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Translation of mitochondrial RNA from yeast cytoplasmic petite mutants in anE. coli cell-free system
Vol. 64, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
TRANSLATION OF MITOCHONDRIAL RNA FROM YEAST CYTOPLASMIC PETITE MUTANTS IN AN E. COLI CELL-FREE SYSTEM Avraham Halbreich*
, Ann Di Franco A , Olga Groudinsky, Jacky Cosson and Piotr P. Slonimski
Centre de G~n~tique Mol~culaire du CNRS 91190 Gif sur Yvette, France
R e c e i v e d A p r i l 25,1975 Summary : Mitochondrial RNA from two cytoplasmic p- mutants of S. c e r e v i s i a e , ~ a v e kept the mitochondrial DNA segment including the ATPase-oligomycin resistance-conferring gene, stimulates protein synthesis in an ~ . e o l i c e l l - f r e e system. SDS-acrylamide gel electrophoresis of the protein product revealed one major peak and two minor ones with apparent molecular weights of around 11,000, 13,500 and 17,000 respectively. The e f f e c t is s p e c i f i c since no stimulation is observed with RNA from a p- mutant devoid of detectable mitochondrial DNA. These results are interpreted to mean that the mitochondrial DNA of these mutants codes for an i n v i t r o t r a n s l a t a b l e mRNA. Several
p- p e t i t e mutants have now been obtained in which large
portions of the mitochondrial DNA had been deleted while the retained segments have been amplified. Genetic information was demonstrated in these "low complexity" DNA mutants, by mating with suitable t e s t e r s t r a i n s , as well as the existence of RNA t r a n s c r i p t s of t h e i r mitochondrial genome ( I ) . these mutants, l i k e a l l other synthetic a c t i v i t y transcripts
However,
p- mutants, are devoid of mitochondrial protein
(2) and i t could not, t h e r e f o r e , be tested whether the RNA
were a c t u a l l y t r a n s l a t a b l e .
The E. c o l i c e l l - f r e e system has been used in recent years for the in vitro
t r a n s l a t i o n of messages from viruses (3, 4, 5), from chloroplasts (6)
and from mitochondria of N. orassa (7). I t was, therefore, decided to adopt this approach for a biochemical characterizai~on of proteins coded by the mitochondria genome. The a v a i l a b i l i t y the
feasibility
of g e n e t i c a l l y well characterized
p mutants enhanced
of these experiments in view of the lower complexity of the
mitRNA populations involved. In a d d i t i o n , i t was deemed possible that the amplif i c a t i o n of the r e l a t i v e l y short mitDNA segments would r e s u l t in an increased concentration of putative mRNA. We chose
p mutants that have retained the gene
conferring oligomycin resistance on mitochondrial ATPase with in view a possi-
,
On leave from the Department of C e l l u l a r Biochemistry, Faculty of Medicine, Hebrew University, Jerusalem. A To whom the correspondance should be sent.
Copyright © 1975 by Academie Press, Inc. All rights of reproduction in any form reserved.
