Mitochondrial transcription complex from Saccharomyces cerevisiae

THE JOURNALOF BIOLOGICALCHEMISTRY Vol. 256, No. 3, Issue of February 10, pp. 1466-1473, 1981 Printed in U.S.A.

Mitochondrial Transcription Complex from Saccharomyces cerevisiae * (Received for publication, September15, 1980)

David Levens$, Richard Morimotog,and Murray Rabinowitz From the Departmentsof Medicine, Biochemistry,and Biology and nThe Franklin McLean MemorialResearch Institute, The University of Chicago, Chicago, Illinois 60637

A DNA protein complex has been isolated from the mitochondria of Saccharomyces cerevisiae. The complex transcribes RNA complementary to mtDNA in a nonrandom manner.The RNA polymerase activity contained in the transcription complex is not dependent on the addition of exogenous template. The activity is rendered template-dependent by autolysis and c a n be further purified by heparin-Sepharose 4B chromatography. The activity is inhibited by heparin, Mn2+, and increasing ionic strength. The activity requires Mg2+ and ribonucleotides. The preferred template for the template dependent activity is poly[d(AT)]. The majority of the RNA synthesized by the transcription complex from endogenous DNA is complementary to the DNA strands directing the synthesis of the large and small ribosomal RNA. In yeast the 21 S and 14 S rRNA genes are widely separated, therefore the transcription of these two regions but not of the intervening regions b y the transcription complexsuggests the existence of at least two transcriptional promoters on the yeast mitochondrial genome.

Yeast mitochondrial biogenesis is a complex process that involves the assembly of components specified by both the nuclear and mitochondrial genomes. Whereas most mitochondrial proteins are translated on cytoplasmic ribosomes from mRNA transcribed inthe nucleus, a small number of peptides essential for oxidative phosphorylation are coded for by mtDNA. These peptides are transcribed by a protein-synthetic apparatus that is itself in part mitochondrially coded. MtDNA contains the genes for a large (21 S) and a small (14 S) rRNA as well as approximately 25 tRNA genes, including at least one for each amino acid. In addition, a peptide (Var1)associated with the small mitochondrial ribosomal subunit is specified by mtDNA (1-3). The capacity for performing genetic analysis of both the nuclear and mitochondrial genomes as well as theease of large scale biochemical manipulations makes yeast particularlyattractive for the study of mitochondrial biogenesis. Since yeast is a facultative anaerobe,it can survive mutations eliminating oxidative phosphorylation if grown on a fermentable carbon *,This study was supported in part by Grants HL 04442 and HL 09172 from the National Institutes of Health, Grant NP281 of the American Cancer Society, andthe Louis Block Fund of The University of Chicago. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. Supported by Public Health Service Grant2 T32 GM07281. 3 Present address, The Biological Laboratories, Harvard University, Cambridge,MA 02138. 1The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the United States Department of

+

Energy under Contract EY-76-C-02-0069.

source. The mitochondrially coded peptides required for electron transport and ATP synthesis have been genetically defined. Three of seven subunits of cytochrome oxidase, two of the subunits of oligomycin-sensitive ATPase, and a peptide associated with the cytochrome bcl complex have been genetically mapped. The genes associated with these peptides are, respectively, 0x1-1, 2, and 3, OLI-1 and 2, and COB-BOX. The COB-BOX gene contains four intervening sequences, and preliminary evidence indicates that the 0x1-3 gene is split as well. In addition the 21 S rRNA gene contains a 1200-base pair intervening sequence (1-3). Yeast nuclear RNA polymerases have been well characterized(4-10) and yeast nuclear transcription is presently the subject of investigation ( l l ) , but theregulatory elements and transcriptional controls in yeast mitochondria have yet to be elucidated. Studies on the interrupted genes and long transcriptional units on mtDNA may defiie transcriptional requirements for the splicing and processing of RNA in addition to contributing to our knowledge of organella biogenesis. The involvement of RNA polymerase in the processing of primary transcripts has been postulated (12). One approach tothe study of mitochondrial transcription is the reconstitution of the RNA-synthetic machinery from purified components. Although our studies are concerned primarily with mitochondrial RNA polymerase and its template, mtDNA, additional factors may be required to mimic in uiuo transcription in vitro, as has been reported for nuclear transcription systems (13-17). Whereas mtDNA has been extensively characterized both physically and genetically (1,2, la), there is no consensus on the properties of the mitochondrial RNA polymerase despite several reports on its purification (19-22). The specific activities are sufficiently low to suggest that alternativeapproaches to mitochondrial transcription must be employed. We have isolated a protein DNA complex from yeast mitochondria that transcribes mtDNA faithfully, partially obviating the need for reconstitution. Similar complexes have been isolated from Euglena chloroplasts (23) and from polyoma virus-infected cells (24). We have also developed a purification scheme for the soluble mitochondrial RNA polymerase, which is described in the following paper. The organization and termini of the yeast mitochondrial transcription units are not known. In HeLa mitochondria, there appears tobe complete symmetric transcription of the mitochondrial genome, followed by degradation of the noncoding RNA complementary to theL strand (25,26). We now present evidence that complete symmetric transcription of the yeast mitochondrial genome does not occur, and that transcription is initiated from at least two promoters. MATERIALS AND METHODS

Strains and Growth of Cells-We used the grande strain of Saccharomyces cerevisiae MH41-7B grown to midlog phase on 2% glucose, 1%yeast extract, and 1%Bacto-peptone unless otherwise noted.

