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Previous Article | Next Article 
Molecular and Cellular Biology, August 2003, p. 5816-5824, Vol. 23, No. 16
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.16.5816-5824.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322-3050
Received 25 February 2003/ Returned for modification 1 April 2003/ Accepted 15 May 2003
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ABSTRACT |
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INTRODUCTION |
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In a series of seminal experiments, Clayton and colleagues defined two essential components required for transcription initiation in human mitochondria, an h-mtRNA polymerase activity and a DNA-binding protein that engages the LSP and HSP directly upstream of the initiation site to stimulate transcription (10, 11, 13). The ability of this protein, now known as h-mtTFA, to activate transcription is presumably linked in some manner to its ability to bend and/or contort the DNA near the initiation site (12). The sequence and spacing of the h-mtTFA binding site relative to the transcription initiation site are also critical (6, 31), suggesting that h-mtTFA must be in proximity to the incoming mtRNA polymerase during initiation. Cloning of the cDNA encoding h-mtTFA revealed that it contains a mitochondrial matrix-localization signal at its amino terminus, two high-mobility group (HMG) box DNA-binding domains that are separated by a short linker region, and a C-terminal tail that is relatively rich in basic amino acids (21). Mutagenesis and domain-swapping experiments subsequently revealed that, while the HMG boxes are required for nonspecific DNA binding properties, the C-terminal tail is absolutely required for specific DNA binding and transcriptional activation (7), indicating that h-mtTFA may make contacts with other factors involved in initiation through this domain.
Studies of Saccharomyces cerevisiae (15, 16, 27, 33) and Xenopus laevis (1, 2) model systems revealed the requirement for a mitochondrial transcription factor (mtTFB) that is distinct from mtTFA, suggesting the existence of a second mitochondrial transcription factor in vertebrates. This prediction was recently realized with the cloning of a cDNA encoding the first human homolog of yeast mtTFB (sc-mtTFB/Mtf1p), which also revealed that this transcription factor is related in primary sequence to a large family of RNA methyltransferases and binds the requisite S-adenosylmethionine (SAM) cofactor used by this class of enzymes (19). Recently, we reported that h-mtTFB1 can indeed methylate tandem adenine residues located within a conserved stem-loop in the small rRNA subunit of ribosomes, demonstrating that it is likely a dual-function protein, acting as both an rRNA methyltransferase and a transcription factor (24). Consistent with these findings is the three-dimensional structure of sc-mtTFB, which reveals striking similarity to the bacterial ErmC' rRNA methyltransferase (23). Isolation of a second human homolog of sc-mtTFB, h-mtTFB2, revealed that it is also related to rRNA methyltransferases and possesses marked transcriptional activation properties in vitro (9).
How the RNA methyltransferase structural features and/or enzymatic activity of h-mtTFB1 impinges on the function of the protein as a transcription factor is unknown. In particular, whether SAM binding and/or RNA methyltransferase activity is associated with the transcriptional activation properties of h-mtTFB1 has not been addressed. Here we report that h-mtTFB1 interacts directly with h-mtTFA and, despite the fact that this protein is also a SAM-dependent rRNA methyltransferase (24), can activate transcription independently of SAM binding and RNA methyltransferase activity. These data support a new model for transcription initiation in human mitochondria in which h-mtTFB proteins act to bridge an interaction between h-mtRNA polymerase and a promoter-bound h-mtTFA complex.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The h-mtTFB2 ORF was isolated by PCR amplification of a human fetal brain cDNA library with the primers 5'-ATGTGGATCCCAGTGGTCGGGCTTCC-3' and 5'-CTACCTATCTTCCAGGGTTTCATC-3' and cloned into the plasmid pGEMT to create the plasmid pGEMT-HB2. This plasmid was subsequently used as a template for PCR with oligonucleotides 5'-TCCCCCCGGGTCATGTGGATCCCAGTGGTCGGG-3' and 5'-ACGCGTCGACCTACCTATCTTCCAGGGTTTC-3', which introduced a SmaI site at the 5' end and a SalI site at the 3' end to facilitate cloning of the h-mtTFB2 ORF into pGEX4T. The resulting plasmid (pGST-HB2) directs expression of the ORF as a fusion protein with GST in E. coli. T4 DNA ligase and restriction enzymes were obtained from either New England Biolabs or Promega.