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bility
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
to correlate the i n v i t r o synthesized products with components of the
enzyme complex. Materials and Methods, IL8-8C/F12 ( a, his I , t r P l ) deleted f o r the mitochondrial loci RIB1, RIB2, OLI2, PARI and carrying in the retained segment the mitochondrial a l l e l e s E514 at the locus RIB3 and the a l l e l e 0
at the locus OLI1 ( i ) ;
KLI4-4A/C211
(a, his, trP2 ) deleted f o r the loci RIB1, RIB2, RIB3, OLI2, PARI and carrying the a l l e l e 01 at the OLII locus (B. Dujon, personal comm.); IL8-8C/H71 deleted for a l l known mitochondrial loci and devoid of detectable mit-DNA (8). For convenience throughout the t e x t , only the numbers a f t e r the bar w i l l be used to designate the d i f f e r e n t s t r a i n s . Yeast c u l t u r e s , t h e i r conversion i n t o spheroplasts, i s o l a t i o n of mitochondria and mitRNA were done as described previously (8, 9). Cytoplasmic RNA from H71 was isolated from the postmitochondrial supernatant by the same procedure. The RNA was then f u r t h e r p u r i f i e d by passage through a hydroxylap a t i t e column. RNA containing f r a c t i o n s , eluted at 0.2 M Ha-phosphate pH 6.8, were pooled, dialyzed extensively against H20, precipitated with 2 volumes of ethanol, 0 . I volume of 20% Na-acetate and kept overnight at -20°C. The p r e c i p i t a t e was collected by c e n t r i f u g a t i o n (30,000 x g f o r 10 min) then dissolved in H20 and dialyzed against H20. Amino acid incorporation i n t o protein in an E. c o l i
c e l l - f r e e system
was tested according to Gold and Schweiger (i0) in i00 ~I reaction mixtures containing 0.I mc/ml 3H-valine. Unless otherwise mentioned, Mg acetate concent r a t i o n s was 12.5 mM. Reaction mixtures were incubated for I hour at 37 ° and protein synthesis was measured ( I I ) . Experimental points were run in duplicate and usually agreed to w i t h i n less than 10%. 15% acrylamide (0.38% bis) gels in 0 . I M Tris pH 8.0 containing 1% SDS (sodium dodecylsu~at~ were polymerized in glass tubes e s s e n t i a l l y according to Laemmli (12). Samples (40 vl of c e l l - f r e e reaction mixture) were treated for electrophoresis according to Model and Zinder (5) bromophenol blue were added before layering on the 8 mA/gel with an electrode buffer containing 0.1M and 0.05 M B-mercaptoethanol. Gels were treated f o r Michel and Neupert (13) except that Bray's s o l u t i o n
and 10% sucrose and
gels. Electrophoresis was at Tris-HCI pH 8.0, 1% SDS counting as described by (14) was employed,
Results. As may be seen in Fig. l a , addition of RNA, extracted from F12 mitomitochondria to the c e l l - f r e e system from E. c o l i resulted in a s i g n i f i c a n t enhancement of 3H-valine incorporation into protein up to a 2-3 fold increase at saturating levels (cf. table I ) . The regular phenol extraction of mitochondrial RNA yielded a product
which i n h i b i t e d the endogenous a c t i v i t y of the
1287
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BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
4
4
$ o _c
o:
2
o L_ o c
I
2
o'5
4
[p-F12 mit-RNA] (mg/ml)
11 ib ~'~ 11 i~ I~ 4s [Mg ++] (mM)
' 2b '
Zo
6'o
Incubahon time (minutes)
Figure 1 : F12 mitRNA stimulated incorporation of 3H-valine into proteins. (a) effect of F12 mitRNA concentration : Mg++, 12,5 mM (b) effect of Mg++ concentration : F12 mitRNA, 0.96 mg/ml ( f u l l circles) (c) time course: Mg++, 12,5 mM; F12 mitRNA, 0.96 mg/ml ( f u l l circles) In b and c the open circles represent the endogenous incorporation.
c e l l - f r e e system. This i n h i b i t o r y a c t i v i t y was removed upon further p u r i f i cation of the RNA by hydroxylapatite chromatography
(see Materials & Methods).
