 2 3 4 5 6 7 8
J. Avfla, J M Hermoso, E Vlfiuela and M Salas, Nature, 226 (197o) 1244 J Avda, J M Hermoso, E. Vlfluela and M Salas, E u r J B~ochem , 21 (1971 ) 526 R Loslck, R G Shorensteln and A L Sonenshem, Nature, 227 (197 o) 91o. J C. C 1V~ala, P Kerlan and J Szulmajster, F E B S Left, 13 (1971 ) 269. VV E Welker and L L Campbell, J. Bacter~ol., 94 (1967) 1124 B E Redly and J. Splzlzen, J Bacterzol, 89 (1965) 782. C Babmet, Btochem B~ophys Res C o m m u n , 26 (1967) 639. 13 M Alberts, F J. Amodlo, M Jenkins, E D G u t m a n and F L Ferrls, Cold Sprzng Harbor Syrup Quant I?~ol., 33 (1968) 2899 0 H Lowry, N J Rosebrough, A L Farr and R J Randall, J Btol Chem , 193 (1951) 265 io E Vlfiuela, I D Algranatt and S. Ochoa, Eur J Bzochem , i (1967) 3i i A L Shapiro, E Vifiuela and J. v 1Vfalzel, Jr, Bzochem Brophys Res Commun , 28 (i967) 815 12 A Ullmann, M E Goldberg, D P e r r m and J Monod, Blochemzstry, 7 (1968) 261. 13 C Tanford, K K a w a h a r a and S Lapanle, J .4m Chem Soc, 89 (1967) 72914 F J Castelhno and R Barker, Bzochem~stry, 7 (1968) 2207 15 U Rutmhauser, 13 A C u m m m g h a m , C /3ennett, W H Konlgsberg and G M Edelman, Proc Nail Acad Scz U S , 61 (1968) 1414 B~och,m B*ophys Acta, 277 (1972) 280 283