The recent identification of two human homologs of yeast mitochondrial transcription factor B, h-mtTFB1 (19
) and h-mtTFB2 (9
), has provided the opportunity to gain a more complete understanding of the mechanism of transcriptional regulation in human mitochondria and its influence on human disease and aging. In our initial characterization of h-mtTFB1 we noted its remarkable primary sequence similarity to a class of rRNA methyltransferase enzymes and demonstrated that it binds the requisite SAM cofactor used by this class of enzymes (19
). We went on to show that this protein actually possesses rRNA methyltransferase activity and is capable of methylating two adjacent adenine residues in a stem-loop structure that is conserved in bacterial 16S and human mitochondrial 12S rRNA molecules (24
). Thus, h-mtTFB1 is apparently a dual-function protein capable of activating transcription and modifying rRNA. The goal of this study was to determine the extent to which these two activities are linked: that is, to determine whether the methyltransferase structural features and/or activity is required for the transcription factor function of h-mtTFB1. In addition, our initial characterizations also revealed a weak and apparently sequence-nonspecific DNA-binding activity of h-mtTFB1 (19
). The relevance of this activity to the ability of h-mtTFB1 to activate transcription was also addressed in this study.
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. ). 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
), and resulted in the predicted defect in cofactor binding (Fig. ), 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. ). The resulting depleted extract is incapable of transcription initiation from the human LSP (Fig. ). The coimmunoprecipitation of these two factors was confirmed by our demonstration that they also interact directly in vitro (Fig. ). 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. ) 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. ) 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. ), 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. ). 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. ) 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
homologs of these proteins, sc-mtTFB (Mtf1p) and mtRNA polymerase (Rpo41p), have been shown to interact (4
). 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.
FIG. 6. Proposed model of the interactions between human mitochondrial transcription proteins during initiation of transcription at the human LSP. Human mtTFA (black) is shown bound to the LSP upstream of the site of transcription initiation (bent arrow). It (more ...)
While this report provides important new information regarding the mechanism of transcription in human mitochondria, many questions remain unanswered. For example, our data indicate that, in terms of SAM binding and its interaction with h-mtTFA (Fig. ), h-mtTFB2 behaves in a manner indistinguishable from h-mtTFB1 in the assays used. Thus, we have not provided an explanation for the observation that h-mtTFB2 activates transcription more efficiently in vitro with a fully recombinant transcription system (9
). Nor have we yet uncovered why the system has evolved a requirement for two h-mtTFB homologs. We would argue that both h-mtTFB1 and h-mtTFB2 are involved directly in transcription and are present to provide a yet-to-be-elucidated mechanism for differential regulation of mitochondrial gene expression. However, it remains a formal possibility that h-mtTFB1 is primarily an rRNA methyltransferase in vivo (24
) and that h-mtTFB2 is primarily a transcription factor in vivo, or vice versa. More detailed studies are needed in the future to decipher the individual or dual roles of these two important factors in mitochondrial gene expression. Clearly, much remains to be learned regarding the regulation of human mitochondrial transcription and its impact on human aging and disease.