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Mitochondrial Transcription Complex from Occasionally, galactose was substituted from glucose with no observable change in results. Cells were harvested by centrifugation either with a Sharples continuous flow centrifuge or by centrifugation at 2000 X g for 5 min in a Sorvall GSA rotor. Preparation of Mitochondria-Cells were washed with cold water and centrifuged a t 2000 X g for 5 min in a Sorvall GSA rotor and were then broken by one of three procedures. (a) The cells were converted into spheroplasts with glusulase and disruptedin a Waring Blendor as described elsewhere (27). This procedure was typically used with 200 g of cells, although it is suitable for 5 to 500 g. ( b ) The cells were resuspended in 1.2 M sorbitol and centrifuged for 5 min at 2000 X g in a Sorvall GSA rotor. The sorbitol wash wasrepeated, and the cells were suspended in an equal volume of breaking buffer (BB, 10 mM Tris-C1, pH 7.5,O.a M sorbitol, and 0.1%bovine serum albumin). Aliquots consisting of 50 ml of suspended cells were poured into 75ml bottles one-thirdfilled with 0.5-mm glass beads, and the suspension was homogenized for 25 s in a Bronwill model MSK mechanical cell homogenizer. The glass beads were allowed to settle for 1 min, and the homogenate was poured off and centrifuged for 10 min at 2000 X g in a Sorvall GSA rotor. The supernatantwas carefully decanted and saved; the pellet consisted of a gelatinous layer of nuclei and broken cells over a more densely packed layer of unbroken cells. The gelatinous layer was resuspended in BB and discarded, and the unbroken cells were suspended in an equal volume of BB.Cell breakage and centrifugation were then repeated. This method allows rapid breakage of 10 to 200 g of cells. (c) The cells were washed with 1.2 M sorbitol as described in procedure b and then suspended in 1.5 volumes of BB and passed through a Manton-Gaulon press a t 6000 p.s.i. The homogenate was centrifuged for 10 min a t 2000 X g in a Sorvall GSA rotor, and the broken cells and nuclei were removed as described in procedure b. The unbroken cells were resuspended in 1.5 volumes of BB, and breakage was repeated cells were passed through the Manton-Gaulon press a total of three times. This procedure is suitable for 500 to 1500 g of cells. All three procedures result in breakage of 60 to 90% of the cells. The cell homogenates from the three procedures were treated identically for the isolation and washing of mitochondria. The homogenates were centrifuged for 10 min at 2,000 X g in a Sorvall GSA rotor, the pellet was discarded, and the supernatants were centrifuged at 40,000 X g for 10 min in a Sorvall SS-34 rotor. The postmitochondrial supernatant was discarded, and the mitochondrial pellets were washed twice by resuspension in BB and centrifugation for 10 min at 40,000 X g. The mitochondrial pellets were resuspended in 500 ml/ 200 g of yeast in 10 mM Tris-Ci, pH 7.5,O.a M sorbitol, 10 mM MgCh, and 100 pg/ml of DNAse I. After incubation on ice for 30 min, the suspension was centrifuged for 10 min a t 40,000 X g,and the mitochondrial pellets were washed twice in BB with 0.01 M EDTA, pH 7.5. The mitochondrial pellets from the final wash were either used immediately or frozen in a dry ice-acetone bath and stored at -7O'C. For preparation of mitochondria collected a t defined stages of growth, cells were grown to a particular optical density and broken according to procedure b, which is the most rapid method for disrupting a small quantity of cells. Mitochondria from petite strain IL8-8c/H71 (a p strain with no detectable mitochondrial DNA) and strain CEO,, were prepared by method a. Preparation of Transcription Complex-Unless otherwise stated, all work was done a t 4OC. Frozen mitochondrial pellets containing approximately I g of mitochondrial protein were suspended in 20 ml of20mM Tris-C1, pH 7.9, 30%glycerol, 10 mM MgClz, 1.0 mM EDTA, 1.0 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Thetotal volume of the suspension was adjusted to 35.5 ml with water. The suspension was homogenized with 10 strokes in a Potter-Elvehjem homogenizer; 2.0 ml of 20% NP-40 were added, and the suspension was homogenized with 10 additional strokes. After addition of 2.5 ml of 4.0 M KCl, the suspension, now referred to as the crude extract, was homogenized with another 10 strokes. The crude extract was centrifuged for 30 min in a Beckman type 30 rotor at 90,000 X g. The amber supernatant was loaded onto a 450-ml (4 x 34 cm) Sepharose 4B column previously equilibrated with TGKMED (10 mM Tris-C1, pH 7.9, 15% glycerol, 0.25 M KCl, 5 mM MgCI2, 0.5 mM EDTA, and 0.5 mM dithiothreitol) containing 0.5% NP-40.20 p~ ATP, and 20 p~ GTP. Ten-milliliter fractions were collected and assayed for RNA polymerase and total protein. Active fractions were pooled and dialyzed against PED (20 mM potassium phosphate, pH 7.8, 0.1 mM EDTA, and 1 mM dithiothreitol) containing 20% glycerol and 0.5 mM MgCl,. For prolonged storage of the transcription complex, the active fractions were dialyzed against PED containing 50% MgC12. For further purification of the RNA glycerol and 0.5 mM