Mitochondrial RNA-directed protein syntheses was optimal at 12.5 - 13~5 mM Mg++ (Fig. lb) and proceeded for about 45 min. (Fig, lc) The c e l l - f r e e system was preincubated prior to the addition of mitRNA,
E. aoli i n i t i a t i o n factors and 3H-valine (table 1). This treatment
diminished considerably the endogenous a c t i v i t y and resulted in a 5-7 fold enhancement of protein synthesis by adding FI2 mitRNA. MitRNA was also extracted from another petite strain-C211, which contains a genetic marker for o l i gomycin resistance. Addition of this RNA to the c e l l - f r e e system resulted in a 50-100% increase of the amino acid incorporation into protein (table I) at a saturating level of 0.2 mg/ml (Fig.2) and an optimal Mg++concentrationof 12.513.5 mM similar to the F12 directed incorporation (Fig. Ib). Preparations of mitRNA were analyzed by gel electrophoresis and shown to contain varying amounts of cytoplasmic RNA (results not shown). Cytoplasmic RNA was, therefore, extracted from a mitochondrial DNA-deficient petite mutant H71 and added to the c e l l - f r e e system, This RNA inhibited the endogenous a c t i v i t y to a varying degree (Fig. 2 and table 1),
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 : The effect of preincubation on the enhancement of valine incorporation into proteins by petite mitochondrial RNA,
~H-valine incorporation into protein (cpm/lO ul reaction mixture) r'
without preincubation RNA added, mg/ml None FI2 mitRNA, 0.96
Exp.l
Exp.1
Exp. 2
5,095
1,055
1,274
16,010
5,162
8,844
1,776
2,559
844
257
C211 mitRNA, 0.30 H71 cytRNA, 1.0
with preincubation
-
ribosomes and supernatant fraction were preincub~ted for 10 min. at 37° in 60 ~l of the normal reaction mixture but without ~H-valine. The tubes were then cooled and the following additions were made : 375 ~g/ml E, eoli i n i t i a t i o n factors (15), 10 ~c °H-valine, Mg-acetate to maintain a constant concentration and, where indicated, the corresponding RNA. At the end of a 60 min incubation at 37° , 10 vl aliquot of each reaction mixture was used for the determination of hot TCA precipitable incorporation as described in Materials and Methods. E. c o l i
Aliquots from reaction mixtures, preincubated and non-preincubated, were analyzed by SDS-acrylamide gel electrophoresis. I t may be seen in Figures 3a and 3d that the endogenous protein synthesis resulted in an essentially single peak with an apparent molecular weight of 9,500 - 10,500. Protein synthesized in the presence of F12 mitRNA exhibited a peak with a maximum at molecular weight of 8,500 and a minor peak at 15,000 molecular weight (Fig. 3b). A plot of the
difference spectrum revealed a major peak of an apparent mole-
cular weight of 7~00 and~ominor ones of 12,500 and 15,000 (Fig.3c). In,the preincubated system, protein synthesized in the presence of FI2 mitRNA (Fig.3e) yielded a major peak at approximately 11,000 molecular weight and a minor one at 16,000. The difference spectrum revealed 3 peaks with apparent molecular weights of 11,500,
14,000 and 17,000 (Fig. 3f). The results are s t i l l
too
scarce to interprete this s h i f t to higher molecular weights. A similar s h i f t was, however, observed under different experimental conditions as well. Finally, i t was calculated from the difference spectra (Figures 3c
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~2 &
o
8
._c
0
100
200 300 ( ~g/ml)
400
[RNA]
Figure 2 : The effect of the concentration of C211 mitRNA (full circles) and H71 RNA (open circles) on 3H. valine incorporation into proteins.
and 3f)
that the incorporation of 340-400 pmoles 3H-valines was induced by
0.38 ~g of F12 mitRNA in the preincubated system and 450 pmoles in the nonpreincubated one. Discussion, The results presented show that RNA from petite mitochondria which contain the oligomycin marker, induces the synthes~s of d i s t i n c t protein species in
E. aoli
cell-free system, The apparent molecular weights of these
species did not change considerably under different experimental conditions (unlike the yield which did change) thus giving a specific aspect to the trans. lation process. This is in agreement with the results of Model and Zinder on the typical translation of viral messages under these conditions (5). Preparations of mitRNA, on the scale employed in this work, are contaminated to a varying degree by cytoplasmic 18 S and 28 S RNA species (8). This was also seen in gel electrophoreses of the present preparations (results not shown). Addition of these cytoplasmic RNA species
to the in vitro system
not only did not stimulate protein synthetic a c t i v i t y , but rather had an inhibitory effect of a varying degree. A mitDNA-less petite strain was chosen for this control in order to avoid a possible contamination of the preparation with messages transcribed on mitDNA. F12 mitochondria do not contain the 16 S RNA (8) and the fact that 16 S RNA was never seen in these RNA preparations argues against the possibil i t y that a bacterial contamination was responsible for the enhancement of protein synthesis.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
d
I
11
m
~I
I$
0 i
i
i
i
i
i
x e
&;
$
&
-tf u 0 0
0
0 L
~o
~'6'o ~
20
40
60
80
number oF gel slice Figure 3 : SDS-acrylamide qel electrophoresis : effect of preincubation on the products syn%hesized in an E. eo~i c e l l - f r e e system stimulated by FI2 mitRNA. Without preincubation (a) endogenous, (b) plus F12 mitRNA, (c) difference calculated by substracting a from b. With preincubation (d) endogenous, (e) plus FI2 mitRNA, (f) difference calculated by substracting d from e. Conditions as described in Table i . The arrows indicate the position of markers that were electrophoresed in p a r a l l e l ; Roman numeral I, l a c t i c dehydrogenase (MW 34,000), I I , bovine B lactoglobulin (MW 18,400), I I I , bovine heart cytochrome c (MW 11,700), IV, pancreatic trypsin i n h i b i t o r (MW 6,500) , V, CIBA's synthetic ACTH (MW 2983) and VI, bromophenol blue.
