TFB2 Is a Transient Component of the Catalytic Site of the Human Mitochondrial RNA Polymerase

Abstract

Summary Transcription in human mitochondria is carried out by a single-subunit, T7-like RNA polymerase assisted by several auxiliary factors. We demonstrate that an essential initiation factor, TFB2, forms a network of interactions with DNA near the transcription start site and facilitates promoter melting but may not be essential for promoter recognition. Unexpectedly, catalytic autolabeling reveals that TFB2 interacts with the priming substrate, suggesting that TFB2 acts as a transient component of the catalytic site of the initiation complex. Mapping of TFB2 identifies a region of its N-terminal domain that is involved in simultaneous interactions with the priming substrate and the templating (+1) DNA base. Our data indicate that the transcriptional machinery in human mitochondria has evolved into a system that combines features inherited from self-sufficient, T7-like RNA polymerase and those typically found in systems comprising cellular multi-subunit polymerases, and provide insights into the molecular mechanisms of transcription regulation in mitochondria.

Mitochondrial DNA mutations in disease and aging

“…TFB1M conditional knockout mice have severely impaired translation and are able to activate mitochondrial transcription, showing that there seems to be no functional redundancy between TFB1M and TFB2M (Metodiev et al, 2009). Biochemical studies further support the conclusion that TFB2M is essential for transcription, as it is a component of the catalytic site of POLRMT necessary for transcription initiation (Sologub et al, 2009;Litonin et al, 2010).…”

TFAM forces mtDNA to make a U-turn

“…Remarkably, TFAM appear to share both the architectural role of the HU/IHF proteins and TBP's role of organizing the transcription initiation complex. In fact, similar to the role of TBP, the specific interaction between TFAM and the mitochondrial promoters, which results in the dramatic bending of mtDNA, is likely of central importance in the activation of mitochondrial transcription, as it results in positioning the carboxy--terminal tail of TFAM in close proximity to the transcription start site occupied by the POLRMT--TFB2M complex 17 (Fig. 1).…”

“…What is clear, though, is that TFB2M interacts directly with promoter DNA near the transcription start site during transcription initiation 17 . The structure of human mitochondrial RNA polymerase (POLRMT) has recently been solved and shows strong similarity to the pol I family of polymerases, in particular to T7 RNA polymerase 20 …”

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

“…However, we demonstrate that POLRMT and h-mtTFB2 can carry out specific initiation from HSP1 and LSP in vitro. Recombinant human transcription systems have been developed previously (14,49,52) that are capable of assessing the individual contributions of the known core components (POLRMT, h-mtTFB1, h-mtTFB2, and h-mtTFA), however the ability of h-mtTFB2 and POLRMT alone (i.e., in the absence of h-mtTFA) to initiate promoter-specific transcription has not been acknowledged previously. In fact, Gaspari et al (53) reported that mitochondrial RNA polymerase/h-mtTFB2 complexes cannot initiate normal or abortive transcription from the LSP in vitro and concluded that h-mtTFA is an obligate member of mitochondrial transcription initiation complexes.…”