A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine

Abstract

A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication. Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA. However, based on studies in model systems, the existence of a second human mitochondrial transcription factor has been postulated. Here we report the isolation of a cDNA encoding h-mtTFB, the human homolog of Saccharomyces cerevisiae mitochondrial transcription factor B (sc-mtTFB) and the first metazoan member of this class of transcription factors to which a gene has been assigned. Recombinant h-mtTFB is capable of binding mtDNA in a non-sequence-specific fashion and activates transcription from the human mitochondrial light-strand promoter in the presence of h-mtTFA in vitro. Remarkably, h-mtTFB and its fungal homologs are related in primary sequence to a superfamily of N6 adenine RNA methyltransferases. This observation, coupled with the ability of recombinant h-mtTFB to bind S-adenosylmethionine in vitro, suggests that a structural, and perhaps functional, relationship exists between this class of transcription factors and this family of RNA modification enzymes and that h-mtTFB may perform dual functions during mitochondrial gene expression.

Human Mitochondrial Ribosomal Protein MRPL12 Interacts Directly with Mitochondrial RNA Polymerase to Modulate Mitochondrial Gene Expression

“…32 P]UTP (3,000 Ci/mmol), 40 units of RNase OUT TM (Invitrogen), 2.5 l of a transcription-competent mitochondrial extract from HeLa cells that was prepared as described previously (2,4), and the indicated concentrations of recombinant h-mtTFA or MRPL12. After 30 min at 32°C, reactions were stopped by adding 200 l of stop buffer (10 mM Tris⅐Cl, pH 8.0, 0.2 M NaCl, and 1 mM EDTA).…”

“…For example, whether other factors interact with POLRMT to regulate transcription initiation and/or elongation or to couple additional RNA-related activities to transcription have not been determined. However, interesting in this regard, h-mtTFB1 and h-mtTFB2 are members of a conserved family of rRNA methyltransferases (3,4) and can methylate small subunit rRNAs at a conserved stem loop (5,6), representing a potential link between the transcription machinery and ribosome biogenesis and/or translation activity. Consistent with such a role, mtTFB1 from Drosophila melanogaster is required for efficient mitochondrial translation in Schneider cells (7).…”

Arabidopsis Phage-Type RNA Polymerases: Accurate in Vitro Transcription of Organellar Genes

“…In mitochondria of the budding yeast Saccharomyces cerevisiae (Greenleaf et al, 1986;Masters et al, 1987), mammals (Tiranti et al, 1997;Falkenberg et al, 2002;Gaspari et al, 2004), and other eukaryotic organisms (Cermakian et al, 1996), a nuclear-encoded T7 phage-type RNAP has replaced the ancestral bacterial-type RNAP. Transcription initiation in the mitochondria of yeast and mammals, however, depends on the transcriptional cofactor mtTFB (MTF1, TFBM), which is related to rRNA dimethyladenosine transferases (Winkley et al, 1985;Schinkel et al, 1987;Falkenberg et al, 2002;McCulloch et al, 2002;Matsunaga and Jaehning, 2004b). Mammalian mitochondrial RNAP additionally requires the high-mobility group-box protein mtTFA (TFAM) for promoter recognition Clayton, 1985, 1988;Falkenberg et al, 2002;Gaspari et al, 2004).…”

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

“…The human mitochondrial RNA polymerase (POLRMT) is a single-subunit enzyme related to the T7 family of bacteriophage RNA polymerases (9,10). However, unlike T7 RNA polymerase, which does not require any transcription factors, efficient promoter-specific initiation by human POLRMT in vitro requires the high mobility group box transcription factor h-mtTFA/TFAM (referred to as h-mtTFA from this point forward) and one of two rRNA methyltransferase-related transcription factors, h-mtTFB1 and h-mtTFB2 (11)(12)(13)(14). Based on work in Drosophila (15,16), cultured human cells (17,18), and mice (19), it is becoming clear that, whereas both h-mtTFB1 and h-mtTFB2 can bind POLRMT and activate transcription in vitro, h-mtTFB2 is probably the primary transcription factor in vivo, whereas h-mtTFB1 is the primary rRNA methyltransferase critical for mitochondrial ribosome biogenesis and translation.…”