I t may thus be concluded that mitochondria of petite mutants, which have retained an oligomycin genetic marker in t h e i r mitDNA, also contain mRNA activity.
This mRNA may be either a t r a n s c r i p t of mitDNA or a species speci-
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fically
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
imported from the nucleus into the organelle. We favor the f i r s t
hypo-
thesis since the mitochondrial RNA's from these strains, extracted by the methods employed in this work, do contain a t r a n s c r i p t of the mitDNA corresponding to t h e i r common genetic marker (G. Faye, unpublished r e s u l t s ) . Work now in progress is aimed at the characterization of the RNA species which are translated and of the products obtained. Petite mutants do not perform mitochondrial protein systhesis. Therefore, the approach
chosen in this work
may allow the characterization of translatable messages in other petite strains which have retained d i f f e r e n t segments of the mitochondrial genome. Acknowledgements. The authors would l i k e to thank M. Grunbe~-Manago for her constant interest in this work. They are also thankful to G. Faye for his advice during the mitRNA preparations , D. Hayes for his g i f t of phage T4 RNA which was used to test the E. e o l l c e l l - f r e e system and J. Dondon for his g i f t of E. e o l i
ini-
t i a t i o n factors. One of us (A. Halbreich) was supported by a short-term fellowship from EMBO. References. 1.
2. 3. 4.
5. 6. 7. 8. 9. i0. II. 12. 13. 14. 15.
Faye,'G., Fukuhara, H., Grandchamp, C~, Lazowska, J , , Michel, F.,Casey, J., Getz, G.S., Locker, J., Rabinowitz, M,, Bolotin-Fukuhara, M., Coen, D., Deutsch, J., Dujon, B., Netter, P,, & Slonimski, P,P, (1973) Biochimie, 55, 779-792. Schatz, G. and Saltzgaber, J, (1969) Biochem. Biophys. Res. Commun. 37, 996-1001. Bryan, R.N.,Sugjur~ M., and Hayashi, M. (1969) Proc. Natl. Acad. Sci. USA, 62, 483-489. Gold, L.M., and Schweiger, M, (1969) Ibid, 62, 892-898. Model, P. and Zinder, N.D. (1974) J. Mol. B ~ I . 83,231-251. Hartley, M.R., Wheeler, A. and E l l i s , R.J. (1975)--J. M o l . B i o l . 91, 67-77. Kuntzel, H. and Blossey, H.C., (1974) Eur. J. Biocnem. 47, 165-I/1. Faye, G. , Kujawa, C.and Fukuhara, H. (1974) J. Mol. B i ~ . 88, 185-203. Petzuch, M. (1971) C.R. Acad. Sci. Paris, 273, 105-108. Gold, L.M. and Schweiger, M. (1971) Methods in Enzymology. Eds Moldave K. and Grossman L. Vol. XX pp. 537-542, Academic Press, New-York. Mans, R.J. and Novelli, G.D. (1961) Arch. Biochem. 9_44, 48-53. Laemmli, U.K., (1970) Nature, 227, 680-685. Michel, R. and Neupert, W. (1973--~- Eur. J. Biochem. 3__66,53-67. Bray, G. (1960) Anal. Biochem. i , 249. Dondon, J., Godefroy-Colburn, T~, Graffe, M. and Grunberg-Manago, M. (1974) FEBS Letters, 45, 82-87.
