(Received for publication, February 6, 1996; and in revised form, March 13, 1996)
From the
Transcription of Xenopus laevis mitochondrial DNA requires mtRNA polymerase and a dissociable factor, xl-mtTFB, that is distinct from the HMG-box factor known as mtTFA. This paper presents the purification of mtTFB and characterizes its DNA binding properties. xl-mtTFB activity copurifies with a 40-kDa polypeptide on silver-stained protein gels. Activity can be recovered following elution of this 40-kDa polypeptide from an SDS-polyacrylamide gel. xl-mtTFB is capable of binding to DNA, but this binding is relatively nonspecific and is easily competed by heterologous DNA.
RNA polymerases typically require additional factors for
recognition of promoter sequences and for initiation of transcription.
This is the case for mtRNA polymerase, which requires at least one
dissociable factor in each system that has been studied to date. Work
from our laboratory has recently provided the first evidence that two
factors are involved in mitochondrial transcription initiation in a
higher eukaryote, Xenopus laevis (Antoshechkin and Bogenhagen,
1995). One of these, which we have designated mtTFB, is required for
basal transcription initiation from core promoters immediately
surrounding the transcription start site. The second factor is the Xenopus homolog of human mtTFA, which is well-established as
an important part of the transcription machinery in human mitochondria
(Fisher et al.(1987), Parisi and Clayton (1991), and reviewed
in Shadel and Clayton(1993)). mtTFA has been characterized as an
abundant HMG box protein that binds upstream from transcription
initiation sites in both human and Xenopus mtDNA. A related
protein has also been observed in yeast mitochondria. sc-mtTFA was
originally characterized as ABF2, a protein purified from whole cell
extracts based on its ability to bind sequences contained in origins of
replication of yeast nuclear DNA (Diffley and Stillman, 1991). Although
sc-mtTFA also has two closely spaced HMG boxes, it does not
significantly stimulate transcription of yeast mtDNA in vitro (Xu and Clayton, 1992). This discrepancy appears to be due mainly
to the fact that sc-mtTFA lacks a C-terminal sequence conserved in xl-
and h-mtTFA that is required for transcription stimulation activity
(Dairaghi et al., 1995). ()All three of the
characterized mtTFA proteins bind DNA with relaxed sequence specificity
and wrap DNA in a fashion that may assist in packaging the mtDNA within
the organelle (Fisher et al., 1992; Diffley and Stillman,
1992; Antoshechkin and Bogenhagen, 1995).
Basal mitochondrial
promoters in both Xenopus and yeast consist of short AT-rich
sequences surrounding the start site for transcription (Biswas et
al., 1987; Bogenhagen and Romanelli, 1988). This similar
organization invites a comparison of the properties of yeast and Xenopus mtTFB. The role of sc-mtTFB in initiation by yeast
mtRNA polymerase has progressed through the efforts of several
laboratories. Shinkel et al.(1987, 1988) provided the first
clear description of sc-mtTFB as a dissociable factor required by
sc-mtRNA polymerase for binding and initiation at core promoters. This
40-kDa factor was shown by Jang and Jaehning (1991) and by Xu and
Clayton(1992) to be the product of the MTF1 gene that had
previously been cloned by Lisowsky and Michaelis(1988). sc-mtTFB
exhibits some sequence homology (Jang and Jaehning, 1991; Jaehning,
1993) and functional similarity to bacterial factors. The
ability of sc-mtTFB to bind mtRNA polymerase and to be released from
transcription complexes shortly after initiation (Mangus et
al., 1994) suggests that it is a cofactor for formation of an
active mtRNA polymerase holoenzyme. However, Shadel and Clayton (1995)
have recently shown that some residues in sc-mtTFB that are conserved
in
factors are not required for biological activity.
The report by Antoshechkin and Bogenhagen(1995) introduced xl-mtTFB as a factor distinct from the HMG box factor mtTFA. The present paper provides the first detailed description of the properties of xl-mtTFB. xl-mtTFB is purified as a 40-kDa protein that is absolutely required for specific transcription initiation. xl-mtTFB can be shown to possess some rather nonspecific DNA binding ability, as has been shown for sc-mtTFB (Riemen and Michaelis, 1993). This work provides the basis for further experiments to examine the mechanism by which xl-mtTFB stimulates transcription initiation by mtRNA polymerase.
