Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved stem-loop.

Human mitochondrial transcription factor B1 (h-mtTFB1) has an unprecedented relationship to RNA methyltransferases. Here, we show that this protein methylates a conserved stem-loop in bacterial 16S rRNA and that the homologous sequence in the human mitochondrial 12S molecule is similarly modified. Thus, h-mtTFB1 appears to be dual-function protein, acting both as a transcription factor and an rRNA-modification enzyme.

Modulation of mitochondrial transcription in response to mtDNA depletion and repletion in HeLa cells.

The steady-state amounts of mitochondrial transcripts and transcription proteins were analyzed during mtDNA depletion and subsequent repletion to gain insight into the regulation of human mitochondrial gene expression. As documented previously, HeLa cells depleted of mtDNA via treatment with ethidium bromide (EB) were found to contain reduced steady-state levels of the mitochondrial transcription factor h-mtTFA. When partially mtDNA-depleted cells were cultured in the absence of EB, h-mtTFA recovered to normal levels at a significantly slower rate than mtDNA. Human mtRNA polymerase exhibited a similar depletion-repletion profile, suggesting that the mitochondrial transcription machinery is coordinately regulated in response to changes in mtDNA copy number. Newly synthesized mitochondrial transcripts were detected early in the recovery phase, despite the fact that mtDNA, h-mtTFA and h-mtRNA polymerase were simultaneously depleted. Although delayed relative to mtDNA, the amounts of h-mtTFA and h-mtRNA polymerase sharply increased during the later stages of the recovery phase, which was accompanied by accelerated rates of transcription and mtDNA replication. Altogether, these data indicate that when mtDNA copy number is low, it is beneficial to prevent accumulation of mitochondrial transcription proteins. In addition, h-mtTFA and h-mtRNA polymerase are either normally present in excess of the amount required for transcription or their activity is up-regulated to ensure continued expression and transcription-dependent replication of the mitochondrial genome during mtDNA-depleted states.

A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine.

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.