A Monomer-to-Trimer Transition of the Human Mitochondrial Transcription Termination Factor (mTERF) Is Associated with a Loss of in Vitro Activity

Abstract

The human mitochondrial transcription termination factor (mTERF) is a nuclear-encoded 39-kDa protein that recognizes a mtDNA segment within the mitochondrial tRNA Leu(UUR) gene immediately adjacent to and downstream of the 16 S rRNA gene. Binding of mTERF to this site promotes termination of rDNA transcription. Despite the fact that mTERF binds DNA as a monomer, the presence in its sequence of three leucine-zipper motifs suggested the possibility of mTERF establishing intermolecular interactions with proteins of the same or different type. When a mitochondrial lysate from HeLa cells was submitted to gel filtration chromatography, mTERF was eluted in two peaks, as detected by immunoblotting. The first peak, which varied in proportion between 30 and 50%, appeared at the position expected from the molecular mass of the monomer (41 ؎ 2 kDa), and the gel filtration fractions that contained it exhibited DNA binding activity. Most interestingly, the material in this peak had a strong stimulating activity on in vitro transcription of the mitochondrial rDNA. The second peak eluted at a position corresponding to an estimated molecular mass of 111 ؎ 5 kDa. No mTERF DNA binding activity could be detected in the corresponding gel filtration fractions. Therefore, we propose that mTERF exists in mitochondria in two forms, an active monomer and an inactive large size complex. The estimated molecular weight of this complex and the fact that purified mTERF can be eluted from a gel filtration column as a complex of the same molecular weight strongly suggest that this inactive complex is a homotrimer of mTERF.

The Human Mitochondrial Transcription Termination Factor (mTERF) Is FullyActive in Vitro in the Non-phosphorylatedForm

“…The mTERF protein has been shown previously to stimulate HSP transcription (5,9). The stimulation is only observed when mTERF binds to its cognate binding site and both the promoter and the mTERF-binding site are in the same orientation.…”

“…Gel Filtration Chromatography-The gel filtration chromatography of Ni 2ϩ -NTA-purified mTERF was performed as described (9) with the following modifications. The column (Superose ® 12; Amersham Biosciences) was equilibrated in running buffer (25 mM HEPES (pH 7.6), 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.1% Tween 20, and 500 mM KCl) at a flow rate of 0.3 ml/min.…”

“…Furthermore, a recent study of recombinant rat mTERF suggested that phosphorylation of four amino acids is required for transcription termination activity (8). Alternatively, mTERF activity may be regulated by its polymerization state, because native human mTERF is present in HeLa mitochondrial lysates in two forms, an active monomer and an inactive polymer (9). Native mTERF has a high tendency to polymerize, thereby explaining a dramatic loss of activity during purification.…”

<italic>hMTERF4</italic> knockdown in HeLa cells results in sub-G1 cell accumulation and cell death

“…Three groups of proteins, mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM), and mitochondrial transcription factor B1 or B2 (TFB1M or TFB2M) are necessary and sufficient for specific transcription initiation of mtDNA in vitro [1]. Although several new regulatory proteins have been identified [2,3], the mechanism of mitochondrial gene regulation in vivo is still poorly defined.…”

MTERF1 regulates the oxidative phosphorylation activity and cell proliferation in HeLa cells

“…Though several mtDNA transcription regulatory proteins have been identified to date, the in vivo regulation of mitochondrial transcription and OXPHOS function remains poorly understood [3,4]. The mitochondrial transcription termination factor (MTERF) family is a member of the mitochondrial transcription regulatory factors.…”