Figure 1:
Cloned DNA templates.
XLMT L945 described by Bogenhagen and Romanelli(1988) is shown.
The mtDNA insert in plasmid pBS- extends from a HindIII (H) adapter ligated to a deletion end point at mtDNA residue
945 to an EcoRI (E) site in an adapter ligated to a
deletion end point at mtDNA residue 1051. The mtDNA insert is contained
in a PvuII (P) fragment with adjacent sequences
derived from plasmid DNA (dashed lines). Transcription of this PvuII fragment from LSP1 and HSP1 gives rise to RNAs 259 and
205 nucleotides in length, respectively. For DNA binding studies, the
plasmid was 3` end-labeled at either the HindIII site or the EcoRI site and recut with RsaI (R) to
provide DNA fragments of 75 or 62 base pairs, respectively. The 75-base
pair fragment contains the bidirectional mtDNA promoter. pXLMT LSP was
constructed using oligonucleotides as described under
``Experimental Procedures'' and contains LSP1 adjacent to the
binding site for mtTFA (oval). The AflIII (A)/NdeI (N) fragment derived from this
clone supports synthesis of 286-nucleotide RNAs in run-off
transcription assays, as shown.
Figure 6: SDS-PAGE purification and renaturation of mtTFB. Panel A, 50 µl of mtTFB purified through fraction V was precipitated with acetone and subjected to electrophoresis on a 10% polyacrylamide gel containing SDS, and proteins were detected by silver staining. The marks on the right indicate the positions of zones 1-11 excised from the adjacent gel lane, which contained twice as much mtTFB protein. Panel B, standard transcription reactions using DNA template XLMT-LSP (Fig. 1) were performed using 2 µl of mtTFB prepared by SDS-PAGE as in panel A and renatured as described under ``Experimental Procedures'' (lanes 1-11) or using 2 µl of mtTFB purified through fraction V as a positive control (lane V). An autoradiogram of a polyacrylamide-urea gel analysis of in vitro transcripts is shown.
Fractions from the Poros PH column containing factor
activity were combined and loaded onto a Sephadex 200 HiLoad (60
1.6-cm) gel filtration column equilibrated with a buffer
containing 300 mM KCl, 5% glycerol, 20 mM Tris, pH 8,
0.1 mM EDTA, 2 mM DTT, 0.02% Triton X-100, 40
µg/ml gelatin, and protease inhibitors. Transcription factor
activity eluted from this column at a volume of approximately 80 ml
(void volume, 48 ml). The active fractions were combined, diluted
2-fold with gel filtration buffer lacking KCl, and loaded on a 1-ml
heparin HiTrap column. Proteins were eluted with an 18-ml gradient of
0.1-1 M KCl in buffer containing 20 mM Tris, pH
8, 10% glycerol, 2 mM DTT, 1 mM EDTA, 0.02% Triton
X-100, and protease inhibitors. Following storage at -80 °C,
active fractions were combined, diluted to a conductivity equivalent to
60 mM NaCl in Q buffer, and loaded onto a 1-ml Mono Q column.
Proteins were eluted with a 17-ml gradient from 20 to 500 mM NaCl in buffer Q (20 mM Tris, pH 8, 1 mM EDTA,
5% glycerol, 7 mM 2-mercaptoethanol, 0.02% Triton X-100, and
protease inhibitors). 2 µl of each fraction was assayed for
specific transcription activity, while the major portion of each
fraction was mixed with volume of storage buffer for storage at
-80 °C. Following identification of the peak of transcription
stimulation activity, 600 µl of the peak fraction was concentrated
to approximately 120 µl by centrifugation in a Centricon 10 device
(Amicon) and layered on a 3.8-ml 20-50% glycerol gradient in
buffer Q containing 50 µg/ml gelatin. The gradient was spun at
58,000 rpm for 20 h in a Beckman SW60Ti rotor. Fractions were collected
from the bottom of the tube, and 4-µl samples were assayed for
transcription activity. Proteins were acetone precipitated from the
remainder of each fraction for gel analysis.
