Coimmunoprecipitation of h-mtTFB1 and h-mtTFA from a transcriptionally active mitochondrial lysate from HeLa cells. A soluble HeLa cell mitochondrial extract was prepared that efficiently initiated transcription from a linear DNA template containing the human LSP (top panel). The lysate (undepleted, lane 1) produced a specific radiolabeled runoff transcript (labeled LSP) that was visualized by autoradiography. Transcription activity was also assayed from lysates that were immunodepleted with affinity-purified h-mtTFB1 antibody (h-mtTFB1 depleted, lane 3) or a control affinity-purified preimmune serum (pre-immune depleted, lane 2). Western immunoblot analysis of the corresponding extracts with an antibody to h-mtTFB1 (middle panel) or h-mtTFA (bottom panel) is also shown.

Direct interaction between h-mtTFB1 or h-mtTFB2 and h-mtTFA requires the C-terminal activation domain of h-mtTFA. The results of solid-phase protein-protein interaction assays are shown. (A) The amount of recombinant gel-purified h-mtTFA that remained stably associated with a GST::h-mtTFB1 fusion protein that was bound to glutathione-Sepharose beads was visualized by Western immunoblotting with an antibody to h-mtTFA (lanes labeled as “bound h-mtTFA”). Full-length h-mtTFA (indicated as such) as well as the following three C-terminal deletion mutants was analyzed: 1-199 (missing C-terminal 5 amino acids), 1-194 (missing C-terminal 10 amino acids), and 1-179 (missing entire C-terminal 25-amino-acid tail). Twenty-five percent of the total amount of protein initially incubated with the beads in each assay is also shown (indicated as “input h-mtTFA”). (B) The results of a direct protein-binding assay between h-mtTFB2 and h-mtTFA C-terminal deletion mutants are shown in the same manner as described for panel A above.

Coimmunoprecipitation of human mtRNA polymerase with h-mtTFB1. A soluble HeLa cell mitochondrial extract was prepared as described in Materials and Methods. This extract was probed for the presence of h-mtRNA polymerase by Western analysis (lane 1). The same extract was immunodepleted with a polyclonal h-mtTFB1 antibody (lane 2) or the control preimmune serum (lane 3) and probed for the presence of the h-mtRNA polymerase polypeptide (∼139 kDa) in the corresponding immunoprecipitates (IP) by Western analysis. Positions of migration of molecular weight standards (in kilodaltons) are shown to the right of the figure.

Effects of h-mtTFB1 RNA methyltransferase motif point mutations on SAM binding. A schematic diagram of the 346-amino-acid h-mtTFB1 protein is shown at the top of the figure with its RNA methyltransferase motifs I to VIII (, ) indicated by the black boxes. The positions of the three point mutations characterized in this study (G65A, motif I; N141A, motif IV; and K220A, motif VIII) are indicated by the arrows. The results of a solid-phase SAM-binding assay that involves the binding of radiolabeled ligand to immobilized GST::h-mtTFB1 proteins are shown in the bar graph. The ordinate represents the amount of labeled SAM (in counts per minute) bound to beads containing GST peptide alone (GST), GST::h-mtTFB1 (h-mtTFB1), and each mutated GST::h-mtTFB1 fusion protein (G65A, N141A, and K220A). That similar amounts of control and h-mtTFB1 proteins were loaded onto the beads was confirmed by Western analysis prior to each assay (data not shown).

Effects of h-mtTFB1 RNA methyltransferase motif point mutations on DNA binding, h-mtTFA binding, and transcriptional stimulation from the human LSP. (A) Electrophoretic mobility shift assays. Recombinant wild-type GST::h-mtTFB1 (B1, lanes 1 and 2) and the indicated point mutants (G65A, lanes 3 and 4; N141A, lanes 5 and 6; and K220A, lanes 7 and 8) were tested for their ability to bind to a radiolabeled linear DNA fragment containing the human LSP in the presence (+; lanes 2, 4, 6, and 8) or absence (−; lanes 1, 3, 5, and 7) of poly(dI-dC) competitor DNA. A shift in mobility of the LSP-containing fragment is indicated by the arrowhead. The position of migration of the unbound, end-labeled probe is shown by the diamond, and its migration in the absence of added protein is shown in lane 9. A slower-migrating band (indicated by the arrow) is commonly observed with this probe under these conditions. The physical nature of this species that causes its altered mobility is unknown. (B) Transcription assays. Wild-type GST::h-mtTFB1 (B1) and the indicated mutated proteins (G65A, N141A, and K220A) were tested in a transcription factor-dependent transcription assay that measures specific initiation from the LSP. The assay results in the production of a specific radiolabeled transcript from the LSP (arrowhead). A human HeLa cell mitochondrial extract deficient in specific LSP activity was used as a source of human mtRNA polymerase (assayed in lane 1). The addition of recombinant GST::h-mtTFB1 alone and recombinant h-mtTFA alone to the extract is indicated in lanes 2 and 3, respectively. The ability of GST::h-mtTFB1 (B1, lanes 4 and 5) and the indicated mutants (lanes 6 to 11) to stimulate LSP transcription was assessed in the presence of h-mtTFA (indicated by the bracket labeled +h-mtTFA). Two amounts (50 ng, lanes 4, 6, 8, and 10, and 250 ng, lanes 5, 7, 9, and 11) of each h-mTFB1 protein were tested. (C) h-mtTFA binding assays. The indicated amount of recombinant h-mtTFA (input h-mtTFA) was incubated with equal amounts of GST::mtTFB1 (B1) or the indicated mutated proteins bound to glutathione-agarose beads. The amount of h-mtTFA bound (bound h-mtTFA) by each was assessed by Western blotting with an antibody to h-mtTFA.

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 is shown bending the DNA (parallel lines) at the promoter as part of its ability to activate transcription initiation as proposed by Fisher et al. (). The C-terminal activation region of h-mtTFA (-C) is shown binding to h-mtTFB1 (or h-mtTFB2) based on the results of this study. In this model, h-mtTFB1 serves an adapter function to bridge an interaction between h-mtTFA and h-mtRNA polymerase at the promoter, which is demarcated by a specific h-mtTFA/DNA complex. The 1:1 complex shown between h-mtTFB1 (or h-mtTFB2) and h-mtRNA polymerase is drawn based on our ability to coimmunoprecipitate these proteins (Fig. ), the report of Falkenberg et al. () that these proteins interact in vitro during copurification, and on the reports of Cliften et al. that sc-mtTFB physically interacts with mtRNA polymerase in S. cerevisiae (, ).