1292
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
TRANSLATION OF MITOCHONDRIAL RNA FROM YEAST CYTOPLASMIC PETITE MUTANTS IN AN E. COLI CELL-FREE SYSTEM Avraham Halbreich*
, Ann Di Franco A , Olga Groudinsky, Jacky Cosson and Piotr P. Slonimski
Centre de G~n~tique Mol~culaire du CNRS 91190 Gif sur Yvette, France
R e c e i v e d A p r i l 25,1975 Summary : Mitochondrial RNA from two cytoplasmic p- mutants of S. c e r e v i s i a e , ~ a v e kept the mitochondrial DNA segment including the ATPase-oligomycin resistance-conferring gene, stimulates protein synthesis in an ~ . e o l i c e l l - f r e e system. SDS-acrylamide gel electrophoresis of the protein product revealed one major peak and two minor ones with apparent molecular weights of around 11,000, 13,500 and 17,000 respectively. The e f f e c t is s p e c i f i c since no stimulation is observed with RNA from a p- mutant devoid of detectable mitochondrial DNA. These results are interpreted to mean that the mitochondrial DNA of these mutants codes for an i n v i t r o t r a n s l a t a b l e mRNA. Several
p- p e t i t e mutants have now been obtained in which large
portions of the mitochondrial DNA had been deleted while the retained segments have been amplified. Genetic information was demonstrated in these "low complexity" DNA mutants, by mating with suitable t e s t e r s t r a i n s , as well as the existence of RNA t r a n s c r i p t s of t h e i r mitochondrial genome ( I ) . these mutants, l i k e a l l other synthetic a c t i v i t y transcripts
However,
p- mutants, are devoid of mitochondrial protein
(2) and i t could not, t h e r e f o r e , be tested whether the RNA
were a c t u a l l y t r a n s l a t a b l e .
The E. c o l i c e l l - f r e e system has been used in recent years for the in vitro
t r a n s l a t i o n of messages from viruses (3, 4, 5), from chloroplasts (6)
and from mitochondria of N. orassa (7). I t was, therefore, decided to adopt this approach for a biochemical characterizai~on of proteins coded by the mitochondria genome. The a v a i l a b i l i t y the
feasibility
of g e n e t i c a l l y well characterized
p mutants enhanced
of these experiments in view of the lower complexity of the
mitRNA populations involved. In a d d i t i o n , i t was deemed possible that the amplif i c a t i o n of the r e l a t i v e l y short mitDNA segments would r e s u l t in an increased concentration of putative mRNA. We chose
p mutants that have retained the gene
conferring oligomycin resistance on mitochondrial ATPase with in view a possi-
,
On leave from the Department of C e l l u l a r Biochemistry, Faculty of Medicine, Hebrew University, Jerusalem. A To whom the correspondance should be sent.
Copyright © 1975 by Academie Press, Inc. All rights of reproduction in any form reserved.