Figure 3:
Purification of mtRNA polymerase. mtRNA
polymerase purified through fraction III was loaded on an analytical
Superose 6 gel filtration column. Fractions were assayed for
nonspecific RNA polymerase activity (panel A) and for specific
transcription from the XLMTL945 PvuII template in the
presence of mtTFB (panel B) as described under
``Experimental Procedures.''
Figure 2: Scheme for purification of mtRNA polymerase and mtTFB. Gradient elution was used for all ion exchange and hydrophobic interaction columns.
mtRNA polymerase was further purified by gel filtration on a Superose 6 fast protein liquid chromatography column in the presence of 0.8 M NaCl and 0.02% Triton X-100. Nonspecific transcription activity and activity in specific transcription reactions complemented by highly purified mtTFB precisely coeluted from this column (Fig. 3, A and B). The mtRNA polymerase was further purified and concentrated by chromatography on heparin-agarose. At this stage, the preparation contains two major polypeptides, the 140-kDa mtRNA polymerase and an abundant 116-kDa protein that does not coelute with polymerase activity (Antoshechkin and Bogenhagen, 1995). DNA binding and RNA polymerase activity also coeluted from heparin-Sepharose (data not shown). A DNA binding activity detected by electrophoretic mobility shift assays also copurified with mtRNA polymerase through gel filtration and heparin-Sepharose chromatography (data not shown).
In order to achieve additional purification and to reduce the salt concentration, the fraction III mtTFB activity was applied to a gel filtration column. Fig. 4shows that mtTFB eluted from the gel filtration column slightly later than a marker of ovalbumin. The native molecular weight for mtTFB was determined to be approximately 37-40 kDa, assuming a globular conformation. This gel filtration behavior is consistent with glycerol gradient sedimentation results presented previously (Bogenhagen and Insdorf, 1988) and below. From this point on in the purification scheme, the activity of mtTFB was found to be linear with time over the course of a standard 20-min reaction and with input protein.
Figure 4: Native sizing of mtTFB. mtTFB purified through fraction III (see Fig. 2) was applied to a preparative gel filtration column, and eluted fractions were assayed for transcription stimulation activity as described under ``Experimental Procedures.'' Arrows indicate the elution positions of calibration proteins aldolase (A, 132 kDa), bovine serum albumin (BSA, 66 kDa), ovalbumin (Ov, 43 kDa), and cytochrome c (C, 12 kDa).
Due to the low yield of total protein at step IV, two standard preparations of mtTFB, each derived from the equivalent of 80 mature frog ovaries, were pooled for subsequent column chromatography on heparin-Sepharose and Mono Q resins. The results are summmarized in Table 1. mtTFB eluted as a single peak of activity from both columns. Analysis of the proteins contained in the active fractions from both columns showed that a 40-kDa protein co-purified with factor activity through both steps (data not shown). Proteins in the Mono Q fraction were concentrated and applied to a 20-50% glycerol gradient. Samples of fractions obtained from the glycerol gradient were assayed for transcription stimulation activity, and the proteins in the remainder of each fraction were precipitated with acetone prior to analysis by SDS-PAGE. Silver staining of the proteins showed that the 40-kDa protein cosedimented with transcription stimulation activity (Fig. 5), suggesting that this is the polypeptide containing mtTFB activity. The experiment shown in Fig. 6confirmed that mtTFB activity can be recovered from a 40-kDa polypeptide eluted from an SDS-polyacrylamide gel. By considering the relative volumes of mtTFB loaded on the protein gel and in the final sample renatured from gel fraction 5, it is possible to calculate that the overall recovery of activity was approximately 20% in this experiment.
Figure 5:
Sedimentation of mtTFB. One-half of the
Mono Q gradient fraction containing the peak of mtTFB was concentrated
and centrifuged in a glycerol gradient as described under
``Experimental Procedures.'' Fractions of 200 µl were
collected from the bottom of the tube. Panel A, 4 µl of
the indicated glycerol gradient fraction or of Mono Q fraction 5 before (L) and after concentration (L) were
assayed for the ability to complement mtRNA polymerase in specific
transcription. An autoradiogram is shown of a polyacrylamide-urea gel
electrophoresis analysis of the run-off transcription products from the
XLMT
L945 PvuII template. Panel B, proteins in
150-µl samples of the indicated glycerol gradient fractions were
concentrated by precipitation with acetone and analyzed on an
8-15% gradient SDS-polyacrylamide gel. The gel was stained first
with Coomassie Blue and then stained with silver. The positions of
molecular mass markers (kDa) run in an adjacent lane are indicated on
the left. The arrowhead on the right shows
the position of the 40-kDa polypeptide that cosedimented with mtTFB
activity.