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polymerase activity from the transcription complex, the pooled peak of activity from the Sepharose 4B column was allowed to incubate for 4 days at 4°C in PED with 20% glycerol and 0.5 mM MgClz, during which time the endogenous DNA was autolyzed. Purification of the RNA Polymerase Activity from the Transcription Complex-When the ratio of poly[d(AT)]-independent to poly(dA-dT)-dependent activity became 1:lO following autolysis, the pooled peak of activity from the Sepharose 4B column was adjusted and loaded onto a 3-ml heparinto 5 mM MgC12 and 20mMKC1 Sepharose 4B column previously equilibrated with PED containing 20% glycerol, 20 mM KC1, and 5 mMMgC12. The column was washed with 10 rnl of PED containing 20% glycerol, 0.23 M KC1, and 5 mM MgC12 and eluted with 10 ml of PED with 20% glycerol, 0.4 M KCl, and 5 mM MgCI2. One-milliliter fractions were collected and assayed for RNA polymerase activity and protein. Active fractions were collected and dialyzed against PED containing 50% glycerol and 0.5 mM MgC12. The dialyzed sample was loaded onto a 0.3-ml DEAEcellulose column previously equilibrated with PED containing 50% glycerol and 0.5 mM MgC12. The column was washed with PED containing 90 mM KC1 and 0.5 mM MgCL and eluted stepwise with 1 rnl each of PED with 0.2 M KCl, 0.5mMMgC12 and 0.3 M KC1, and 0.5 m~ MgC12.Fractions of 0.3 ml were collected, and the active fractions were pooled and stored at -2OOC. RNA Polymerase Assays-RNA polymerase assays were performed in 10 mM Tris-C1, pH 7.9, mM MgCL, 1mg/ml of bovine serum albumin, 5% glycerol, and 150 p~ concentration each of ATP, GTP, and CTP.r3H]UTP (2 Ci/mmol) was included at 5 p ~ When . required, exogenous template, usually poly[d(AT)] (Miles),was added to 50 pg/ ml. The total reaction volume was 100 pl. Generally, 5- to 10-pl aliquots of enzyme fractions were assayed. The samples were incubated at 37°C for 20 min; incubations were terminated by the addition of 0.5 ml of 7% perchloric acid and 0.1 M sodium pyrophosphate. The reaction mixtures were then diluted with 5 ml of 0.1 M HC1 and 0.01 M sodium pyrophosphate and filtered through Whatman GF/C filters presoaked in 1 M HCl and 0.1 M sodium pyrophosphate. The filters were washed three times with 5 ml of 0.1 M HC1 and 0.01 M sodium pyrophosphate and once with 95% ethanol, dried, and counted in a toluene-based scintillation fluid. Preparation of RNA Transcribed by the Transcription ComplexThe peak fraction from the excluded volume of the Sepharose 4B column was dialyzed against PED containing 20% glycerol and 0.5 mM MgClz. A 500-pl portion of the dialysate was transferred to a tube in which 5 pmol of Tris-C1, pH 7.9, 5 pmol ofMgC12,75 nmol each of ATP, GTP, and GTP, as well as 6 nmol of [32P]UTP (200 Ci/mmol) had been lyophilized. Reactions were incubated for the requisite time at 37"C, boiled for 1 min to abolish RNA polymerase activity, and cooled in ice. RNase-free DNase I (Worthington or Calbiochem) and Escherichia coli tRNA (Calbiochem) were added to 20 pg/ml and 200 pg/ml, respectively, to the cooled reaction mixtures, and the mixtures were incubated for an additional 15 minat 37°C. They were then adjusted to 0.2% sodium dodecyl sulfate and 20 mM EDTA and extracted with an equal volume of phenol equilibrated with 0.1 M Tris-C1, pH 7.5, and 10 mM EDTA. The aqueous phase was chromatographed on a 10-ml Sephadex G-50 column equilibrated with 10 mM Tris-C1, pH 7.5, 1 mM EDTA,and 0.1% sodium dodecyl sulfate. Fractions (0.4 m l ) were collected and counted for Cherenkov radiation. The radioactive fractions in the excluded volume were pooled, adjusted to 0.3 M sodium acetate, and then precipitated overnight at -20°C with 2.5 volumes of absolute ethanol. After centrifugation, the resulting pellets were dried under vacuum, resuspended in H20, and stored at -2OOC. Preparation of Mitochondrial RNA-Mitochondrial RNAwas isolated from mitochondria prepared from yeast treated with glusulase. Mitochondria were washed and lysed with sodium dodecyl sulfate, and the RNA was extracted with phenol/chloroform/isoamyl alcohol as described elsewhere (28, 29). Radioiodination ofRNA-Mitochondrial RNA wasradioiodinated with Iz5I as described previously (30). Preparation of Mitochondrial DNA-MitochondrialDNAwas prepared with the use of isopyknic gradients in CsC1, as described by Lewin et al. (31). For analytical CsCl isopyknic centrifugation, mitochondria were lysed with sodium dodecyl sulfate, extracted with phenol, precipitated with ethanol asdescribed above, and centrifuged in a Beckman model E centrifuge. Preparation ofcRNA-cRNA was transcribed from mtDNA template with E . coli RNA polymerase, as described earlier (32). Determination of Protein-Protein was determined either by the method of Lowry (33) or with the Bio-Rad protein reagent.