1286
Vol. 64, No. 4, 1 9 7 . 5
bility
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
to correlate the i n v i t r o synthesized products with components of the
enzyme complex. Materials and Methods, IL8-8C/F12 ( a, his I , t r P l ) deleted f o r the mitochondrial loci RIB1, RIB2, OLI2, PARI and carrying in the retained segment the mitochondrial a l l e l e s E514 at the locus RIB3 and the a l l e l e 0
at the locus OLI1 ( i ) ;
KLI4-4A/C211
(a, his, trP2 ) deleted f o r the loci RIB1, RIB2, RIB3, OLI2, PARI and carrying the a l l e l e 01 at the OLII locus (B. Dujon, personal comm.); IL8-8C/H71 deleted for a l l known mitochondrial loci and devoid of detectable mit-DNA (8). For convenience throughout the t e x t , only the numbers a f t e r the bar w i l l be used to designate the d i f f e r e n t s t r a i n s . Yeast c u l t u r e s , t h e i r conversion i n t o spheroplasts, i s o l a t i o n of mitochondria and mitRNA were done as described previously (8, 9). Cytoplasmic RNA from H71 was isolated from the postmitochondrial supernatant by the same procedure. The RNA was then f u r t h e r p u r i f i e d by passage through a hydroxylap a t i t e column. RNA containing f r a c t i o n s , eluted at 0.2 M Ha-phosphate pH 6.8, were pooled, dialyzed extensively against H20, precipitated with 2 volumes of ethanol, 0 . I volume of 20% Na-acetate and kept overnight at -20°C. The p r e c i p i t a t e was collected by c e n t r i f u g a t i o n (30,000 x g f o r 10 min) then dissolved in H20 and dialyzed against H20. Amino acid incorporation i n t o protein in an E. c o l i
c e l l - f r e e system
was tested according to Gold and Schweiger (i0) in i00 ~I reaction mixtures containing 0.I mc/ml 3H-valine. Unless otherwise mentioned, Mg acetate concent r a t i o n s was 12.5 mM. Reaction mixtures were incubated for I hour at 37 ° and protein synthesis was measured ( I I ) . Experimental points were run in duplicate and usually agreed to w i t h i n less than 10%. 15% acrylamide (0.38% bis) gels in 0 . I M Tris pH 8.0 containing 1% SDS (sodium dodecylsu~at~ were polymerized in glass tubes e s s e n t i a l l y according to Laemmli (12). Samples (40 vl of c e l l - f r e e reaction mixture) were treated for electrophoresis according to Model and Zinder (5) bromophenol blue were added before layering on the 8 mA/gel with an electrode buffer containing 0.1M and 0.05 M B-mercaptoethanol. Gels were treated f o r Michel and Neupert (13) except that Bray's s o l u t i o n
and 10% sucrose and
gels. Electrophoresis was at Tris-HCI pH 8.0, 1% SDS counting as described by (14) was employed,
Results. As may be seen in Fig. l a , addition of RNA, extracted from F12 mitomitochondria to the c e l l - f r e e system from E. c o l i resulted in a s i g n i f i c a n t enhancement of 3H-valine incorporation into protein up to a 2-3 fold increase at saturating levels (cf. table I ) . The regular phenol extraction of mitochondrial RNA yielded a product
which i n h i b i t e d the endogenous a c t i v i t y of the
1287
Vol. 64, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
4
4
$ o _c
o:
2
o L_ o c
I
2
o'5
4
[p-F12 mit-RNA] (mg/ml)
11 ib ~'~ 11 i~ I~ 4s [Mg ++] (mM)
' 2b '
Zo
6'o
Incubahon time (minutes)
Figure 1 : F12 mitRNA stimulated incorporation of 3H-valine into proteins. (a) effect of F12 mitRNA concentration : Mg++, 12,5 mM (b) effect of Mg++ concentration : F12 mitRNA, 0.96 mg/ml ( f u l l circles) (c) time course: Mg++, 12,5 mM; F12 mitRNA, 0.96 mg/ml ( f u l l circles) In b and c the open circles represent the endogenous incorporation.
c e l l - f r e e system. This i n h i b i t o r y a c t i v i t y was removed upon further p u r i f i cation of the RNA by hydroxylapatite chromatography
(see Materials & Methods).
