A unique mitochondrial transcription factor B protein in Dictyostelium discoideum

Abstract

Unlike their bacteriophage homologs, mitochondrial RNA polymerases require the assistance of transcription factors in order to transcribe mitochondrial DNA efficiently. The transcription factor A family has been shown to be important for transcription of the human mitochondrial DNA, with some of its regulatory activity located in its extended C-terminal tail. The mitochondrial transcription factor B family often has functions not only in transcription, but also in mitochondrial rRNA modification, a hallmark of its α-proteobacterial origin. We have identified and characterised a mitochondrial transcription factor B homolog in the soil dwelling cellular slime mould Dictyostelium discoideum, an organism widely established as a model for studying eukaryotic cell biology. Using in bacterio functional assays, we demonstrate that the mitochondrial transcription factor B homolog not only functions as a mitochondrial transcription factor, but that it also has a role in rRNA methylation. Additionally, we show that the transcriptional activation properties of the D. discoideum protein are located in its extended C-terminal tail, a feature not seen before in the mitochondrial transcription factor B family, but reminiscent of the human mitochondrial transcription factor A. This report contributes to our current understanding of the complexities of mitochondrial transcription, and its evolution in eukaryotes.

Figure 1. Domain architecture of Tfb1m.

Bioinformatic analysis revealed that the 56 kDa protein has an N-terminal mitochondrial targeting signal (MTS), an S-adenosyl-methionine (SAM) binding site, and a rRNA adenosine dimethyltransferase domain, features characteristic of most mtTFB homologs.

Figure 2. Amino acid sequence alignment of Tfb1m from Dictyostelium discoideum (DdmtTFB) with mtTFB homologs.

Included are mtTFB homologs from Acanthamoeba castellanii (Ac, accession no. ABB97063), Homo sapiens (Hs, accession no. NP_057104 and NP_071761), Mus musculus (Mm, accession no. NP_666186 and NP_032275), Xenopus (Silurana) tropicalis (Xt, accession no. NP_001016494 and AAI27364), Drosophila melanogaster (Dm, accession no. Q9VTM5 and Q9VH38) and KsgA from Escherichia coli (Ec, accession no. YP_488357). Amino acids shaded in black indicate identical amino acids, while conserved and semi-conserved amino acids are depicted in dark and light grey, respectively.

Figure 3. Mitochondrial localisation of Tfb1m.

Fluorescence microscopy of (A) D. discoideum cells expressing a Tfb1m:GFP fusion protein and (B) stained with Mitotracker, indicating that the fusion protein and the mitochondria co-localise (C).

Figure 4. Analysis of kasugamycin resistance to evaluate rRNA adenosine methyltransferase activity.

Cell densities of an E. coli ksgA mutant (KsgA[pQE30], second column), compared to its parental strain K-12 BW25113 (first column) and the same mutant expressing either the D. discoideum mitochondrial transcription factor Tfb1m (KsgA[pQE30:tfb1m], third column), or a C-terminally truncated variant of Tfb1m (KsgA[pQE30:tfb1mΔC], fourth column). Cells were grown in LB supplemented with ampicillin (100 µg/mL) and kasugamycin (800 µg/mL) at 37°C for 15 h, and growth was analysed by measuring the optical density of the cultures.

Figure 5. Schematic representation of vector constructs used for in bacterio transcription experiments.

a) Vector pZ-NCRrnl contains the non-coding region (NCR) including the transcription initiation site of the D. discoideum mitochondrial genome, and the partial sequence of the rnl gene downstream of the initiation site, which serves as a transcriptional reporter. The vector confers resistance to kanamycin. b) Vector pQ-rpmA contains the rpmA gene under the control of a T5_lac promoter/operator for heterologous protein expression. The vector confers resistance to ampicillin. c) Vector pET-tfb1m contains the full length tfb1m gene under the control of a T7 promoter. The vector confers resistance to tetracycline. d) Vector pET-tfb1mΔC contains the tfb1m gene lacking the 3′ end of the gene encoding the C-terminus. The vector confers resistance to tetracycline. e) Vector pBS-rnl contains the partial sequence of the mitochondrial rnl gene, which was used for in vitro transcription to generate a radiolabelled antisense rnl RNA probe transcribed from the T3 promoter. Constructs pZ-NCRrnl, pQ-rpmA and pBS-rnl were obtained from Le et al., 2009 .

Figure 6. Analysis of in bacterio transcribed RNA.

Northern hybridisation using an antisense rnl probe against total RNA isolated from E. coli cells harbouring vector pZ-NCRrnl (lanes 1 and 2), vectors pZ-NCRrnl and pQ-rpmA (lanes 3 and 4), vectors pZ-NCRrnl, pQ-rpmA and pET-tfb1m (lanes 5 and 6), or vectors pZ-NCRrnl, pQ-rpmA and pET-tfb1mΔC (lanes 7 and 8). Heterologous expression of the D. discoideum mitochondrial RNA polymerase (rpmA) from pQ-rpmA and of the mitochondrial transcription factor Tfb1m from pET-tfb1m or pET-tfb1mΔC is indicated by+(induced) or – (non-induced). The top bands (∼ 420 nucleotides long) indicated by the arrow represent genuine transcription products initiated at the mitochondrial transcription start site. The smear below may be the result of partially degraded or prematurely terminated transcription products. RNA size markers are indicated in kilo bases to the left of the panel.

Figure 7. Coiled-coil motif prediction analysis for Tfb1m.

Analysis was performed using the bioinformatic program Coils at 14 (green), 21 (blue) and 28 (red) amino acid frames.

Figure 8. Comparison of the D. discoideum mitochondrial transcription factor to the human mitochondrial transcription factors.

Displayed are the domain architectures of human mitochondrial transcription factors TFAM (A), h-mtTFB1 (B) and h-mtTFB2 (C) and the D. discoideum mitochondrial transcription factor Tfb1m (D). Purple overlapping regions denote coiled-coil regions.