The mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells

Abstract

Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position -1 to -2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.

Figure 1

Reconstitution of the mouse mitochondrial transcription system in vitro. (A) SDS–PAGE gel stained with Coomassie blue showing the recombinant mouse proteins used for in vitro transcription reaction. Whereas mTFAM is expressed on its own, mPOLRMT is co-expressed and co-purified with mTFB2M. (B) Transcription from mLSP only occurs when mTFAM (2.5 pmol), mPOLRMT (500 fmol) and mTFB2M (500 fmol) are present simultaneously. (C) The human and mouse transcription machinery cannot initiate transcription from heterologous promoters. Linear templates (85 fmol) containing the mouse or hLSP were used for in vitro run-off transcription assays. The reactions were performed with the following pure recombinant proteins: human or mouse POLRMT (500 fmol), human or mouse TFB2M (500 fmol) and human or mouse TFAM (2.5 pmol).

Figure 2

DNA sequences governing species specificity are localized upstream of the transcription initiation site. (A) Schematic representation of the LSP templates harboring a chimeric human LSP-mouse transcript (hLSP/mT) or a mouse LSP-human transcript (mLSP/hT). The transcription initiation site (+1) is indicated with an arrow. (B) The templates were assayed for their ability to support in vitro transcription with the pure recombinant human system. The transcription reaction mixtures contained hPOLRMT (500 fmol), hTFB2M (500 fmol), hTFAM (2.5 pmol) and the indicated mtDNA template (85 fmol). (C) The templates were assayed for their ability to support in vitro transcription with the pure recombinant mouse system. The transcription reaction mixtures were as under (B), but with the corresponding mouse proteins. (D) A sequence comparison between hLSP and mLSP. Conserved nucleotides are indicated with asterisks.

Figure 3

Mutational analysis of the hLSP promoter. (A) A series of LSP promoter constructs in which 2 bp at the time were mutated from position −1 to −20 from the transcription initiation site. The introduced mutations made the following changes in the hLSP sequence: A to C, C to A, T to G and G to T. The transcription initiation site is (+1) indicated with an arrow. Only the hLSP noncoding strand is represented. (B) The ability of the mutant promoter constructs to support transcription was investigated in the complete human in vitro transcription system. The in vitro transcription reaction mixtures contained hTFAM (2.5 pmol), hPOLRMT (500 fmol), hTFB2M (500 fmol) and 85 fmol of human LSP template. (C) Two mLSP promoter constructs were mutated at positions −1/−2 and −3/−4 as described for hLSP under (A). The ability of the mutant mLSP constructs to support transcription was investigated in the complete mouse in vitro transcription system. The in vitro transcription reaction mixtures contained mTFAM (2.5 pmol), mPOLRMT (500 fmol), mTFB2M (500 fmol) and 85 fmol of mLSP template.

Figure 4

Introduction of the human TFAM-binding sites enables the human transcription machinery to initiate transcription from mouse LSP. (A) Series of hybrid promoter constructs in which the TFAM-binding site of mouse LSP was replaced with corresponding sequences from the human LSP. Shaded boxes correspond to the introduced human sequence. The transcription initiation site (+1) is indicated with an arrow. (B) The ability of the hybrid promoter constructs to support transcription was investigated in the complete human in vitro transcription system. The in vitro transcription reaction mixtures contained hTFAM (2.5 pmol), hPOLRMT (500 fmol), hTFB2M (500 fmol) and 85 fmol of human LSP template.

Figure 5

(A) Mouse TFAM can efficiently support transcription on hLSP. Template containing the LSP (85 fmol) of human mtDNA was used for in vitro run-off transcription assays. The reactions were performed with the following pure recombinant proteins: hPOLRMT (300 fmol), hTFB2M (300 fmol) and hTFAM or mTFAM in increasing amounts (0, 0.025, 0.1, 0.25, 1.0, 2.5, 10 pmol). (B) Mouse POLRMT cannot initiate transcription at hLSP. In vitro transcription from hLSP was performed with the proteins indicated (500 fmol) in the presence of (2.5 pmol) of hTFAM.

Figure 6

Promoter recognition by mPOLRMT is critically dependent on the PPE. (A) A series of hybrid promoter constructs in which the −1 to −10 hLSP region was gradually (2 bp) replaced with the corresponding sequences from mLSP. (B) Hybrid promoter constructs were used for in vitro run-off transcription assays. The reactions were performed with the following pure recombinant proteins: mPOLRMT (500 fmol), mTFB2M (500 fmol), mTFAM (2.5 pmol) and 85 fmol of template. (C) Sequence comparison between hLSP and mLSP. Conserved nucleotides are marked with asterisks and the A to T transversion at position −1 is in bold font.

Figure 7

Promoter recognition by hPOLRMT/hTFB2M is strictly dependent on hTFAM (A). DNase I footprinting reveals that neither hPOLRMT nor hTFB2M (lanes 2 and 3) in isolation interacts with hLSP. The binding site for hTFAM (lane 4) is indicated with a solid line. (B) The hPOLRMT/hTFB2M heterodimer interacts with the transcription start site in the presence (lane 3), but not in the absence (lane 4), of hTFAM. The region protected by hPOLRMT/hTFB2M is indicated with a dashed line. (C) The hPOLRMT/hTFB2M complex (lane 3), but not the mPOLRMT/hTFB2M complex (lane 4), interacts with the hLSP transcription start site in the presence of hTFAM. (D) A schematic representation of protein interactions with hLSP. Human TFAM protects the −15 to −38 region (solid line) and the hPOLRMT/hTFB2M heterodimer protects the +10 to −4 region (dashed line).

Figure 8

hTFAM and hTFB2M are required for abortive transcription initiation. hTFAM, hPOLRMT and hTFB2M were added as indicated. Transcription reactions were analyzed in parallel on 6% and 25% denaturing polyacrylamide gels.