Purification of recombinant proteins. GST-tagged and untagged (thrombin-cleaved) h-mtTFB1 and h-mtTFB2 and the h-mtTFB1 site-directed variants were each produced in bacteria and purified by glutathione-Sepharose chromatography as described previously (19). Full-length and C-terminally truncated recombinant h-mtTFA proteins were expressed in E. coli, gel purified, and renatured as described previously (7).
Antibody production. GST-tagged h-mtTFB1 was produced in bacteria, purified by glutathione-Sepharose chromatography, and cleaved with thrombin as previously described (19). The recombinant, untagged protein was resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after which the gel was stained with 0.25 M KCl and the h-mtTFB1 protein was excised and eluted into dilution buffer (0.1% SDS, 50 mM Tris [pH 7.9], 0.1 mM EDTA, 150 mM NaCl, 5 mM dithiothreitol) as described (14). The eluted protein was sent to Rockland Immunochemicals for the production of rabbit polyclonal antibodies. Antibodies were affinity purified from the provided rabbit crude serum by protein A-Sepharose (Amersham Pharmacia Biotech, Inc.) chromatography. The protein A-purified antibody was then affinity purified on an Aminolink column (Pierce, Inc.) that had recombinant untagged h-mtTFB1 cross-linked to it as described by the manufacturer.
Immunodepletion of mitochondrial extracts and mitochondrial transcription assays.
Mitochondrial protein extracts were prepared as described previously (20) from 
3 x 109 HeLa cells grown in Joklik's modified minimal essential medium (Sigma Chemical, Inc.) plus 10% fetal calf serum. Protein A-purified h-mtTFB1 antibodies or the corresponding preimmune serum was bound to protein A-Sepharose in mitochondrial lysis buffer (MLB; 25 mM HEPES-KOH [pH 7.6], 5 mM MgCl2, 10% glycerol, 0.125% Tween 20, 0.125 M KCl, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride). Unbound antibodies were removed by sequential washes with MLB after incubation. Mitochondrial extracts were diluted 1:4 with MLB to a final volume of 200 µl and incubated with 50 µl of antibody-bound beads for 1 h at 4°C with rotation. The beads were removed by centrifugation at 2,000 x g for 2 min at room temperature. The singly depleted extract was subjected to a second depletion with a new batch of beads under conditions identical to those for the first depletion. The resulting h-mtTFB1 immunodepleted extract was used in the transcription assays and for Western blot analysis (Fig. 1).
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The conditions used for the detection of h-mtRNA polymerase by coimmunoprecipitation with h-mtTFB1 were similar to those used above, except that the soluble HeLa cell mitochondrial extracts were diluted 1:8 in MLB to a final concentration of 62.5 mM KCl and 0.06% Tween 20. The extract was then precleared by incubation with rabbit preimmune serum, which was bound to protein A-Sepharose beads (Amersham Pharmacia) at 4°C for 1 h with rotation. The extract was then centrifuged at 3,000 x g for 2 min to pellet the beads, and the supernatant was transferred to new 1.5-ml tubes. A portion of this precleared soluble mitochondrial protein extract (75 µg of total protein) was incubated with protein A-Sepharose bound to either rabbit preimmune serum or anti-h-mtTFB1 antibody at 4°C for 2 h with rotation. The preimmune serum and h-mtTFB1 antibody used at this step in the experiment were purified over a protein A-Sepharose column and then cross-linked to the protein A-Sepharose beads with dimethyl pimelimidate (Pierce) according to the manufacturer's instructions. The beads were then centrifuged at 3,000 x g for 2 min, and the supernatant was removed. The beads were then washed five times with 1 ml of MLB (with 62.5 mM KCl and 0.0625% Tween 20). Finally, an equal volume of 2x Laemmli gel loading buffer was added to the beads. The eluted proteins corresponding to the immunoprecipitate were separated by electrophoresis on an 8% polyacrylamide gel and subjected to Western analysis with an anti-h-mtRNA polymerase peptide antibody (25).
