Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

Abstract

Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.

Fig. 1.

HSP2 transcription in vitro. (A) Schematic of mtDNA promoters: LSP, HSP1 and HSP2, interpromoter region, TRE for LSP and HSP1, and nearby genes. This region of mtDNA was used to produce LONG and SHORT templates capable of producing the indicated runoff transcripts. (B) Runoff transcription from the LONG or SHORT templates and indicated factors. All experimental details for this experiment and subsequent experiments are provided in SI Materials and Methods. M designates size markers. (C) Sequence of DNA oligonucleotide representing HSP2; the proposed TSS is indicated by +1. (D) Runoff transcription from the HSP2-1 template and indicated factors.

Fig. 2.

Specificity of HSP2 transcription in vitro. (A) HSP2 transcription templates designed to assess the specificity of initiation. (B) Runoff transcription from the templates shown in A by POLRMT-TFB2M in the absence or presence of TFAM. (C) Sequence surrounding HSP2; shown are the expected start sites and sizes of RNA transcripts when using pApApC, pApApA, or pApCpA trinucleotide primer for initiation with the indicated nucleotide(s). (D) Transcription by POLRMT-TFB2M from the HSP2-1 template in the presence of pApApC and the indicated nucleotide(s). (E) Runoff transcription from the HSP2-1 template in the presence of pApApA, pApApC, or pApCpA trinucleotide primer and all four nucleotides. (F) Alignment of human HSP2 with corresponding mouse and bovine sequences reveals a region of high interspecies differences from −18 to −6. (G) Mouse POLRMT-TFB2M fails to produce a promoter-specific runoff transcript from human HSP2 indicating specificity of human POLRMT-TFB2M.

Fig. 3.

Repression of HSP2 transcription by TFAM in vitro. (A) TFAM uses two HMG boxes (Boxes A and B) for DNA binding and CTD for transcriptional activation. The activation portion of the CTD is deleted in TFAM-ΔCT26. (B) TFAM-ΔCT26 does not inhibit runoff transcription from the HSP2-1 template by POLRMT-TFB2M. (C) EMSA of HSP2-1 template over a range of TFAM concentrations shows formation of four complexes (I–IV), with complex I present from 0.5 nM. Formation of complex I is impaired for TFAM-ΔCT26. (D) DNase I footprinting of TFAM-HSP2-1 complexes reveals protection in the regions designated as A₁, A₂, A₃, and D without protection in regions B and C. A schematic of HSP2-1 is shown on the left, and size markers (M) are on the right. (E) Runoff transcription from the HSP2-1 template (100 nM) in the absence or presence of 100 or 10 nM TFAM, which are values that are 10-fold higher than or equivalent to the equilibrium dissociation constant, respectively. In this experiment, product formed at 30 min over the indicated range of POLRMT-TFB2M concentrations.

Fig. 4.

Repression of HSP2 transcription by TFAM orthologs in vitro. (A) Alignment of carboxyl-terminal sequences of TFAMs from human, mouse, and bovine species. (B) TFAMs from mouse and bovine inhibit runoff transcription from the HSP2-1 template by POLRMT-TFB2M. (C) EMSA of HSP2-1 template over a range of mouse and bovine TFAM concentrations shows formation of four complexes (I–IV), with complex I present from 0.5 nM as observed for human TFAM in Fig. 3C.

Fig. 5.

Interactions of the TFAM CTD. Structural models were produced using Protein Data Bank ID code 3TQ6 (7). (A) TFAM residues 232–236 (green) interact with the phosphodiester backbone of bound DNA (red). (B) Two TFAM-DNA complexes are present in the asymmetric unit and designated here as chains A (dark green) and B (light green). Structural integrity of the CTD of each monomer benefits from interaction of Arg-227 in each monomer with both Asp-229 and Glu-148 of the same monomer. The CTD of one monomer packs against the CTD of a second monomer, perhaps creating a mechanism for association between TFAM-DNA complexes.