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FIG. 1. Sepharose 4B chromatography of mitochondriallysates from grande and petite yeast strains. Mii

tochondria were prepared with glusulase, lysed, and subjected to Sepharose 4B chromatography as described under “MaterialsandMethods.” O “ 0 , [3H]UMP incorporated; O---O, protein, milligrams/ml. a, Mitochondria from grande strain MH41-7B; b, mitochondria from petite strainCEOII;c, mitochondria from the DNA-minus (PO) petite strain I&&, HF71.

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FIG. 2. Sepharose 4B chromatography of mitochondrial lysates from grande yeast strain harvested at differentstages of growth. Mitochondria were isolated with the Bronwill model MSK mechanical cell homogenizer from yeast harvested at the specified Am, lysed, and subjected to Sepharose 4B chromatography as described under “Materials andMethods.” a, Yeasts were harvested a t Am = 0.62 (midlog phase). One-half milliliter of mitochondrial lysate containing 23.5 mg/ml of protein was chromatographed on a 10-ml Sepharose 4B column. 0.5-ml fractions were collected. b, Yeasts were harvested at A610 = 4.0 (late log phase). One-half milliliter of mitochondrial lysate containing 25.1 mg/ml of protein was subjected to chromatography as above. c, Yeasts were harvested at AM = 6.0 (stationary phase). One-half milliliter of mitochondrial lysate containing 31.5 mg/ml of protein was subjected to Sepharose 4B chromatog[3H]UMP incorporated; 0- - -0, raphy as described above. M, protein, milligrams/ml. Preparation of Heparin-Sepharose 4B-Heparin-Sepharose 4B was prepared by coupling of heparin to cyanogen bromide-activated Sepharose 4B according to Sternbach et al. (34). Hybridizations-Hybridizations to DNA immobilized on nitrocellulose filters either directly (35) or after agarose gel electrophoresis (36) were performed as described. RNA-RNA hybridizations were performed by incubation of [3H]cRNA or 32P-transcriptioncomplexsynthesized RNA to a vast excess of unlabeled mitochondrial RNA in a hybridization mixture containing 0.23 M sodium chloride and 0.04 M sodium phosphate (pH 6.8), unlabeled mitochondrial RNA, and radioactive RNA transcribed in uitro. The mixtures were sealed into glass capillaries and completely denatured by heating to 110°C for 5 min, followed by quick coolingin an ice-salt bath. Samples to be hybridized were then incubated at 60°C for 16 h to the specified Rot values. At the specified time, the contents of the capillaries (total volume less than 20 pl) were emptied into 2.0 ml of 50 mM Tris-C1, pH 7.4. One-half of this sample was precipitated with 5% trichloroacetic acid, and the other half was incubated for 1 h a t 37OC with 10 pg/ml each of pancreatic RNase and T, RNase. Precipitates were collected and counted in a toluene-based scintillation fluid. We compared ribonuclease-treated samples with nonribonuclease-treated