Direct protein-protein interaction assays. Recombinant wild-type or mutant GST::h-mtTFB1 fusion proteins were isolated from soluble crude E. coli extracts and bound to glutathione-Sepharose beads as described previously (19). The protein-bound beads were then incubated with full-length protein or each of the C-terminal deletion mutants of h-mtTFA in MLB with rocking at 4°C for 2 h. The samples were then washed five times with 1 ml of MLB. After washing, an equal volume of gel loading buffer was added to the beads and the eluted proteins were separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with an antibody directed against h-mtTFA. When the h-mtTFB1 mutants were tested for h-mtTFA binding (see Fig. 5C), the amounts of each h-mtTFB1 variant bound to the beads were checked by Western blotting to ensure that similar amounts of protein were analyzed.
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RESULTS |
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We reported previously that h-mtTFB1 activates transcription only in collaboration with h-mtTFA (19), suggesting to us that loss of transcription in the h-mtTFB1 immunodepletion experiment described above could be explained by coimmunoprecipitation of h-mtTFA from the extract. Indeed, Western analysis revealed that treatment with h-mtTFB1 antibody also resulted in severe depletion of h-mtTFA from the extract that was not observed in the preimmune control depletion (Fig. 1, compare lane 3 to lane 2). Thus, h-mtTFB1 and h-mtTFA appear to be present in a complex in this transcriptionally active mitochondrial extract.
h-mtTFB1 and h-mtTFB2 interact with the C-terminal activation region of h-mtTFA. Given that h-mtTFB1 is capable of forming an immunoprecipitable complex containing h-mtTFA, we next determined whether the two proteins interact directly in an in vitro protein-binding assay. Recombinant GST::h-mtTFB1 fusion protein was produced in E. coli and bound to glutathione-Sepharose beads (see Materials and Methods). When gel-purified, renatured recombinant h-mtTFA protein was incubated with the h-mtTFB1-bound beads, a significant fraction remained bound to the beads after several stringency washes (Fig. 2A).
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Antibodies to h-mtTFB1 also coimmunoprecipitate h-mtRNA polymerase. Given that h-mtTFB1 and h-mtTFA were coimmunoprecipitated from our transcriptionally active mitochondrial extract (Fig. 1), we attempted to reconstitute transcription activity in the depleted extract by adding back both recombinant h-mtTFB1 and h-mtTFA. However, we were still unable to reconstitute transcription activity in the extract in this manner (data not shown). This suggested to us that, in addition to h-mtTFA, h-mtRNA polymerase may also be coimmunoprecipitated from the extract with h-mtTFB1 antisera. Unfortunately, we were unable to confidently detect the h-mtRNA polymerase polypeptide in the immunoprecipitate from this experiment (data not shown). Therefore, to address this possibility, we prepared a new mitochondrial extract that contained amounts of h-mtRNA polymerase detectable by Western analysis (Fig. 3, lane 1) and repeated the immunoprecipitation with h-mtTFB1 antibodies under conditions better optimized for detecting h-mtRNA polymerase in the immunoprecipitate (see Materials and Methods). Under these conditions, we were able to demonstrate that h-mtRNA polymerase can be coimmunoprecipitated with h-mtTFB1 (Fig. 3, lane 2). This interaction was specific for h-mtTFB1 in that no coimmunoprecipitation of h-mtRNA polymerase was observed with the corresponding control preimmune serum (Fig. 3, lane 3). These data demonstrate that h-mtTFB1 and h-mtRNA polymerase can exist as a part of a complex in the soluble mitochondrial extract and also likely explain our inability to reconstitute transcription activity by adding back recombinant h-mtTFB1 and h-mtTFA to the original h-mtTFB1 immunodepleted extract.
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By the use of a solid-phase SAM-binding assay described previously (19), the ability of the three mutated h-mtTFB1 proteins to bind the cofactor was assessed. In accordance with previous results, wild-type GST::h-mtTFB1 fusion protein bound approximately fivefold-more SAM than did a GST peptide used as a negative control (Fig. 4). Similar results were obtained with the N141A and K220A mutant proteins (Fig. 4). In contrast, the G65A mutant in motif I resulted in no detectable SAM-binding activity in this assay. The inability of this mutant to bind SAM provides a logical explanation for the previously reported loss of RNA methyltransferase activity as a result of this substitution (24). Lastly, h-mtTFB2 was found to bind SAM in a similar manner as h-mtTFB1 in this assay (data not shown).
