Organization of the human mitochondrial transcription initiation complex

Abstract

Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human mitochondrial transcription initiation complex on the light-strand promoter (LSP) through solution X-ray scattering, electron microscopy (EM) and biochemical studies. Our EM results demonstrate a compact organization of the initiation complex, suggesting that protein–protein interactions might help mediate initiation. We demonstrate that, in the absence of DNA, only POLRMT and TFAM form a stable interaction, albeit one with low affinity. This is consistent with the expected transient nature of the interactions necessary for initiation and implies that the promoter DNA acts as a scaffold that enables formation of the full initiation complex. Docking of known crystal structures into our EM maps results in a model for transcriptional initiation that strongly correlates with new and existing biochemical observations. Our results reveal the organization of TFAM, POLRMT and TFB2M around the LSP and represent the first structural characterization of the entire mitochondrial transcriptional initiation complex.

Structural models of mammalian mitochondrial transcription factor B2

“…As shown in Figure 5c, Similarly, the longest -helix that can be accommodated in the protrusion has a maximum of 15 residues, accounting for only half of the missing N-terminal residues. The compact conformation of the N-terminus observed in our SAXS model was not observed in a recently published SAXS model of h-TFB2M [57]. In the published model, the N-terminus forms a tail structure reminiscent of that seen in the crystal structure of the translation factor TFB1M from mouse [58].…”

“…Although TFB2M has been shown to interact with other transcription components in the context of cell-free systems [19], similar interactions have not been observed using highly purified proteins [57]. Based on our models, we speculate that DNA may be essential for interaction of TFB2M with factors such as POLRMT.…”

“…This is in agreement with our model which predicts Lys-325 to interact with DNA (Figure 7b); the loss of transcription activity is possibly due to the inability of the KHR/AAA h-TFB2M mutant to bind DNA promoter. Additional evidence in favor of model 2 over the other two models is provided by comparing our models to the cryo-EM map generated for the initiation complex that included h-POLRMT in complex with DNA promoter, h-TFB2M and h-TFAM[57]. While all three models could be accommodated by the cryo-EM envelope, model 2 works best(Figure 10).…”

Maintenance and Expression of Mammalian Mitochondrial DNA

“…Next, TFAM interacts with POLRMT and recruits the protein to the promoter. POLRMT recruitment depends on direct interactions with TFAM, but it also involves structural changes in the promoter region, which facilitate POLRMT-DNA interactions (47)(48)(49). In agreement with this notion, conditions that induce promoter breathing can circumvent the strict TFAM requirement for transcription initiation (38).…”

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

“…Recombinant human mitochondrial transcription factor A (residues 43-246) was expressed with an N-terminal His-tagged maltose-binding protein fusion and was purified untagged as described previously (supplemental Fig. S1) (50). The catalytically inactive BamHI-E111A restriction endonuclease, used in a previous study (32), was provided by New England Biolabs (Ipswich, MA).…”