Figure 7: Mobility shift analysis of the interaction of mtTFB with a bidirectional mitochondrial promoter. Panel A, 4-µl samples of fractions from the Mono Q column (step VI; peak transcription activity in fractions 4, 5, and 6) were assayed for the ability to bind a 75-base pair DNA fragment containing a single bidirectional promoter in an electrophoretic mobility shift assay performed as described under ``Experimental Procedures.'' The binding reactions for the two right lanes labeled 4+ and 6+ contained 30 ng of poly(dI-dC) as a nonspecific binding competitor. Panel B, to test the DNA sequence specificity of DNA binding by mtTFB, 4 µl of Mono Q fraction 6 was incubated with the labeled probe in reactions shown in lanes 2-10. Binding reactions shown in lanes 3-6 included 5, 20, 80, or 160 ng of a 153-base pair mtDNA fragment containing both promoter regions 1 and 2. Binding reactions shown in lanes 7-10 included the same concentrations of a 187-base pair fragment containing the Xenopus borealis somatic 5 S RNA gene as a nonspecific competitor.
This paper presents the purification of xl-mtTFB and an analysis of its contribution to transcription initiation by mtRNA polymerase. Antoshechkin and Bogenhagen(1995) showed that xl-mtTFB is required for transcription and cannot be replaced by xl-mtTFA. However, the presence of a low concentration of mtTFA can stimulate transcription from certain templates, while high concentrations can repress transcription. Therefore, the transcription templates used in this study to follow mtTFB purification (Fig. 1) were selected to lack mtTFA binding sites to permit study of basal transcription without the influence of xl-mtTFA. xl-mtTFB was recovered as a single peak of transcription stimulation activity on each of several columns and copurified with a 40-kDa protein (Fig. 5). The protein eluted from the 40-kDa region of an SDS-polyacrylamide protein gel recovered factor activity following renaturation (Fig. 6). At this time, there is no evidence for any additional dissociable factors required for transcription of Xenopus mtDNA other than mtTFA and mtTFB.
Both xl-mtTFB (Fig. 7) and xl-mtRNA polymerase (data not shown) possess some DNA binding ability, although the specificity of interaction of the separate proteins is rather limited. A number of electrophoretic mobility shift assays were performed with combinations of mtRNA polymerase and mtTFB in attempts to observe a tertiary complex containing both proteins bound to promoter sequences (data not shown). To date, these experiments have not revealed any evidence of supershifting of the mtRNA polymerase-DNA complex upon addition of mtTFB. Indeed, no convincing evidence has been obtained to show that xl-mtTFB actually binds along with mtRNA polymerase at promoter sequences.
The results presented here further extend the parallel between the cis- and trans-acting elements involved in transcription of X. laevis and S. cerevisiae mtDNA. In both organisms, transcription initiation requires a core consensus sequence surrounding the start site (Biswas et al., 1987; Bogenhagen and Romanelli, 1988). The X. laevis mitochondrial RNA polymerase is similar in size to the cloned yeast mtRNA polymerase (Bogenhagen and Insdorf, 1988; Kelley et al., 1986; Masters et al., 1987). In both systems, the HMG-box factor mtTFA cannot substitute for mtTFB. Although it seems likely that further experiments will confirm a close functional and structural relationship between xl-mtTFB and sc-mtTFB, this is still a matter of conjecture. The data accumulated on the role of sc-mtTFB summarized in the Introduction generally support a model in which this protein binds to mtRNA polymerase to form a holoenzyme that recognizes promoter sequences (Biswas, 1992; Mangus et al., 1994). A definitive comparison of xl-mtTFB and sc-mtTFB will require cloning and sequencing of the gene encoding xl-mtTFB. Cloning the gene for xl-mtTFB will also permit overexpression of the protein in order to facilitate additional studies of its role in promoter binding, open complex formation, and transcription initiation.