Mitochondrial RNA-directed protein syntheses was optimal at 12.5 - 13~5 mM Mg++ (Fig. lb) and proceeded for about 45 min. (Fig, lc) The c e l l - f r e e system was preincubated prior to the addition of mitRNA,
E. aoli i n i t i a t i o n factors and 3H-valine (table 1). This treatment
diminished considerably the endogenous a c t i v i t y and resulted in a 5-7 fold enhancement of protein synthesis by adding FI2 mitRNA. MitRNA was also extracted from another petite strain-C211, which contains a genetic marker for o l i gomycin resistance. Addition of this RNA to the c e l l - f r e e system resulted in a 50-100% increase of the amino acid incorporation into protein (table I) at a saturating level of 0.2 mg/ml (Fig.2) and an optimal Mg++concentrationof 12.513.5 mM similar to the F12 directed incorporation (Fig. Ib). Preparations of mitRNA were analyzed by gel electrophoresis and shown to contain varying amounts of cytoplasmic RNA (results not shown). Cytoplasmic RNA was, therefore, extracted from a mitochondrial DNA-deficient petite mutant H71 and added to the c e l l - f r e e system, This RNA inhibited the endogenous a c t i v i t y to a varying degree (Fig. 2 and table 1),
1288
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 : The effect of preincubation on the enhancement of valine incorporation into proteins by petite mitochondrial RNA,
~H-valine incorporation into protein (cpm/lO ul reaction mixture) r'
without preincubation RNA added, mg/ml None FI2 mitRNA, 0.96
Exp.l
Exp.1
Exp. 2
5,095
1,055
1,274
16,010
5,162
8,844
1,776
2,559
844
257
C211 mitRNA, 0.30 H71 cytRNA, 1.0
with preincubation
-
ribosomes and supernatant fraction were preincub~ted for 10 min. at 37° in 60 ~l of the normal reaction mixture but without ~H-valine. The tubes were then cooled and the following additions were made : 375 ~g/ml E, eoli i n i t i a t i o n factors (15), 10 ~c °H-valine, Mg-acetate to maintain a constant concentration and, where indicated, the corresponding RNA. At the end of a 60 min incubation at 37° , 10 vl aliquot of each reaction mixture was used for the determination of hot TCA precipitable incorporation as described in Materials and Methods. E. c o l i
Aliquots from reaction mixtures, preincubated and non-preincubated, were analyzed by SDS-acrylamide gel electrophoresis. I t may be seen in Figures 3a and 3d that the endogenous protein synthesis resulted in an essentially single peak with an apparent molecular weight of 9,500 - 10,500. Protein synthesized in the presence of F12 mitRNA exhibited a peak with a maximum at molecular weight of 8,500 and a minor peak at 15,000 molecular weight (Fig. 3b). A plot of the
difference spectrum revealed a major peak of an apparent mole-
cular weight of 7~00 and~ominor ones of 12,500 and 15,000 (Fig.3c). In,the preincubated system, protein synthesized in the presence of FI2 mitRNA (Fig.3e) yielded a major peak at approximately 11,000 molecular weight and a minor one at 16,000. The difference spectrum revealed 3 peaks with apparent molecular weights of 11,500,
14,000 and 17,000 (Fig. 3f). The results are s t i l l
too
scarce to interprete this s h i f t to higher molecular weights. A similar s h i f t was, however, observed under different experimental conditions as well. Finally, i t was calculated from the difference spectra (Figures 3c
1289
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BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
~2 &
o
8
._c
0
100
200 300 ( ~g/ml)
400
[RNA]
Figure 2 : The effect of the concentration of C211 mitRNA (full circles) and H71 RNA (open circles) on 3H. valine incorporation into proteins.
and 3f)
that the incorporation of 340-400 pmoles 3H-valines was induced by
0.38 ~g of F12 mitRNA in the preincubated system and 450 pmoles in the nonpreincubated one. Discussion, The results presented show that RNA from petite mitochondria which contain the oligomycin marker, induces the synthes~s of d i s t i n c t protein species in
E. aoli
cell-free system, The apparent molecular weights of these
species did not change considerably under different experimental conditions (unlike the yield which did change) thus giving a specific aspect to the trans. lation process. This is in agreement with the results of Model and Zinder on the typical translation of viral messages under these conditions (5). Preparations of mitRNA, on the scale employed in this work, are contaminated to a varying degree by cytoplasmic 18 S and 28 S RNA species (8). This was also seen in gel electrophoreses of the present preparations (results not shown). Addition of these cytoplasmic RNA species
to the in vitro system
not only did not stimulate protein synthetic a c t i v i t y , but rather had an inhibitory effect of a varying degree. A mitDNA-less petite strain was chosen for this control in order to avoid a possible contamination of the preparation with messages transcribed on mitDNA. F12 mitochondria do not contain the 16 S RNA (8) and the fact that 16 S RNA was never seen in these RNA preparations argues against the possibil i t y that a bacterial contamination was responsible for the enhancement of protein synthesis.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
d
I
11
m
~I
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0 i
i
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i
i
i
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-tf u 0 0
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number oF gel slice Figure 3 : SDS-acrylamide qel electrophoresis : effect of preincubation on the products syn%hesized in an E. eo~i c e l l - f r e e system stimulated by FI2 mitRNA. Without preincubation (a) endogenous, (b) plus F12 mitRNA, (c) difference calculated by substracting a from b. With preincubation (d) endogenous, (e) plus FI2 mitRNA, (f) difference calculated by substracting d from e. Conditions as described in Table i . The arrows indicate the position of markers that were electrophoresed in p a r a l l e l ; Roman numeral I, l a c t i c dehydrogenase (MW 34,000), I I , bovine B lactoglobulin (MW 18,400), I I I , bovine heart cytochrome c (MW 11,700), IV, pancreatic trypsin i n h i b i t o r (MW 6,500) , V, CIBA's synthetic ACTH (MW 2983) and VI, bromophenol blue.