Using an electrophoretic mobility shift assay, we showed previously that wild-type h-mtTFB1 binds to an LSP-containing DNA fragment in an apparently nucleotide sequence-independent manner (19). Using this same assay, we next determined the ability of the three mutated h-mtTFB1 proteins to bind DNA. The results of this analysis were similar to that of the SAM-binding experiment. That is, when added at equal concentrations, the N141A and K220A mutants bound DNA in a similar manner as the wild-type GST::h-mtTFB1 fusion protein (Fig. 5A), while the G65A protein was deficient in DNA binding (Fig. 5A, lane 3). Notably, the G65A protein was unable to significantly bind the probe even when substantially higher protein concentrations were tested (data not shown). These data indicate that, in addition to eliminating SAM binding, the G65A mutation in h-mtTFB1 also eliminated its double-stranded DNA-binding activity.
Next, we tested the ability of mutant h-mtTFB1 proteins to activate transcription from the mitochondrial LSP in a transcription factor-dependent transcription assay. In this assay, recombinant h-mtTFA and h-mtTFB1 were added to a soluble mitochondrial extract from HeLa cells that served as a source of human mtRNA polymerase (see Materials and Methods). As described previously (19), addition of recombinant GST::h-mtTFB1 protein alone to the extract did not result in stimulation of transcription initiation (Fig. 5B, compare lane 2 with lane 1), while addition of recombinant h-mtTFA alone resulted in a minimal, yet significant, amount of specific transcription initiation (Fig. 5B, compare lane 3 with lane 1). When wild-type protein or any one of the mutant GST::h-mtTFB1 fusion proteins was added in addition to recombinant h-mtTFA, transcription initiation was significantly increased above that seen with h-mtTFA alone (Fig. 5B, lanes 4 to 11). In addition, stimulation by all the mutant h-mtTFB1 proteins was dose dependent, similar to that observed with the wild-type protein (Fig. 5B, lanes 4 to 11). Finally, consistent with their ability to activate transcription, all of the mutant h-mtTFB1 proteins retained the ability to bind h-mtTFA in vitro in a manner similar to the wild-type protein (Fig. 5C). Altogether, these data indicate that mutations in three conserved RNA methyltransferase motifs do not affect the transcriptional activation properties of h-mtTFB1, despite the facts that a mutation in motif I (G65A) resulted in loss of SAM binding and the ability to interact with DNA and that the G65A and K220A mutations both resulted in lack of significant RNA methyltransferase activity (24).
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DISCUSSION |
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The first main conclusion from this work is that the ability of h-mtTFB1 to activate transcription in collaboration with h-mtTFA is largely, if not completely, independent of its activity as an rRNA methyltransferase. The data supporting this conclusion are as follows. First, point mutations in three sequence motifs that are conserved between rRNA methyltransferases and h-mtTFB1 had no effect on the ability of h-mtTFB1 to activate transcription from the mitochondrial LSP in vitro (Fig. 5B). Two of these, K220A and G65A, were shown previously to greatly reduce and eliminate RNA methyltransferase activity, respectively (24). The G65A mutation is in conserved motif I, which is implicated in binding SAM directly (3, 17), and resulted in the predicted defect in cofactor binding (Fig. 4), thus providing a logical explanation for why this mutation results in loss of RNA methyltransferase activity. We conclude from these data that the ability of h-mtTFB1 to activate transcription does not require RNA methyltransferase activity, SAM binding, or an intact SAM-binding pocket.
The second main conclusion from this work is that the C-terminal domain of h-mtTFA is a physical and likely functional interaction point for h-mtTFB1 and h-mtTFB2. The data supporting this conclusion are as follows. First, immunodepletion of h-mtTFB1 from a transcription-competent HeLa cell mitochondrial extract not only immunoprecipitates h-mtTFB1 but also coimmunoprecipitates most of the h-mtTFA as well (Fig. 1). The resulting depleted extract is incapable of transcription initiation from the human LSP (Fig. 1). The coimmunoprecipitation of these two factors was confirmed by our demonstration that they also interact directly in vitro (Fig. 2). Of particular significance is the fact that at least one important interaction point for h-mtTFB1 is the C-terminal tail of h-mtTFA that has been implicated previously in the transcriptional activation function of this HMG box, mtDNA-binding protein (7). When we tested a series of h-mtTFA C-terminal tail deletion mutations for their interaction with h-mtTFB1 in vitro, the results were in remarkable correspondence with the ability of these mutant proteins to activate transcription in vitro reported by Dairaghi et al. (7). That is, a deletion of 5 amino acids (h-mtTFA 1-199) was still capable of interacting with h-mtTFB1 (Fig. 2) and maintained wild-type transcriptional activation function (7). Deletion of 10 (h-mtTFA 1-194) or 25 (h-mtTFA 1-179) C-terminal residues resulted in a severe reduction, if not complete loss, of interaction with h-mtTFB1 in vitro (Fig. 2) that correlates with the reported loss of transcriptional activation function (7). These data strongly suggest that the direct interaction between h-mtTFA and h-mtTFB1 reported here is an important determinant of the ability of these two proteins to cooperate during transcription initiation in human mitochondria.
