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M o l. Cells, Vol. 5. N o.3, pp. 2 17-223 A Single-step Purification of the Saccharomyces cerevlslae Mitochondrial RNA Polymerase Specificity Factor Overproduced in Escherichia coli Eun Ah Lee, Jae Sub Yang and Sei H eon Jang* DepGl1ment oj Molecular Biology, Taegu University, Ky ungsan 713-714, Korea (Received on March 27, 1995) Mitochondrial RNA polymerase of Saccharomyces cerevisiae consists of two different proteins: a catalytic core RNA polymerase of 145 kDa (Rp041p) and a specificity factor of 43 kDa (Mtflp) required for recognizing promoters of the various genes encoded in the mitochondrial genome. A recombinant MtfIp fusion protein was previously partially purified from E. coli by combination of conventional column chromatographies [Mangus, D. A., Jang, S. H., and Jaehning, J . A. (1994) J. Bioi. Chern. 269, 26568-26574J . However, the expression level was low and the purification involved multiple steps. Thus, here we have constructed a new expression plasmid coding for the yeast specificity factor and purified it to > 95% purity in a single step. The MTFl gene was sub cloned into Novagen's pET15b, which contains an N-terminal six-histidine tag. The resulting plasmid, pET-MTF1, was overexpressed in E. coli, and the fusion protein in inclusion bodies was solubilized with 6 M urea and purified in one step by NiH -nitrilotriacetic acid agarose chromatography. The recombinant specificity factor (44 kDa) purified in the presence of urea was refolded by dialysis against buffers containing decreasing concentrations of urea. Four mg of the renatured specificity factor were obtained from one gram of cells. The renatured specificity factor in the presence of yeast core polymerase showed promoter selective activity comparable to that purified from yeast in an in vitro selective transcription assay. The Saccharomyces cereVlszae mitochondrial genome is transcribed by a nuclea r-encoded mitochondrial RNA polymerase. This enzyme is composed of two subunits; a co re polymerase and a specificity factor, which are sufficient for selective initiation at the nonanucleotide mitochondrial promoter, 5'-ATATAAGTA-3', (Osinga et al., 1982; Winkley et al. , 1985; Biswas et al., 1987). The core polymerase is a 145-kDa protein encoded by the RP041 gene (Kelly et al., 1986; M aster et al., 1987). The sequence of the RP041 gene is simila r to those of the RNA polymerases of bacteriophages T3 and TI, but not to th at of Escherichia coli RNA. polymerase (M aster et al., 1987; McAllister and Raskin, 1993). The specifi city factor encoded by the MTFl gene is a 43-kDa polypeptide (Lisowsky a nd Michaelis, 1988; l ang a nd laehning, 1991) which co nfers promoter selectivity on the co re polymerase to initi ate transcription only at mitochondria l promoters. The Mtflp sha res regions of amino acid similar to the eubacterial sigma factors (la ng and laehning, 1991) and recycles like sigma factors to mediate the core polymerase function (Mangus et al., 1994). * To whom correspondence should be addressed. Previously, recombinant form s of MtD p fusion protein were pa rtially purified from E. coli and proven to be able to synthesize correct transcript in a runoff tra nscription (Xu and Clayton, 1992; Mangus el at., 1994). However, the protocols described by the above authors for isolation of recombinant Mtn p are arduous, requiring many conventional column chromatogaphic steps and yielding low amounts of protein because of its toxicity to bacteria. To overcome these problems, we have constructed a new expression plasmid coding for the yeast specificitY fa ctor and purified it to near homogeneity in a single step. As with ma ny proteins overexpressed in E. coli, mitochondrial RNA polymerase specificity factor was found to be insoluble. In this report we describe one-step purification and