Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

Abstract

A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated

Mammalian transcription factor A is a core component of the mitochondrial transcription machinery

“…In addition, TFAM could also stimulate inefficient promoter melting, by recruiting the transcription machinery via direct protein-protein interactions that positions POLRMT and TFB2M in direct proximity of the transcription start site. TFAM has been shown to interact directly with TFB2M and its paralogue, TFB1M (32). The functional importance of this finding is still unclear, because TFB1M is not involved in mitochondrial transcription, but rather plays a role in ribosomal biogenesis (33).…”

TFAM forces mtDNA to make a U-turn

“…1). The carboxy--terminus of TFAM plays a critical role in transcription activation 5 and has been reported to interact with both TFB2M and the structurally similar TFB1M protein 18 . These putative interactions must be evaluated further, particularly as TFB1M likely is not involved in transcription, because TFB1M functions as an rRNA methyltransferase essential to mitochondrial ribosomal biogenesis in vivo 19 .…”

Mitochondrial Ribosomal Protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription

“…Relevant to this study, h-mtTFB2 is homologous to the bacterial KsgA family of rRNA methyltransferases, an ortholog of which was inherited from the original bacterial endosymbiont ancestor of mitochondria (15,16). This core two-component system is activated by the HMG-box transcription factor, h-mtTFA (or TFAM) (17)(18)(19), that binds at the mtDNA promoters LSP and HSP1 and interacts with h-mtTFB2 (20). The role of h-mtTFA as a transcriptional activator is well documented, but it also has proposed roles in packaging (21)(22)(23) and repair (24) of mtDNA.…”