I t may thus be concluded that mitochondria of petite mutants, which have retained an oligomycin genetic marker in t h e i r mitDNA, also contain mRNA activity.
This mRNA may be either a t r a n s c r i p t of mitDNA or a species speci-
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fically
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
imported from the nucleus into the organelle. We favor the f i r s t
hypo-
thesis since the mitochondrial RNA's from these strains, extracted by the methods employed in this work, do contain a t r a n s c r i p t of the mitDNA corresponding to t h e i r common genetic marker (G. Faye, unpublished r e s u l t s ) . Work now in progress is aimed at the characterization of the RNA species which are translated and of the products obtained. Petite mutants do not perform mitochondrial protein systhesis. Therefore, the approach
chosen in this work
may allow the characterization of translatable messages in other petite strains which have retained d i f f e r e n t segments of the mitochondrial genome. Acknowledgements. The authors would l i k e to thank M. Grunbe~-Manago for her constant interest in this work. They are also thankful to G. Faye for his advice during the mitRNA preparations , D. Hayes for his g i f t of phage T4 RNA which was used to test the E. e o l l c e l l - f r e e system and J. Dondon for his g i f t of E. e o l i
ini-
t i a t i o n factors. One of us (A. Halbreich) was supported by a short-term fellowship from EMBO. References. 1.
2. 3. 4.
5. 6. 7. 8. 9. i0. II. 12. 13. 14. 15.
Faye,'G., Fukuhara, H., Grandchamp, C~, Lazowska, J , , Michel, F.,Casey, J., Getz, G.S., Locker, J., Rabinowitz, M,, Bolotin-Fukuhara, M., Coen, D., Deutsch, J., Dujon, B., Netter, P,, & Slonimski, P,P, (1973) Biochimie, 55, 779-792. Schatz, G. and Saltzgaber, J, (1969) Biochem. Biophys. Res. Commun. 37, 996-1001. Bryan, R.N.,Sugjur~ M., and Hayashi, M. (1969) Proc. Natl. Acad. Sci. USA, 62, 483-489. Gold, L.M., and Schweiger, M, (1969) Ibid, 62, 892-898. Model, P. and Zinder, N.D. (1974) J. Mol. B ~ I . 83,231-251. Hartley, M.R., Wheeler, A. and E l l i s , R.J. (1975)--J. M o l . B i o l . 91, 67-77. Kuntzel, H. and Blossey, H.C., (1974) Eur. J. Biocnem. 47, 165-I/1. Faye, G. , Kujawa, C.and Fukuhara, H. (1974) J. Mol. B i ~ . 88, 185-203. Petzuch, M. (1971) C.R. Acad. Sci. Paris, 273, 105-108. Gold, L.M. and Schweiger, M. (1971) Methods in Enzymology. Eds Moldave K. and Grossman L. Vol. XX pp. 537-542, Academic Press, New-York. Mans, R.J. and Novelli, G.D. (1961) Arch. Biochem. 9_44, 48-53. Laemmli, U.K., (1970) Nature, 227, 680-685. Michel, R. and Neupert, W. (1973--~- Eur. J. Biochem. 3__66,53-67. Bray, G. (1960) Anal. Biochem. i , 249. Dondon, J., Godefroy-Colburn, T~, Graffe, M. and Grunberg-Manago, M. (1974) FEBS Letters, 45, 82-87.
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