refolding procedures fo r this protein. The renaturation process of the recombina nt Mtflp restored its selective transcriptional activity in vitro on the mitochondrial DNA template in the presence of yeast core polymerase. T he abbreviations used are: IPTG, i sopro py l-~ -t hi oga l acto pyranoside; NTA, nitrilotri acetic acid; PAGE, polyacrylamide gel electrophoresis: PMSF, phenylmethylsulfonyl flu oride: SDS, sodiu m dodecyl sulfate. © 1995 The Korea n Society for Molecul ar Biology 218 Recombinant Mitochondrial RNA Polymerase Specificity Factor Materials and Methods Chemicals and enzymes The electrophoresis reagents were from Bio-Rad. Urea (electrophoresis grade), isopropyl-~-D-thiogalac topyranoside (IPTG), imidazole, goat anti-rabbit antibody conjugated with horseradish peroxidase, color reagent (4-chloro-l-naphtol) and NiH -nitrilotriacetic acid (NTA) agarose were from Sigma. Deoxy- and ribonucleotide triphosphates were from Phamacia LKB Biotechnology. [a- 32 pJUTP was from Amersham Corp. Restriction enzymes, Klenow fragment and bacteriophage T4 DNA ligase were from New England Biolabs. Sequenase was from the United States Biochemical Corp. The pETlSb expression vector and E. coli strain BL21(DE3) were from Novagen. The oligonucleotide primers were synthesized in the Institute for Molecular and Cellul ar Biology, Indiana University. Construction of the expression plasmid pET-MIFl The 1040-bp fragment containing the entire coding sequence of MTFl was excised from plasmid pJJ525 (Mangus et a/., 1994) by EcoRI digestion and subcloned with anti-sense forming orientation into Bluescript SK( +) plasmid vector to give pJHl44. Since there is no convenient restriction site for subcloning the MTFl into the pET1Sb expression vector, oligonucleotidedirected mutagenesis reactions (Kunkel, 1989) were carried out to introduce an Ndel recognition site at the protein initiation codon of the MTFl in pJHI44 (resulting plasmid called pJHlS9). With pJH1S9 as the template, site-directed mutagenesis was also used to destroy both internal NdeI and BamHI sites without changing amino acid codons (called pJH17S). Briefly, an overnight culture of CJ236 cells transformed with pJHl44 was diluted 1 : SO into SO ml of YT containing both SO J..Iglml of ampicillin a nd 20 J..Iglml of chloramphenicol. After growth at 37 °C for 30 min, helper phage R408 was added to 2 X 109 plaque-forming units/ ml and cultures were grown with vigorous shaking for 12 h. Uracil-containing single-stranded DNA was isolated from the clarified supernatant using standard methods (Sambrook et al., 1989) and used as a template in mutagenesis reactions: O.2S J..Ig of NdeI oligonucleotide S' -CCTGATATCGAACATATGTCTGTTCCA-3' encoding the NdeI site, the initiation Met and flanking sequences, and 0.1 J..Ig of single-stranded DNA were denatured at 70 t for S min and allowed to anneal slowly to room temperature. The strand synthesis and ligation were performed at the same tube in IX synthesis buffer (20 ruM Tris-HCI, pH 7.5, 10 mM MgCh, SO mM NaCl, 1 mM dithiothreitol, 0.5 mM ATP and 0.5 mM deoxyribonucleotide triphosphates) with S units of Sequenase and 1 unit of T4 DNA ligase at 20 t for 1 h. Transformation of E. coli NMS22 cells with ligation products gave a number of transformants. Plasmids were isolated from eight transformants and examined for a new NdeI site on Mol. Cells the agarose gel electrophoresis. The resulting plasmid, pJH1S9 was transformed into E. coli CJ236 cell s, and then uracil-containing single stranded DNA were isolated and used as a template for constructing pJH17S which lost both internal BamHI and NdeI sites by the same mutagenesis reaction as described for constructing pJH1S9. The oligonucleotide MamHI (5' AGAAGAATGGGACCCCATTTT-3') corresponds to bases 663-683 of MTFl with a single mismatch at base 67S. The oligonucleotide MVdeI (S'-AAAAGAGATCCTTATGACTGGTCA-3') corresponds to bases 29S-318 of MTFl and contains a single mismatch at base 306. All constructs were sequenced by the dideoxy chain termination method to confirm nucleotide changes. The lOS7-bp NdeI-BamHI fragment of pJHl7S was subcloned into pETlSb digested with the same two enzymes to give the expression plasmid pET-MTFl. This plasmid expresses Mtfl p with 6 N-terminal histidines. Overexpression in E. coli E. coli strain BL21(DE3) was transformed with the plasmid pET-MTFI. Transformants were selected on an LB-agar plate containing SO J..Ig/ml of ampicilli n. For monitoring and induction of gene expression, an overnight culture was prepared from a single colony, and I ml of an overnight culture was used to inoculate SO ml LB medium containing ampicillin. The cell s were grown to ~ of 0.6 and IPTG was added to a final concentration of 1 mM. Aliquots of 500 J..1l of the cu lture were harvested by centrifugation at 1, 2 and 3 h after the initiation of induction. The cell pellets obtained were resuspended in SO J..1l of Laemmli SDS-sample buffer (65 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol and 3% 2-mercaptoethanol), boiled for 3 min, and microcentrifuged for 2 min. The supernatants were resolved electrophoretically in a 10% SDSpolyacrylamide gel. For checking the solubility of the produced protein and its subcellular localization, cells from SO-ml culture were sonicated in S ml of extraction buffer [20 mM Tris-HCI, pH 7.9, 1 mM EDTA, S% glycerol, 10 mM MgCh, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM ~-mercaptoethanoI J and centrifuged. The supernatant and pellet were a nalyzed by SDS-PAGE. Purification of recombinant Miflp by Nickel chelation chromatography For the preparative purification of Mtflp, I ml of an overnight culture was used to inoculate SOO m1 of LB medium containing 50 J..Iglrnl ampicillin. Th e cells were induced with IPTG for 3 h as described above and harvested by centrifugation at 5,000 X g at 4 t for 5 min. Cell s were resuspended in 20 ml of extraction buffer, lysed by three cycles of sonication for 1 min each a nd centrifuged at 12,000 X g and 4 t for 10 min. The supernatant was decanted and the insoluble material was resuspended in 20 ml of binding buffer (20 mM Tris-HC1, pH 7.9, 5% glycerol, S Vol. 5 (1995) Eun Ah Lee mM imidazole, 500 mM KCI, 1 mM PM SF a nd 6 M urea) to disperse the inclusion bodies and clarified by centrifugation at 39,000 X g a nd room temperatu~e for 20 min . The supernata nt was loaded onto a S-ml NiH -NTA chelation column equilibrated with binding buffer (Ja nknecht el al., 1991). Th e column was washed with 4 column volumes of binding buffer, followed by S column volumes of washing buffer (20 mM TrisHCI, pH 7.9, S% glycerol, 20 mM imidazole, SOO mM KCI, 1 mM PMSF a nd 6 M urea). Th e protein was eluted with 20 ml of elution buffer (20 mM Tris-HCl, pH 7.9, S% glycerol, I M imidazo le, SOO mM KC l, I mM PMSF a nd 6 M urea) a nd a nalyzed for purity by SOS-PAGE. Antibody production Po lyclonal antibodies were generated in rabbits. NTA column fractions containing recombinant Mtflp were fractionated on 10% SOS- PAGE, a nd visualized by staining with Coomassie blue R-2S0. Bands containing approximately ISO J.lg of the respective antigen were excised from the gel, crushed between plates, emulsified with complete Freu nd's adj uvant a nd injected into white rabbits. The rabbi ts were boosted at least three times with 100 J.lg o f antigen in incomplete Freund's before obtain ing serum (Harlow and Lane, 1988). SDS-PAGE and Western blots Protein concentrations were determined by the method of Bradfo rd (1976) using bovine serum albumin as a sta nda rd. SDS-PAGE was ca rried out as previously described by Laemmli (1970). P rotein bands were visualized by staining with Coomassie-blue R-2S0 or with silver (Wray et al., 1981). For