. 2015 Apr 20;43(7):3726-35.
Epub 2015 Mar 23.
A Model for Transcription Initiation in Human Mitochondria
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A Model for Transcription Initiation in Human Mitochondria
Nucleic Acids Res
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Regulation of transcription of mtDNA is thought to be crucial for maintenance of redox potential and vitality of the cell but is poorly understood at the molecular level. 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.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Identification of the key interactions between mtRNAP and transcription factors. (
A) Schematic model of transcription initiation in mitochondria. TFAM recruits mtRNAP to promoter to form a pre-initiation complex (pre-IC). TFB2M binding to the pre-IC results in promoter melting and formation of an ‘open’ IC. Protein–protein interactions in pre-IC and IC were probed by pBpa cross-linking (blue stars); interactions between TFB2M variants and mtRNAP—with DSG cross-linker (yellow star); DNA–protein interactions—with photo reactive base analogs, 4-thioUMP and 6-thioGMP (red stars). ( B) Structure of TFAM showing major cross-link sites in the C-terminus. Conserved ‘RKD loop’ in the C-terminal domain of TFAM (PDB ID 3TMM) is shown illustrating location of residues which substitution to pBpa resulted in cross-link with mtRNAP. ( C) Mapping of TFAM-233pBpa cross-link to mtRNAP. The pre-ICs were assembled using mutant mtRNAPs having a single asparagine-glycine (NG) pair at position 408, 443, 462 or 493 and 32P-labeled 233pBpa-TFAM, UV-irradiated and treated with hydroxylamine (lanes 2, 4, 6 and 8). Cleavage pattern is consistent with location of the major cross-linking site in the region 444–462 of the D helix of mtRNAP. (D) Mapping of TFAM-227pBpa cross-link to mtRNAP. The pre-ICs were assembled using mutant mtRNAPs having a single NG pair at position 150 and 32P-labeled 217pBpa-TFAM or 227pBpa-TFAM, UV-irradiated and treated with hydroxylamine (lanes 3 and 4). In both reactions the N-terminal mtRNAP fragments were labeled (lanes 3 and 4), suggesting that the cross-link is to region 44–150 in mtRNAP. These data, taken together with the finding that Δ119 mtRNAP efficiently cross-links to the 227pBpaTFAM (13), suggest that the cross-linking is to the interval 120–150. Lanes 1–3 represent essential controls and have been published previously (13). ( E, F) Scanning cross-linking of pBpa-containing mtRNAP and TFB2M. The ICs were assembled using 32P-labeled TFB2M, LSP, TFAM and mtRNAP having pBpa at the position indicated. ( G) Pre-IC interacts with TFB2M when assembled on promoter DNA. The ICs were assembled as above using 591pBpa-mtRNAP and the LSP promoter (lane 2) or non-specific DNA (NS, lane 3).
Identification of the binding sites in mtRNAP and TFB2M.
(A, B) Mapping of mtRNAP–TFB2M cross-link. MtRNAP–TFB2M cross-link (lanes 1–3) and 32P-TFB2M (lanes 4–6) were treated with CNBr for the time indicated. Methionine cleavage pattern reveals two band representing labeled peptides 352–396 and 365–396 in TFB2M that were not shifted by the cross-linking to mtRNAP and is consistent with cross-link location between M315 and M352 in the α8 helix of TFB2M (lower panel and Supplementary Figure S3). (C) TFB2M mutant having substitutions in the α8 helix cannot support transcription. Transcription assay was performed with the wild-type (WT) (lanes 1–4) and mutant TFB2M (KHR/AAA, lanes 5–8). (D) Deletion of the B-loop of mtRNAP results in decrease of IC assembly efficiency. The pre-ICs were assembled using WT (lanes 1–6), B-loop deletion (lanes 7–11) or intercalating hairpin deletion (lanes 12–15) mtRNAP and incubated with TFB2M in the presence of DSG. (E) DNA–mtRNAP interactions in pre-IC and IC. The complexes were assembled on the LSP promoter containing photo reactive 4-thio UMP or 6-thio dGMP at the positions indicated and UV-irradiated. ( F) Both TFB2M and mtRNAP interact with the −5 template base of promoter. The LSP promoter template containing 6-thio dGMP was incubated with the proteins indicated and UV irradiated. Note the increase of DNA–mtRNAP cross-link upon addition of TFB2M to the pre-IC (lane 4). (G) Mapping of mtRNAP interaction with the −49 template base in the pre-IC. The cross-linking was performed using Δ119 mtRNAP having NG pair at position 369 (lanes 1 and 2), 408 (lane 3), 443 (lane 4), 493 (lane 5) and 556 (lane 6). Cleavage of the NG369 and NG408 mtRNAP mutants generates a single labeled fragment corresponding to the C-terminal region of mtRNAP (lanes 2 and 3). The NG443 mtRNAP cleavage produces two fragments. One fragment corresponds to the N-terminal region (residues 44–443, 80% efficiency), the other fragment corresponds to the C-terminal fragment (residues 444–1230, 20% efficiency), suggesting that the major DNA cross-linking site is located between residues 409–443 (lane 4). Consistent with this, cleavage of NG493 and NG556 mtRNAPs verified that the location of the major cross-linking site was N-terminal to these cleavage site positions (lanes 5 and 6). Mol. weights of the protein markers (Mark12, Invitrogen) are indicated to the left of the panel (kDa).
Structural model of human mitochondrial transcription IC. The model combines data obtained by mapping of protein–protein and DNA–protein interactions (this work) as well as mapping of TFB2M interactions with the priming substrate and +1 template DNA base (13). Large horizontal bars denote mtRNAP (with major domain and structural elements indicated), TFB2M (blue) and TFAM (red). Lines and arrows connect contacts identified by cross-links from specific nucleotide positions or amino acids and protein segments (yellow shaded boxes) of mtRNAP or transcription factors. The positions of nucleotide residues are numbered relative to the promoter start site, which is denoted +1.The extent of promoter melting (region −4 to +3) is shown according to pre-melted promoter template assay (13). TFAM makes interactions with the −15/−35 region of promoter (not shown).
Model of the pre-IC. Two experimental data sets (mtRNAP, PDB ID 3SPA and DNA/TFAM complex, PDB ID 3TMM) were used to model the pre-IC based on biochemical data. The upstream and downstream promoter DNA regions (depicted as ribbons, T-strand, blue, NT-strand, cyan) were extended to emulate trajectory of the nucleic acid in the pre-IC. MtRNAP is depicted as a ribbon (color codes as in Figure 3). A Mg
2+ ion (orange) was placed according to a T7 RNAP structure. TFAM is shown in red (ribbon). The TFAM and −49 DNA cross-linking sites in mtRNAP are indicated in yellow.
A model of transcription initiation process in human mitochondria. The models of the pre-IC and IC are shown as a surface representation. The IC model was generated using mtRNAP ‘open’ conformation found in the elongation complex (PDB ID 4BOC). MtRNAP subdomains are colored according to Figure 3, TFB2M is in light blue. The model suggests that the N-terminus of TFB2M descents into the active site of mtRNAP by passing through the opening between the thumb subdomain and the intercalating hairpin.
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Mitochondria / metabolism*