FIG. 3. Autolysis of DNA in transcription complex.a, Correlation between endogenous/poly[d(AT)]-stimulated activated and DNA content of the transcription complex as a function of time. RNA polymerase assays and DNA measurements were performed as described under “Materials and Methods.” O”--O, Endogenous/ poly[d(AT)]-stimulated activity of transcription complex stored in 50% glycerol at -20°C; O”-O, endogenous/poly[d(AT)]-stimulated activity of transcription complex stored at 4OC. Bars indicate DNA content. b, DNase profile of the Sepharose 4B column. RNA polymerase and DNase assays were performed as described under “MateRNA polymerase activity, [3H]UMP rials and Methods.” -, incorporated; 0- - -0,DNase activity. samples to determine the per cent hybridization. In order to determine the amount of self-annealing occurring with the transcription complex-synthesized RNA, we performed hybridizations as described above, except that no cold mitochondrial RNA was included in the hybridization mixture. Determination of DNA Content in the Transcription ComplexAliquots (50 p1) of the transcription complex in PED in either 20% or 50% glycerol and 0.5 mM MgC12 were diluted to 1 ml with 10 mM TrisC1, pH 7.5, and 10 mM MgCL, and the fluorescence in the presence of 1 pg/ml of ethidium bromide was determined (excitation at 548 nm, emission at 590 nm). An identical sample was treated for 0.5 h at 37OC with 250 pg/ml of RNase-free DNase I prior to the addition of ethidium bromide and fluorescence determination. The difference in fluorescence between the two samples was compared to a standard curve generated by identical treatment of known quantities of mtDNA. In all cases, digestion with DNase reduced the fluorescence to background levels. DNase Assays-Aliquots (25 pl) of Sepharose 4B fractions were incubated in 100 p1of 100 mM Tris-C1, pH 7.9, 10 mM MgCL, 2 mM dithiothreitol, and 1 pg of [3H]E. coli DNA (l0,OOOcpm/pg). Samples were incubated for 15 min at 37OC, precipitated with 5%trichloroacetic acid, and spun for 5 min in an Eppendorf microcentrifuge. The supernatants were counted in Instagel (Packard). RESULTS

Due t o the low specific activities and instability of most reported preparations of yeast mitochondrial RNA polymerase (19-22), we purified an RNA polymerase-mtDNA complex. The bufferused to lyse the mitochondria contains a number of components which stabilize the RNA polymerase

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activity and its interaction with the endogenous template. In particular, Mg2+and the nucleotides ATP and GTP, which are known to stabilize the interaction between E. coli RNA polymerase and promoters, were included in the buffer (37). Mitochondria lysed with 0.25 M KC1 and 0.5%NP-40 appear to lose 90% of their RNA polymerase activity. This loss is recovered following Sepharose 4B chromatography, indicating that the RNA polymerase is either inhibited or masked by nucleases in the crude extract. The RNA polymerase activity elutes from the Sepharose 4B column in two peaks (Fig. la). One peak of activity is found in the fractions excluded from the column and contains less than 1%of the mitochondrial protein. The otherpeak elutes with the bulk of the protein in the included fractions. Synthesis of RNA in the absence of exogenously added template is supported by the fractions from the excluded volume,whereas the second peakis totally dependent on added template. The distribution of activity between the complexed and soluble peaks has been found to be a function of the stage of growth at which the cells used for preparation of the polymerase are harvested. As is shown in Fig. 2, the amount of activity in the transcription complex decreases as thecells progresstoward the stationary phase. It is difficult to assess the amount of RNA polymerase actually present in the second peak, since soluble nucleases also elute in the same fraction. It is possible that thedecrease in activity in the transcription complex reflects a physiological regulational event. The transcription complex has been isolated from a p- strain, CEOrr, but, as predicted, is not found in a DNA-less (p’) strain (Fig.1, b and c ) . The RNA polymerase activity in the transcription complex is very tightly associated with DNA. Attempts toliberate the activity by use of phase partition (38), streptomycin or Polymin P precipitations (39) followed by high salt washes, treatment with micrococcal orpancreatic nucleases (40),and chromatography on hydroxylapatite followed by highsalt elutions (41) were all unsuccessful. In all of the above procedures, the activity either remained associated with the endogenous template or was lost. Uponstorage of the complex for 2 or 3 days at 4°C inPED with 20% glycerol and 0.5 mM MgCl,, the RNA polymerase activity became increasingly template dependent, with a concomitant decrease in the DNA content of the complex (Fig. 3a). The time necessary to render the activity 90% template dependent could behastened if the leading edge of the peak of soluble proteins from the Sepharose 4B column is pooled with the transcriptionally active fractions. The leading edge of the peak of soluble protein contains a DNase activity which may be responsible for the degradation of the endogenous DNA and the release of RNA polymeraseactivity from the template (Fig. 321). The rate at which the complex becomes template dependent is decreased if the f i s t peak of activity is stored in 50% glycerol with 0.5 mM MgC1, at -20°C. Once released from the DNA, the RNA polymeraseactivity was further puritied by heparin-Sepharose and DEAE-cellulose chromatography. Stepwise elution of the activity from the columns was utilized, since gradient elution resulted in dilution of the RNA polymerase activity and subsequent loss of activity. Only small amounts (less than 50 pg, optimally) of FIG. 4. Properties of the RNA polymerase activity in the transcription complex. Transcription complex and RNA polymerase fractions were preparedand assayed as describedunder “Materials and Methods” in the presence of the indicated additions. a, Divalent cation dependence, 0- -0, MnC12; U, MgC12. b, Inhibition of RNA polymerase activity by MnC12 and KCI. 0- -0, MnC12, assayed in the presence of 5 mM MgC12; U, KCI. c, Inhibition of the RNA polymerase activity by heparin. U, template-dependent activity, upper scale; 0- -0, template-independent activity, lower scale.