Western blot a nalysis, the electrophoresed protein s were blotted onto lmmobilin-P (Millipore) membranes, and hybridized with 1/200 diluted primary a ntibody a nd ho rseradish peroxidase-conjugated secondary a ntibody using conditions previously described by Harlow and Lane (1988). Ba nds were visualized by color with 4-chloroI-naphtol. Non-fat dry milk was used in the blocking steps and fo r a ntibody dilution. Renaturation of purified recombinant Miflp The ('('Iumn fractions having a pure form of recombinant Mtfl p were pooled and dialyzed sequentially for renaturation. The samples were first dialyzed against dialysis buffer (20 mM Tris-HC1, pH 7.9, S% glycerol, 0.1 mM dithiothreito l, SOO mM KCI and 1 mM PMSF) co ntaining 4 M urea at 4 °c for 8 h and then sequentially dialyzed in the sa me buffer with decreasing urea concentration. The final buffer did not contain urea. These sa mples were fro zen in liquid N 2 a nd stored at - 70 °C. In vitro transcription Run-off transcription assays were carried ou t on promoter co ntaining ONA (Sc48 I 6) for the holo- el al. 219 en zym e as previously described by Ja ng and Jae hning (1991). To reconstitute holoenzyme activity, renatured recombinant Mtflp was mixed with 300 ng of parti ally purified core polymerase (SP60). Transcripts were a nalyzed o n 7 M urea-lO% polyacrylamide gel. Results and Discussion Construction of expression plasmid pET-MIFf Porath et al. (197S) have shown th at histidine, tryptophan, a nd cystein are liga i1ds for the immobilized tra nsition metal io ns such as cobalt, nickel, zinc a nd copper. A recent work indicated th at histidine residues o n protein surface are the predomina nt electron donor groups (Hemdan et al., 1989). Thu s th e pETlSb vector developed by Studier et al. (1990) was used for the co nstruction of a plasmid expressing Mtflp with 6 histidines at its N-termin al end . The construction of the expression' pl asmid pETMTFI is summa rized in Figu re I. The plasmid pJH 144 was generated by subcloning th e entire coding region o f MTFl into the EcoRI site of the pBluescript SK( +) vector. Since the MTFl gene in plasmid pJH 144 does not contain convenient restriction sites for ubcloning into the pETISb vector, oligo nucleotidedirected mutagenesis reaction s were ca rried out not only to create a new Ndel site at the transl ation initiation codon of the MTFl , but also to destroy the internal BamHI a nd NdeI recognitio n sites of th e gene without changing amino acid codo ns as described in M ateri als and Methods. The plasmid pJH17S generated from pJHI44 by two steps of oligonucleotide-directed mutagenesis was digested with Ndel and BamHI restriction en zymes. The lOS7-bp NdeI-Bam HI fragment o f pJH17S was subcloned into the same sites o f the pETlSb to give expression plasmid pET::MTFl. H ence, as shown in Figu re I, the expression plas mid pET-MTFI contains a T7 promoter element, a ribosomal binding site, six histidines followed by MTFl, the gene for p-lactamase a nd the replication origin of pl asmid pBR322. Overexpression and purification of Miff p in E. coli E. coli strain BL21(DE3) cells contai ning plasmid pET-MTFI were grown in LB medium at 37 t for th e a nalysis of the express ion kinetics and the subcel lua r localization and solubility o f the M tfl p. Figure 2A shows a Coomassie Blue-stai ned polyacrylamide gel of the time course of expression of total proteins in E. coli BL21(OE3). There was no expressio n of the MTFl gene in the absence of IPTG. After th e additio n of IPTG, a band corresponding to th e predicted size of the yeast mitochondrial RNA po lymerase specificity facto r accumul ated, reaching a maxi mum level in 3 h (Fig. 2A, lanes 2-4). Further inductio n o f cells (up to 8 h) did not increase the accumul ation of Mtnp (data not shown). The 3 h-induced E. coli cell s expressing the MIFl gene were disrupted by sonication. Soluble a nd insoluble fractio ns were sepa rated by centri- Mol. Cells Recombinant Mitochondrial RNA Polymerase Specificity Factor 220 1. 