Finally, we found that the G65A mutation also resulted in dramatic loss of DNA-binding activity in an electrophoretic mobility shift assay (Fig. 5A), leading us to conclude that the transcription factor function of h-mtTFB1 is independent of not only RNA methyltransferase activity but also its double-stranded DNA-binding ability. This suggests that the formation of a closed transcription complex at the mitochondrial LSP can occur without a DNA binding contribution from h-mtTFB1. However, we do acknowledge that we have not eliminated the possibility that h-mtTFB1 may contribute to activation of initiation through binding of single-stranded DNA during initiation (e.g., during open complex formation). A potential function involving single-stranded DNA binding is perhaps more likely for a transcription factor that binds RNA, a nucleic acid substrate with at least some single-stranded character. If such an interaction is occurring, it is possible that the G65A mutation has differentially affected single-stranded and double-stranded DNA binding of h-mtTFB1, perhaps indicating the existence of two separate nucleic acid binding sites on the molecule. In the absence of this speculation, however, the simplest explanation of our results is that transcriptional activation by h-mtTFB1 is independent of its only documented DNA-binding activity.
Based on the data presented in this report and the present state of knowledge, we propose the following model describing the fundamental interactions required for transcription initiation at a human mitochondrial promoter (Fig. 6). The basic premise of this model is that h-mtTFB1 (and by analogy h-mtTFB2) functions to bridge an interaction between h-mtTFA and h-mtRNA polymerase at the promoter to facilitate transcription initiation. We propose that the interaction between h-mtTFB1 and h-mtTFA is mediated in large part by the C-terminal tail of h-mtTFA based on the data presented herein and on the documented strict requirement for this domain in transcriptional activation (7). The proposed direct interaction between h-mtTFB1 and mtRNA polymerase is supported by our data showing that antibodies to h-mtTFB1 coimmunoprecipitate h-mtRNA polmerase (Fig. 3) and on the indirect, yet compelling, evidence reported by others that a one-to-one complex can form between h-mtTFB1 or h-mtTFB2 and h-mtRNA polymerase (9). Such a complex is also consistent with the fact that the S. cerevisiae homologs of these proteins, sc-mtTFB (Mtf1p) and mtRNA polymerase (Rpo41p), have been shown to interact (4, 5). A final aspect of this model is based on our observation that activation of transcription by h-mtTFB1 is independent of its normal double-stranded DNA-binding activity. This suggests that promoter recognition by h-mtRNA polymerase (i.e., closed complex formation) per se is facilitated not by h-mtTFB1 DNA-binding activity but rather by the sequence-specific DNA binding of h-mtTFA at the promoter and/or its generation of a specific protein-DNA conformation that is accomplished at that site through its ability to bend and wrap DNA (12). According to this model, h-mtTFB1 has an "adapter" function that facilitates delivery of mtRNA polymerase to the promoter, which is demarcated by a specific configuration of the C-terminal domain of h-mtTFA bound at the promoter. The ability of h-mtTFB1 to activate transcription independently of its RNA methyltransferase activity is consistent with the proposed adapter function in that it suggests that this function involves the regions of the protein that are unique to this class of transcription factors and absent in the related RNA methyltransferase proteins (23). While this model directly implicates h-mtTFA as an important player in the promoter recognition process, our data do not discount the possibility that h-mtRNA polymerase and/or h-mtTFB1 and h-mtTFB2 also contribute to promoter specificity in some manner. Additional experiments are needed to determine precisely how these four factors cooperate to achieve promoter-specific transcription initiation from the LSP and HSP.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL-59655) awarded to G.S.S. and an NRSA (HL-68459) awarded to V.M.
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FOOTNOTES |
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