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the yeast mitochondrial RNApolymerase have been purified amanitin and is inhibited by rifampicin onlya t extremely high by the procedure described above; quantities of the enzyme concentrations. sufficient for structural and transcriptional studies are better Seventy per cent of the activityof the transcriptioncomplex purifiedby theprocedure describedin theaccompanying is inhibited by heparin (Fig. 4 4 , whereas 30% remained repaper (42). Although both preparations have similar specific sistant even to large amounts of heparin. We decided to activities (300 nmol of UMP incorporated/mg of protein/lO determine whether the heparin-sensitive activity represented that portion of transcription resulting from in vitro initiation. min whenpoly[d(AT)]is used astemplate),theactivity isolated from the transcription complex is more labile and RNA was synthesized by the complex in the presence of [ y variable. "'PIATP or [Y-''~P]GTP.No incorporationof radioactivity was The RNApolymerase purified from the transcription com- detected byacid precipitation of the RNA. Furthermore, plex possesses enzymatic properties and requirements similar analysis of an alkaline hydrolysate of newly synthesized RNA to those of the highly purified mitochondrial RNA polymer- revealed a lackof the expected adenosine tetraphosphate and ase. The activity is inhibited by salt. Concentrations aslow as guanosine tetraphosphate. We concludethat thecomplex does 30 mM KC1 caused significant inhibition (Fig. 46). The polym- not initiate with ATP or GTP. The absence of initiation in erase activity requires Mg2+;Mn2+will not substitute and is, the complex must beconsidered tentative, however. The in fact, inhibitory even in the presence of a large excess of presence of very large mitochondrial RNAs (29, 43, 44) sugM$+ (Fig. 4, a and 6). The activity prefers poly[d(AT)] 15- gests that the mitochondrial transcription units are long, and fold to natural DNAs. The activity is not sensitive to a- that initiation may be a rare event. Furthermore, the possibility that initiation does not occur with a purine deserves special consideration in view of the recent discovery of a transcription unit initiating with CTP in E . coli (45). If the complex does not initiate, then the heparin inhibition may reflect a complex mode of action such as that occurring with bacterial RNApolymerases (46,47). Once it becomes template dependent, the RNApolymerase activity is 100% inhibited by heparin (Fig. 4c). The net incorporation of UMP by the transcriptioncomplex continues for only 20 min. This is consistent with the lack of

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FIG.5. Time courseof RNA synthesis by transcription complex. Transcription complex was prepared and allowed to synthesize RNA for the stated timein the presence of [:1H]UMP. The RNA was acid-precipitated and counted for radioactivity as described under "Materials and Methods."

TABLE I Hybridization of RNA transcribed fromRNA polymerase. DNA complex RNA transcribed by the RNA polymerase activity in the transcription complex or by E. coli RNA polymerase inthe presence of ["'PIUTP as described under "Materials and Methods" was hybridized with excess DNA immobilized on nitrocellulose circles as described, or was annealed with excess DNA immobilized on nitrocellulose circles as described, or was annealed with excess unlabeled mitochondrial RNA prepared as described for 16 h to an fit of 2.4 X lo". For all hybridizations, the input activity ranged from 5 to 10 X 10" cDm.

Yeast mtDNA Yeast nuclear DNA E. coli DNA Salmon sperm DNA Yeast mitochondrial RNA Not determined.

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FIG.6. Determination of punty of mtDNA from mitochondria used in preparation of the transcription complex. Mitochondria were prepared with glusulase as described under "Materials and Methods," suspended in 10 mM Tris-CI and 10 mM EDTA, lysed with 1% sodium dodecyl sulfate, extracted with an equal volume of phenol, and precipitated with the addition of sodium acetate to 0.3 M and 2.5 volumes of ethanol. The pellet was resuspended in distilled Hz0 and subjected to analytical or preparative CsCl centrifugation as described. Analytical centrifugation of 8 pg of DNA yielded the peak shown. The buoyant density was determined tobe identical with that of mtDNA. Top, densitometricscan of photograph of analytical buoyant density ultracentrifugation of mtDNA. Left side is the light side of the gradient. Bottom, photograph of 8 pg of mtDNA centrifuged as described above.