2. 3. 4. Helper phage R408 Oligonucleotide NdeI Sequenase T4 DNA ligase BamBI 1.Helper phage R408 2.0ligonucleotides ABamHI and ANdel 3.Sequenase 4.T4 DNA ligase ",,_Md. I I ~ B.m.H lac I \' pl!:T1Sb f1 orl. 1. NdeI + BamHI 1. NdeI + BamHI 2. Open Vector 2. Gel purify NdeI/BamHI fragment T4 DNA ligase Figure 1. Construction of expression plasmid pET-MTFI. To introduce an NdeI recogrutJon site at the initiation codon of the MTFJ gene, a phosphorylated oligonucleotide containing a NdeI restriction site was annealed with single-stranded DNA from pJHl44 and mutagenesis reactions were carried out as previously described by Kunkel et at. (1989)_ The internal BamHI and NdeI restriction sites on the MIFl gene were destroyed by oligonucleotide-directed mutagenesis as described in Materials and Methods. The expression plasmid pET-MTFI was constructed by inserting the NdeI-BamHI fragment from pJH175 into pETl5b which contains a 17 promoter, the ribosomal binding site, 6 histidines and multiple cloning sites as indicated. Vol. 5 (1995) A. Eun Ah Lee et at. 221 A. kDa Ul 1 IP B W kDa 116 116 97 97 66 - -- - -- 45 45 - 44 - 29 .. 44 ----~ 29 1 2 1 2 3 4 B. 3 4 S 6 kDa 116 B. kDa 97 1 16 66 4S 43 45 - 29 - .44 29 - 123 4 56 7 Figure 2. Time course of production and subcelluar localization of recombinant Mtflp. (A) Time course of induction of total proteins on a Commassie Blue-stained 10% SDSpolyacrylamide gel. Cultures of E. coli BL21(DE3) transformed with pET-MTFI were induced with I mM IPTG for o h (lane I), I h (lane 2), 2 h (lane 3) and 3 h (lane 4). (B) Partition of recombinant Mtfl p between soluble and insoluble fractions. Bacterial Iysates of E. coli cultures induced with I mM IPTG for 0 h (lane I), I h (lanes 2 and 3), 2 h (lanes 4 and 5) and 3 h (lanes 6 and 7) were separated into soluble (lanes 2, 4 and 6) and insoluble (lanes 3, 5 and 7) fractions by centrifugation and analyzed by 10% SDSPAGE. Lane I shows total Iysates of uninduced cultures. The arrow on the right side indicates the migration of the recombinant Mtflp. The positions of the molecular weight standards in kDa are shown by the bars on the left side (1 16, ~-galactosidase ; 97, phosphorylase b; 66, bovine serum albumin; 45, ovalbumin; 29, carbonic anhydrase). fugation and analyzed by SDS-PAGE as shown in Figure 2B. The recombinant Mtflp is present mostly in an insoluble form and migrated at approximately 44 kDa. It is I kDa larger than the native yeast pro- 1 2 3 4 5 Figure 3. SDS-PAGE and Western blot analysis of fractions collected during purification of Mtfl p by the Ni2+ -NTA column. (A) A silver-stained 10"10 SDS-polyacrylamide gel of recombinant Mtflp purified from E. coli BL21(DE3) transformed with pET-MTF1. Lane I, uninduced total bacterial lysate (Iabled UI); lane 2, total insoluble extracts of bacteria lysate after 3 h of IPTG induction (lab led I); lane 3, samples of the input (Iabled IP); lane 4, 20 mM imidazole washing fraction (labled W); lanes 5 and 6, 1 M imidazole eluate (Iabled E). The sample in lane I corresponds to 40 IJ.l of culture. Lane 2, inclusion bodies from a 500-ml culture were solubilized in 20 ml of 6 M urea and 40 IJ.l were loaded on the gel. Amounts of I M imidazole eluates loaded in lanes 5 and 6 were 1.5 /lg and 200 ng, respectively. An arrow head on the right side indicates the purified recombinant Mtflp. Molecular weight standards indicated on the left side are the same as described in Figure 2. (8) Western blot probed with polyclonal antibodies raised against the recombinant Mtflp. Lane 1, 30 IJ.l of a partially purified specificity factor from yeast; lanes 2 and 3, 0.8 /lg of the recombinant Mtflp; lanes 4 and 5, 2 /lg of the recombinant Mtflp. An arrow head on the right side indicates the purified recombinant Mtflp, and an arrow head on the left side indicates the native yeast Mtflp. 