©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of mtTFB and mtRNA Polymerase at Core Promoters for Transcription of Xenopus laevis mtDNA (*)

(Received for publication, February 6, 1996; and in revised form, March 13, 1996)

Daniel F. Bogenhagen (§)

From the Department of Pharmacological Sciences, University at Stony Brook, Stony Brook, New York 11794-8651

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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). (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Xenopus laevis females were obtained from Xenopus I (Ann Arbor, MI). DEAE Sephacel and S-Sepharose resins, heparin-Sepharose HiTrap, Mono Q (HR5/5), and Sephadex 200 HiLoad (60 times 1.6-cm) columns as well as ultrapure nucleotides were obtained from Pharmacia Biotech Inc. The Poros Phenyl PH (7.5 times 50-mm) column was obtained from Perceptive Biosystems (Cambridge, MA). Protease inhibitors leupeptin, aprotinin, and E-64 were obtained from Boehringer Mannheim. Nitrocellulose membranes were obtained from Millipore Corp. (Bedford, MA). Benzamidine HCl, dithiothreitol (DTT), (^2)bovine serum albumin, and pepstatin were obtained from Sigma. Radioisotopes were obtained from ICN Radiochemicals (Irvine, CA). Restriction endonucleases were obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim. Other reagent grade chemicals were obtained from Fisher Scientific (Springfield, NJ), Boehringer Mannheim, or Sigma.

Methods

DNA Templates

Three plasmids containing mtDNA sequences are diagrammed in Fig. 1. The X. laevis mtDNA subclone XLMTDeltaL945 containing a 136-base pair HindIII/EcoRI fragment cloned in the pBS- vector (Stratagene) has been described (Bogenhagen and Romanelli, 1988). Digestion of XLMTDeltaL945 with PvuII produced a fragment of 467 base pairs containing only promoter region 1 of the mtDNA that was purified by anion exchange chromatography on a Gen-Pak FAX column and used for routine transcription complementation assays. The DNA template used in the experiment in Fig. 6was derived from a clone designated pXLMT LSP containing residues 926-987 of X. laevis mtDNA (Cairns and Bogenhagen, 1986), including promoter region 1 adjacent to a single mtTFA binding site, as a BamHI to HindIII insert in pUC9. This plasmid was generated using synthetic oligonucleotides to provide a minimal promoter fragment in which three base pair changes were introduced at residues 971, 972, and 973 to inactivate HSP1. Therefore, LSP1 is the only active promoter in this plasmid.


Figure 1: Cloned DNA templates. XLMT DeltaL945 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.



In Vitro Transcription Reactions

Run-off transcription assays were performed in 40-µl reactions containing 2-4 µl of mtRNA polymerase, 1-4 µl of mtTFB fraction, and 20 ng of a promoter-containing DNA restriction fragment. When mtRNA polymerase purified through fraction III was used, the enzyme was microdialyzed to reduce the salt concentration against a buffer containing 20% glycerol, 20 mM Tris, pH 8.4, 0.1 mM EDTA, 5 mM DTT, and protease inhibitors. When mtRNA polymerase purified through fraction V was used, microdialysis was not performed. Reactions included 25 mM Tris, pH 8, 5 mM MgCl(2), 3 mM DTT, 1 mM ATP, 100 µM CTP, 100 µM UTP, 10 µM GTP, and 5-10 µCi of [alpha-P]GTP. Following incubation at 30 °C for 20 min, reactions were terminated by thed addition of 180 µl of 0.3 M sodium acetate, 10 mM EDTA, 10 mM Tris, pH 7.6, 0.5% SDS, and extraction with phenol-chloroform. Nucleic acids were precipitated with ethanol and fractionated by polyacrylamide gel electrophoresis in the presence of 8 M urea. Autoradiography was performed using Kodak XAR-5 film. Radioactivity incorporated into the run-off transcript initiated at LSP1 was determined by direct scanning of dried gels in a Beta imager (Betascope) or PhosphorImager (Molecular Dynamics). 1 unit of mtTFB-specific transcription activity was defined as the incorporation of 100 cpm into the LSP1 transcript under standard conditions.

Purification of xl-mtTFB

Fractions eluting from S-Sepharose at slightly higher salt than mtRNA polymerase were found to stimulate specific transcription (Antoshechkin and Bogenhagen, 1995). These fractions were combined, adjusted to contain 1.2 M (NH(4))(2)SO(4), and loaded on a Poros PH column at a flow rate of 1.5 ml/min. Proteins were eluted with HIC buffer (20 mM Tris, pH 8, 1 mM EDTA, 2 mM DTT) using a gradient of (NH(4))(2)SO(4) decreasing from 1.2 to 0 M. A protease inhibitor mix used in this case and in all column buffers included 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml each of leupeptin, aprotinin, and E-64, 1 µM pepstatin A, 0.5 mM benzamidine HCl. 10-µl samples were microdialyzed for 20 min on ice against a buffer containing 25 mM Tris, pH 8.4, 5 mM DTT, 20% glycerol, and protease inhibitors to reduce the concentration of (NH(4))(2)SO(4) prior to the addition to transcription reactions. 2- or 4-µl samples of the microdialyzed samples were assayed for transcription factor activity as described above. The transcription factor activity typically eluted at approximately 1 M (NH(4))(2)SO(4). After this column and all subsequent columns, the remaining volume of each fraction was stored at -80 °C after the addition of volume of glycerol storage buffer (75% glycerol, 20 mM Tris, pH 8.0, 1 mM EDTA, 5 mM DTT, 0.02% Triton X-100, and protease inhibitors) pending the results of specific transcription assays.