Mitochondrial Transcription Complex from S. cerevisiae detectable initiation (Fig. 5). The size of the in vitro product isolated after 1 min of incubation is about 11 S and decreases with time despite an increasein acid-precipitable RNA (data not shown). Thisis probably due to the presence of contaminating ribonucleases. Thus far, it has not been possible to inhibittheRNaseswithoutsimultaneouslyinhibitingthe RNA polymerase activity. The RNA synthesized by the transcriptioncomplex hybridizes with mtDNA, but not with heterologous DNAs, thus attesting to the mitochondrial origin of the transcription complex (TableI).Thisresultisconsistentwiththe lack of detectable nuclear DNA contaminating the mitochondria as analyzedby analyticalbuoyantdensityultracentrifugation (Fig. 6). The RNA synthesized in vitro neither self-anneals nor anneals with a large excess of cold mitochondrial RNA, showing that the transcription supported by the transcription complex is asymmetric and is transcribed from the same DNA strand that specifies the RNA synthesized in vivo. The RNA

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FIG.8. Comparison of RNA synthesized from the transcription complex for 1, 5, or 20 min in vitro with mitochondrial RNA synthesized invivo andwith RNA transcribed from mtDNA by E. coli RNA polymerase. a, ( I ) Agarose gel of mtDNA

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FIG.7. Comparison of RNA synthesized by the transcription complex with RNA synthesized in vivo. RNA was synthesized by the transcription complex for 1 min in uitro in the presence of [:‘“P]UTP, as described under “Materials and Methods.” The RNA was isolated and hybridized to an Hha I digest of MH41-7B RNA as described under “Materials and Methods.” Unlabeled RNA was extracted from mitochondria, radioiodinated, and hybridized to anHha I restriction enzyme digestas described under “Materials and Methods.” a, Densitometric scan of RNA transcribed in uitro, hybridized to mtDNA.b, Autoradiogram of RNA transcribed inuitro, hybridized to mtDNA. c, Ethidium bromide fluorescence of an agarose gel of mtDNA,restrictedwithrestrictionendonuclease Hha I prior to transfer of nitrocellulose. d, Densitometric scan of radioiodinated mitochondrial RNA hybridized to mtDNA. e, Autoradiogram of radioiodinated mitochondrial RNA hybridized to mtDNA. Ethidium bromide fluorescence of an agarose gel of mtDNA, restricted with Hha I prior to transfer to nitrocellulose. g, Restriction mapof mtDNA fromMH41-7B treated with Hha I; thickblack bars indicate the most heavily transcribed regions and the locations of the rRNAgenes. Upper line indicates the order of the restriction fragments; lower line indicates fragment size and physical map position relative to the single Sal I restriction endonuclease sitein mtDNA.

treated with restriction endonuclease Hha I and Barn HI prior to transfer to nitrocellulose. (2) Hybridization of RNA transcribed during a1-min incubation of the transcription complex as described under “Materials and Methods” to digest shown in I . (3) Hybridization of RNA transcribed duringa 5-min incubation. ( 4 ) Hybridization of RNAtranscribedduring a 20-min incubation. The fragments containing the14 S and 21 S rRNA genes areindicated. b, ( I ) Agarose gel of the digest shown in a-I. (2) Hybridization of mitochondrial RNA synthesized in uiuo, extracted, and radioiodinated as described under “Materials and Methods.” c, ( I ) Agarose gel of same digest shown in a - I . (2) Hybridization of RNA transcribed from mtDNA by E. coli RNApolymerase as described under “Materials and Methods.”

transcribed in vitro hybridizesprincipally to those regions that code for the 14 S and 21 S rRNAs. RNAs from other regions of the genome are present in reduced amounts (Figs. 7 and 8,a and 6). The patternof hybridization is the samefor RNAs synthesized for 1, 5, or 20 min in vitro (Fig. 8a). In contrast, when E . coli RNA polymerase is used to transcribe mtDNA, all regions of the genome are represented equally (Fig. 8c). In addition, at least 30%of the RNA synthesized by the bacterial enzyme anneals with excess mitochondrial RNA, indicating that transcriptionby that enzyme is nonphysiologic (Table I). DISCUSSION

The purification of RNA polymerase from themitochondria of yeast (19-22), rat liver (48, 49), Xenopus laevis (50),and Neurospora crassa (51)has thus farfailed to yield an enzyme that has allowed faithful transcription in vitro of mitochonis stillno consensus onthe drial RNA. Foryeast,there properties or characteristics of the mitochondrialRNA polymerase. We have isolated a transcription complex that promotes

