Download A Single-step Purification of the Saccharomyces cerevlslae

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.
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