Human Mitochondrial Transcription Initiation Complexes Have Similar Topology on the Light and Heavy Strand Promoters

Abstract

Transcription is a highly regulated process in all domains of life. In human mitochondria, transcription of the circular genome involves only two promoters, called light strand promoter (LSP) and heavy strand promoter (HSP), located in the opposite DNA strands. Initiation of transcription occurs upon sequential assembly of an initiation complex that includes mitochondrial RNA polymerase (mtRNAP) and the initiation factors mitochondrial transcription factor A (TFAM) and TFB2M. It has been recently suggested that the transcription initiation factor TFAM binds to HSP and LSP in opposite directions, implying that the mechanisms of transcription initiation are drastically dissimilar at these promoters. In contrast, we found that binding of TFAM to HSP and the subsequent recruitment of mtRNAP results in a pre-initiation complex that is remarkably similar in topology and properties to that formed at the LSP promoter. Our data suggest that assembly of the pre-initiation complexes on LSP and HSP brings these transcription units in close proximity, providing an opportunity for regulatory proteins to simultaneously control transcription initiation in both mtDNA strands.

Keywords: LSP; RNA polymerase; TFAM; TFB2M; mitochondrial DNA (mtDNA); mtRNAP; promoter; protein cross-linking; transcription; transcription factor.

FIGURE 1.

Characterization of the initiation complex assembled on human HSP promoter. a, schematic illustration of the footprinting data on HSP. Black numbers indicate positions of the bases in promoter DNA relative to the start site (+1), and gray numbers indicate the positions according to the reference (Cambridge) mtDNA. The −11 base in the template DNA stand that interacts with mtRNAP is highlighted in yellow. b, Y162A TFAM mutant is defective in run-off transcription assays on both mitochondrial promoters. nt, nucleotides. c, TFAM variants Y162A and L6 are deficient in transcription on both LSP (black bars) and HSP (red bars). Error bars indicate means ± S.E. d, TFAM variants deficient in transcription on LSP are also defective on HSP.

FIGURE 2.

Topology of the pre-initiation complexes assembled on LSP and HSP. a, schematics of TFAM interactions with mtRNAP on LSP. Two major interactions involve TFAM region next to residue 217 and Trp-122 in the N-terminal extension region of mtRNAP (residues (res.) 120–134, predicted α-helix) and TFAM region next to residue 228 and mtRNAP region 444–463. b, the C-terminal region of TFAM interacts with the N-terminal extension region of mtRNAP in the pre-ICs assembled on LSP or HSP promoter. The pre-ICs were UV-irradiated, treated with hydroxylamine (NH₂OH), and resolved using SDS-PAGE. The radioactive species (∼37 kDa) that appear upon the treatment of the pre-IC with hydroxylamine represent TFAM cross-link to the region 44–150 of mtRNAP. XL, cross-link. c, MtRNAP residue Trp-122 is a primary interaction target for TFAM in complexes assembled on both promoters. Upper panel, cross-linking performed using ³²P-labeled Bpa217 TFAM and WT (lanes 1 and 2) or Trp-122 mtRNAP (lanes 3 and 4). Lower panel, cross-linking performed as above but using ³²P-labeled Bpa228 TFAM. d, MtRNAP interacts with the −49 base of the HSP promoter. TS, DNA template strand; NT, DNA non-template strand. e, transcription initiation complexes assemble in close proximity on human mtDNA. Interactions probed in this study are indicated by the yellow stars.