222 Mol. Cells Recombinant Mitochondrial RNA Polymqase: Specificity Factor teins, because it additio nally contains 6 histidines a nd thro mbin recognitio n a mino acid residues. Even though it was repo rted th at inductio n of cells at a low temperature (30 °C) helps the solubility of recombina n t p roteins (Schein a nd Noteborn, 1989), we did not notice the difference in solubility of the Mtfl p a t a low temperature incubation (data not sh own). Fo r purification of the recombina nt mitocho ndrial RNA polyme rase specificity factor a t the prepa rative scale, E. coli BL21(OE3) cells containing pET-MTFl were grown in 500 ml of LB medium at 37 °C a nd were disrupted by three cycles of sonication. Inclusion bodies coll ected by centrifugation a t 12,000 X g for 10 m in were solubilized in 6 M urea, clarified a nd loaded o n a NiH -NTA agarose column. After washing with th e binding buffer a nd 20 mM imidazole in which some Mtflp was eluted, the remainder of the Mtfl p was eluted with I M imidazole buffer. As shown in F igure 3A, the recombinant Mtflp conta ining six histidines was purified in o ne step a nd migrated as a single ba nd of 44 kDa. The recombina nt Mtflp in la ne 5 of Figure 3A was >95% pure as estim ated fro m the siver staining of the protein gel. Approxi ma tely 6 mg of purified Mtflp was recovered from o ne gra m of wet cells. It is generally believed that E. coli does not possess enzymes fo r ca talyzing posttra nsla tional modificatio n such as phospho ryl atio n, glycosyla tio n, amidatio n a nd acetyl ation. At least glycosyla tio n a nd phosphorylation, which are co mmo n modifica tio ns of tra n scriptio n facto rs in euca ryo tic cells (Jackso n and Tjia n, 1988; Sadows ki et al., 1991 ), do no t occur to a ny great extent in E. coli (Ha rris, 1983). Co nsidering the size of the recombinant Mtfl p on SOS-PAG E, post-tra nslational modificatio n does no t a ppea r to be requi red for the function of Mtflp in yeast. Th e pu ri fied Mtfl p was used as a n a ntigen to produce rab bit a nti-mitocho ndrial RNA polymerase specificity facto r po lyclonal a ntibodies. As shown in Figure 3B of the W estern blo t, a ntibodies reacted solely with a 44-kOa recombina nt Mtflp from E. coli, whereas, the sa me a ntibodies we re directed exclusively against a 43-kDa polypeptide among pa rti ally purified yeast pro teins. The I kDa increase in the size of recombina nt Mtflp co mpa red with the n ative yeast Mtfl p resulted from the presence o f six histidines a nd thro m b in recognitio n a mino acid residues a t its N-termin al e nd. Thus, the results indica te th at the recombina nt Mtfl p is the mitochondrial RNA polymerase specificity facto r overexpressed in E. coli. . f; {FaCtions + - - - - - - - Core(spfll) - + - + + + + + rM til p IDenatured Renatured - - - - + + +++ - - ++ + - + , - - .. 260 I 2 3 4 567 nt 8 Figure 4. Refolded recombinant Mtflp is functional in in vitro transcription assay. Recombinant forms of Mtn p fro m E. coli were purified in the presence of 6 M urea and refolded as described in Materials and Methods. Purified recombinant Mtflp (denatured and renatured) was assayed individually and in combination with core polymerase from yeast for selective run-off transcription . activity using Sc4816 template. Core polymerase (SP60) was partially purified from yeast on D EAE/Phospho-cellulose and Sephacryl-300 columns (l ang and l aehning, 1991). Holo forms of yeast mitochondrial RNA polymerase (P fractions) were purified by DEAE/Phospho-cellulose columns (lang and l aehning, 199 1). The arrow indicates the position of the 260-nucleotide run-off transcript. Lane I, P fractions only (5 /-lg); lane 2, core only (300 ng); lane 3, refolded Mtnp only (1 50 ng); lanes 4, 6 and 8, core and refolded Mtfl p; lanes 5 and 7, core and denatured Mtflp. The equal amount of core polymerase used in lane 2 was also employed in reconstitution assay, and amounts of refolded Mtflp used in this assay were 150 ng (lane 4), 75 ng (lane 6) and 20 ng (lane 6). Amounts of denatured Mtflp in lanes 5 and 7 were 500 ng and I iJg, respectively. Transcripts were analyzed on 7 M urea-lO% polyacrylamide gel. The M tflp purified o n the NiH -NTA column was in a den atured fo rm without activity. Thi s recombina nt protein was rena tured by three dia lysis ch a nges by th e a mount of soluble fractions. Inc reased os motic pressure at higher ratios may cause urea to be removed too rapidly, forcing prema ture refolding into a n insoluble form . U sing these conditions, we recovered 4 mg of the renatured Mtfl p from 6 mg of the column-purified den atured Mtfl , resulting in a 50-fo ld increase in yield compa red with tha t previously de- at 4 t as described in Materials and Methods. The scribed by Manguset al.(1994). maximum re natura tio n yield was o btai ned when the ratio o f d ialysis bu ffe r volume to sa mple volume was 50 : I, wi th higher ra tios giving poorer yields as judged In vitro transcription with the renatured Miflp The recombina nt fo rms of Mtfl p (denatured a nd Refolding of recombinant Miff p Vol. 5 (1995) Eun Ah Lee et al. renatured) were assayed individually or constitutively with core polymerase from yeast by in vitro transcription reactions (Fig. 4) as described in Materials and Methods. A transcript of 260 nucleotides was detected in the control reaction (lane 1) which was carried out with the phosphocellulose column fractions of yeast mitochondrial RNA polymerase as previously described in Jang and Jaehning (1991). These fractions contained core polymerase and specificity factors which were required for the selective transcription on mitochondrial promoter-containing DNA template. The core polymerase alone prepared from yeast can not selectively initiate transcription (lane 2) and neither can the recombinant forms of Mtflp themselves (lane 3). When the renatured Mtfl p was mixed with core polymerase, transcription was reconstituted (lanes 4, 6 and 8). However, selective transcription did not occur in the reactions with core polymerase and denatured forms of Mtflp (lanes 5 and 7). These results indicate that the process of refolding is required and sufficient to restore the activity of the denatured Mtflp. It has previously been demonstrated that core polymerase and specificity factor renatured from gel slices could reconstitute selective transcription (Schinkel et ai., 1988). The His-tag on recombinant Mtflp can be removed by thrombin treatment. However, the selective transcription shown in Figure 4 was performed without removing the His-tag, indicating that the fusion tag has little effect on the activity of Mtflp. Hence, the recombinant Mtflp can be used directly for the reconstitution assay of mitochondrial RNA polymerase. The approach described here is a rapid and efficient way to produce the mitochondrial RNA polymerase specificity factor. The purified M tfl p is to be used for studying the transcriptional initiation mechanism in yeast mitochondria and will facilitate the understanding of mitochondrial RNA processing, tum-over rate and coordination of nuclear and mitochondria interaction. Acknowledgment This paper was supported by a grant from the Genetic Engineering Research Fund (1993) of the Ministry of Education to S. H. Jang. References Biswas, T. K , Ticho, B., and Getz, G. S. (1987) J Bioi. Chern. 223 262, l3690-136%. Bradford, M. M. (1976) Anal. Biochern. 72, 248-254. Harlow, E , and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Harris, T. 1. R. (1983) Genet. Eng. 4, l30-131. Hemdan, S. 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