Fractions from the Poros PH column containing factor activity were combined and loaded onto a Sephadex 200 HiLoad (60 times 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.

Recovery of mtTFB Activity following SDS-PAGE

The procedure for gel elution and renaturation of mtTFB was adapted from that of Hager and Burgess(1980). 100 µl of fraction V mtTFB was mixed with 6 µg of lysozyme as a carrier protein, precipitated with 5 volumes of acetone, resuspended in SDS-PAGE sample loading buffer, heated at 37 °C for 5 min, and subjected to electrophoresis on a 6-cm-tall 10% polyacrylamide minigel containing 0.1% recrystallized SDS. The lysozyme carrier, which migrated near the dye front, and the presence of 1 mM thioglycolic acid in the gel running buffer were included to react with any residual oxidizing agents remaining in the gel following polymerization. Using an adjacent lane containing prestained molecular mass markers as a guide, the gel lane was divided into 11 2-3-mm-wide slices spanning the interval from 15 to 180 kDa. Gel pieces were soaked for 10 min in 5 mM DTT and then crushed in elution buffer containing 50 mM Tris (pH 8), 20 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 0.1% SDS, 10 µg/ml bovine serum albumin, and protease inhibitors. Following incubation for 3 h on a rotator, each sample was spin-filtered through a 0.22-µm membrane (Millipore), and proteins were precipitated with acetone. The pellet was resuspended in 20 µl of 6 M guanidinium HCl in dialysis buffer (25 mM Tris, pH 8.4, 25 mM NaCl, 5 mM DTT, 20% glycerol, and protease inhibitors) and incubated at room temperature for 15 min. The sample was dialyzed for 90 min against the same buffer lacking guanidinium-HCl, and 2 µl was used in an in vitro transcription assay using the XLMT LSP AflIII-NdeI fragment as template.

Preparation of mtRNA Polymerase

The nonspecific assay for mtRNA polymerase was performed as described (Bogenhagen and Insdorf, 1988). Purification of mtRNA polymerase through the hydrophobic interaction chromatography step was as described in Antoshechkin and Bogenhagen(1995). This fraction was used for routine transcription complementation assays of mtTFB activity. The peak fractions from the Poros PH column were applied to a gel filtration column, either Superose 6 (for analytical purposes, as in Fig. 3) or Sephadex 200 HiLoad (for preparative purposes), as described for mtTFB, except that the buffer included 0.8 M NaCl in place of 0.3 M KCl. Fractions containing the peak of RNA polymerase activity were diluted to a salt concentration of 0.1 M with S column buffer lacking salt and loaded on a heparin-Sepharose HiTrap column. mtRNA polymerase was eluted with a gradient of 0.1-0.8 M KCl in S column buffer (Antoshechkin and Bogenhagen, 1995).


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 XLMTDeltaL945 PvuII template in the presence of mtTFB (panel B) as described under ``Experimental Procedures.''



Electrophoretic Mobility Shift Assays for DNA Binding

The promoter-containing DNA fragment used in electrophoretic mobility shift protein binding assays was prepared from XLMTDeltaL945 by cleavage with HindIII adjacent to the deletion end point at map residue 945, labeling with [alpha-P]dATP and avian myeloblastosis virus reverse transcriptase, and recutting with RsaI at map residue 1006. A control fragment not containing promoters was prepared from the same plasmid by 3` labeling an EcoRI end adjacent to map residue 1050 and recutting with RsaI. The resulting 75-base pair promoter-containing fragment and 62-base pair control fragment were purified by electrophoresis on a 6% polyacrylamide gel. The bidirectional LSP1/HSP1 promoter is located in the center of this 75-base pair fragment. Binding reactions contained 2.5 fmol of probe in 12-µl reactions containing 1 times transcription buffer, 0.05% Triton X-100, and, where indicated, 30 ng of poly(dI-dC) nonspecific competitor. Some reactions were performed in parallel with and without 1 mM ATP. The addition of ATP was found to have no effect on the extent of binding. Reactions were incubated at 25 °C for 10 min and loaded onto 7 times 10 times 0.1-cm minigels containing 6% acrylamide (50:1 acrylamide:bisacrylamide), 5% glycerol, 20 mM Hepes, pH 8, 0.1 mM EDTA, 2 mM thioglycolic acid, and 0.02% Triton X-100. Following electrophoresis at 70 V for approximately 50 min at room temperature the gels were dried and exposed to x-ray film.