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the transcription in vitro of coding regionsof the yeast mitochondrial genome, and we have partially purified the RNA polymerase from this complex. Transcription complexes similar to that which we described have been useful inthe examination of transcription in Euglena gracilischloroplasts (23) and in polyoma virus (24). In addition, agarose gel fdtration of protein DNA complexes has been used for analysis of DNA-binding proteins from T4-infectedE. coli cells (52). Several lines of evidence show that the transcription complex and its associated RNA polymerase activity are mitochondrial in origin. 1) DNA isolated from the same mitochondria that are usedin the preparation of the transcription both preparative and complex bands as a singlepeakby analytical CsCl ultracentrifugation and has the buoyant density of mtDNA. Furthermore, the DNA exhibits a restriction enzyme fragment pattern characteristic of mtDNA (data not shown). Hence, we conclude that the DNA recovered in the excluded volume of the Sepharose 4B column is mitochondrial, and this strongly suggests that the associated RNA polymerase activity is also mitochondrial. 2) The RNA transcribed by the complex hybridizes to mtDNA with the correct strand specificity and primarily to regions known to code for the mitochondrial rRNAs. The possibility that yeast nuclear RNA polymerasefortuitously transcribes mtDNA with appropriate biologic specificity would seem to be remote. 3) The RNA polymerasemDNAcomplex is not found in the mitochondria of po petite strains; this suggests an association between the transcribing activity and mtDNA. 4) The properties of the RNA polymerase activity isolated from the transcription complex are very different from the properties of any known yeast nuclear RNA polymerase. The RNA polymerase found in the transcription complex is fully resistant to a-amanitin and is inhibited by Mn2' and by ionic strengths not inhibitory to the nuclear enzymes. The mitochondrial RNA polymerase uses poly[d(AT)] as template with much greater efficiency relative to other templates than do the nuclear RNA polymerases (4-10). 5) Finally, the RNA polymerase activity isolated from the complex is inhibited by antibodies raised to the purified soluble mitochondrial RNA polymerase; this is distinct from the nuclear enzymes (see accompanying paper). The presence of the transcription complex inthe mitochondria of petite strains of yeast indicates that the RNA polymerase is the product of a nuclear gene. Since the RNA polymerase is specified by the nucleus, it is reasonable to expect that the regulation and synthesis of this enzyme playa central role in the depression of the mitochondrial genome. In fact, South and Mahler (53) found that the RNApolymerase activity present in whole mitochondria rises prior to theexpression of induced enzymes such as malate dehydrogenase and cytochrome oxidase and falls as the cells progress toward stationary phase. We have observed that thetranscription complex from logarithmically growing yeast possesses more RNA polymerase activity than does transcription complex from stationary yeast, indicating that the transcription we observe may reflect the physiologic state of the mitochondria. The RNA synthesized by the transcription complex has properties similar to those of mitochondrial RNA synthesized in vivo. The RNA synthesized in vitro neither self-anneals nor anneals with excess coldmitochondrial RNA, it is, therefore, likely that this material is transcribed asymmetrically. The alternative possibility that thecomplex exclusivelytranscribes regions of the mitochondrial genome which are not transcribed in vivo is unlikely, since the material synthesized invitro and radioiodinated mitochondrial RNA hybridize similarly to nitrocellulose blots of restriction fragments of mtDNA. In both cases, hybridization occurs predominantly to

the fragments that code forthe 21 S and 14 S rRNAs. Analysis of total mitochondrial RNA on agarose-urea or methyl mercury hydroxide-agarose gels reveals that the 21 S and 14 S rRNAs are themost abundant mitochondrial species (29,43). We conclude that thebulk of the RNA synthesized in vitro is specified by the same strand and the same regions that serve as template for RNA synthesis in uivo. In S.cereuisiae, the large and small rRNA genes are separated by one-third of the mitochondrial genome (25 kb) (5361). Since we observed extensive transcription of the two rRNA regions relative to the transcription of the sequences between these genes, we conclude that the two rRNAs are probably transcribed from separate promoters. The lack of detectable initiation in vitroindicates that thesynthesis which we observed reflects the in vivosituation. If both the 21 S and 14 S rRNAs were processed from the same primary transcript, the DNA protein complex wouldhave to degrade specifically the RNA coded for by the nonribosomal regions of the genome. One would expect absent or minimal hybridization of the RNA to the3' distal sequences with short incubations and increasing hybridization to these sequences as the time of transcription in vitro is increased. In fact, acid-precipitable RNA continues to accumulate for 15 min, and the same hybridization patterns are observed with RNAs isolated after incubations of 1, 5, or 20 min. If transcription from a single promoter had occurred, the sequences between the rRNAs would have been degraded instantaneously by an activity in the protein-DNA complex. We believe this possibility to be much less likely than the existence of separate promoters for the 14 S and 21 S rRNA genes. This view is supported by the observation that petites containing only one or the other rRNA gene transcribe and process that rRNA normally. The possibility of nonspecific transcription followed by specific processing also appears unlikely considering the specific nature of the hybridization of the RNA transcribed by the complex. The existence of separate promoters for each of the RNA genes makes it likely that additional transcriptional regulation occurs. Our conclusion that the rRNA genes are over-transcribed relative to theother sequences in the genome implies that thenon-rRNA genes are under a different transcriptional control. One possibility is that regulation occurs at tennination. Thus, if there were read-through transcription at the terminators for the rRNA genes, the sequences downstream would be underrepresented relative to the rRNA sequences. Alternatively, the non-rRNA genes may have completely independent transcriptional controls, at both termination and initiation sites. Once again,the existence of apparently normal transcription in petites suggests that the controls for each gene are separate. Characterization of RNA synthesis from transcription complexes isolated from various petites might prove useful for the analysis of the transcriptional organization of the mitochondrial genome. The transcription complex provides a reference point for the examination of transcriptional activity obtained by reconstitution in vitro of the RNA synthetic apparatus from its components.

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