A model for transcription initiation in human mitochondria.
Bottom Line: In this study we mapped the binding sites of the core transcription initiation factors TFAM and TFB2M on human mitochondrial RNA polymerase, and interactions of the latter with promoter DNA.This allowed us to construct a detailed structural model, which displays a remarkable level of interaction between the components of the initiation complex (IC).The architecture of the mitochondrial IC suggests mechanisms of promoter binding and recognition that are distinct from the mechanisms found in RNAPs operating in all domains of life, and illuminates strategies of transcription regulation developed at the very early stages of evolution of gene expression.
Affiliation: Department of Cell Biology, School of Osteopathic Medicine, Rowan University, 2 Medical Center Dr., Stratford, NJ 08084, USA.Show MeSH
Mentions: The cross-linking data indicate that in the absence of the promoter TFB2M is unable to bind to mtRNAP efficiently suggesting that binding of TFAM and promoter DNA causes structural changes in mtRNAP. Since the N-terminus of TFB2M is directed toward the active site of mtRNAP, where it interacts with both the priming ATP and a templating (+1) DNA base, such changes should provide a passage within mtRNAP in order to accommodate TFB2M. The opening of a nucleic acid binding cavity and movement of the N-terminal domain relative the palm domain was observed in the structure of mtRNAP elongation complex (16). We therefore used this structure to model interactions within the IC (Figure 5). To model the melted promoter DNA region proximal to the start site, we used homology modeling with the T7 RNAP IC (24). The DNA duplex in this complex is melted in the region from +3 to −4, which is consistent with the data for mtRNAP promoter complex (15). TFB2M was docked manually to the B loop of mtRNAP so it would come in a close proximity to the α8 helix of TFB2M. We further considered that, since the N-terminal extension region of TFB2M interacts with both −5 and +1 bases of the template strand of promoter DNA, the only way for TFB2M to reach the active site of mtRNAP would be via the opening between the thumb (res 720–760) and the intercalating hairpin of mtRNAP. This restraint sets the orientation of TFB2M relative to mtRNAP and results in minimal clashes between these proteins (Figure 5). As we noted previously when modeling the melted promoter region into mtRNAP structure, the intercalating hairpin clashes with the promoter DNA indicating that its location is not fixed in the apo enzyme (18). In phage RNAPs, this structural element is inserted between DNA stands and maintains the trailing edge of the transcription bubble during initiation of transcription (30,31). Our cross-linking data indicate that the intercalating hairpin interacts with the −5 base of promoter only when TFB2M is present (Figure 2F and Supplementary Figure S4). We therefore propose that binding of TFB2M to the adjacent B-loop pushes the intercalating hairpin toward the DNA duplex and forces it to assume a position identical to the position of the corresponding loop in T7 RNAP IC. The repositioning of the intercalating loop may open the passage between the N-terminal domain and the thumb allowing access of TFB2M to the active site of mtRNAP.
Affiliation: Department of Cell Biology, School of Osteopathic Medicine, Rowan University, 2 Medical Center Dr., Stratford, NJ 08084, USA.