Protein Gel Electrophoresis

Protein gels were run with the Tris/glycine buffer system (Laemmli, 1970) on either 10% polyacrylamide or 8-15% polyacrylamide. Gels were stained with Coomassie Blue, extensively destained, and stained with silver (Wray et al., 1981).


RESULTS

Purification and DNA Binding Properties of mtRNA Polymerase

Purification of mtTFB depended upon a consistent source of mtRNA polymerase activity. Although our laboratory published a scheme for purification of X. laevis mtRNA polymerase (Bogenhagen and Insdorf, 1988) additional experience with this enzyme showed that chromatography and storage of the mtRNA polymerase under low salt conditions led to a rapid loss of activity. In particular, mtRNA polymerase purified by chromatography on phenyl-Superose eluted at very low salt and was quite unstable to further handling. The mtRNA polymerase exhibited very different chromatographic behavior on an HIC column prepared by another manufacturer. The mtRNA polymerase activity used in the experiments reported here was purified on a Poros PH Phenyl column, which allowed elution of the mtRNA polymerase at 1 M (NH(4))(2)SO(4). The different flow rate properties and different phenyl ligand densities on these commercial columns may contribute to the variable behavior of the mtRNA polymerase. The revised procedure for preparation of mtRNA polymerase is diagrammed in Fig. 2.


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).

Purification of xl-mtTFB

Antoshechkin and Bogenhagen(1995) recently showed that mtTFB is efficiently separated from the HMG-box factor mtTFA by chromatography on DEAE-Sephacel and S-Sepharose. mtTFB was purified as a factor required to complement mtRNA polymerase in transcription of the XLMTDeltaL945 template diagrammed in Fig. 1following the procedure shown in Fig. 2. As described by Antoshechkin and Bogenhagen(1995), mtTFB was separated from mtRNA polymerase by chromatography on S-Sepharose. The second highly resolving step in the purification of mtTFB was chromatography on the Poros PH column. As noted above for mtRNA polymerase, this column was used as a substitute for phenyl-Superose used in the earlier preparation (Bogenhagen and Insdorf, 1988). A single peak of transcription-stimulatory activity eluted from this column. Following hydrophobic chromatography, the fractions with mtTFB activity retained less than 0.1% of the protein present in the initial mitochondrial lysate (Table 1). However, it is difficult to determine the specific activity of the transcription factor at early stages in purification. Specific transcription reactions with fractions derived from S-Sepharose generally showed a high background on autoradiographs of gel analyses of the transcription products, as previously reported (Bogenhagen and Insdorf, 1988). Control experiments showed that the peak fractions containing mtRNA polymerase and mtTFB activity also contained a Mg-stimulated protease activity that was not completely suppressed by use of a combination of protease inhibitors. (^3)Thus, it is possible that proteolysis of the factor and/or RNA polymerase occurred in the course of transcription reactions conducted in the presence of Mg with partially purified proteins. Similarly, specific transcription reactions with fractions derived from hydrophobic chromatography, step III, were difficult to quantify, either due to the presence of a high concentration of (NH(4))(2)SO(4) or to the presence of an uncharacterized inhibitor. For both the S-Sepharose and Poros PH column steps, the transcription products did not increase linearly with increasing volume of the factor fraction or with increasing time. Therefore, Table 1does not report the estimates of the recovery of activity at early stages of purification.



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(c)) 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 XLMTDeltaL945 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.



xl-mtTFB Possesses Nonspecific DNA Binding Ability

Electrophoretic mobility shift assays were performed to determine whether mtTFB binds specifically to sequences within or surrounding the minimal core consensus sequence that serves as a mitochondrial promoter in X. laevis. These binding assays employed a 75-base pair fragment containing the overlapping LSP1/HSP1 promoters diagrammed in Fig. 1. Fig. 7A shows that mtDNA binding activity was observed in Mono Q column fractions containing transcription factor activity. However, the binding was greatly reduced by the inclusion of poly(dI-dC) as a nonspecific competitor in the binding reactions. A more complete analysis of the DNA sequence specificity of binding is shown in Fig. 7B. In this experiment, various concentrations of either a specific mtDNA promoter fragment or an unrelated DNA fragment were added to binding reactions. A DNA fragment containing the Xenopus 5 S RNA gene was selected as a relatively GC-rich nonspecific competitor. These fragments competed approximately equally for binding of mtTFB.


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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM29681. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 516-444-3068; Fax: 516-444-3218; Dan{at}Pharm.Sunysb.edu.

(^1)
I. Antoshechkin, unpublished results.

(^2)
The abbreviations used are: DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; LSP, light strand promoter; HSP, heavy strand promoter.

(^3)
D. Bogenhagen, unpublished observation.


ACKNOWLEDGEMENTS

I thank Mariana Margarit and Kevin Pinz for technical assistance, Preeti Gokal Kochar for interest in the early stages of this project, and Jose Carrodeguas and Igor Antoshechkin for comments on the manuscript.


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