A novel intermediate in transcription initiation by human mitochondrial RNA polymerase

Abstract

The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP), structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation-TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate-a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein-protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription.

Figure 1.

TFAM makes direct interactions with mtRNAP. (A) TFAM is absolutely required for efficient transcription of both mtDNA promoters. In vitro transcription assay was performed with the nucleotide sets lacking CTP using PCR amplified templates with the HSP1 (lanes 1,2) or LSP (lanes 3,4) promoters. The gel image is overexposed to dramatize lack of transcription initiation on the LSP and trace activity (<0.5%) observed on the HSP1 in TFAM absence. (B) TFAM-mtRNAP interactions do not require TFB2M but depend on the DNA presence. The complexes were assembled using MBP-modified Cys217TFAM and ³²P-labeled mtRNAP and TFB2M (where indicated) and UV-irradiated in the absence (lanes 1,2) or in the presence (lanes 3,4) of DNA. (C) Location of the residues probed in photo cross-linking experiments using MBP or pBpa (yellow spheres) on TFAM-DNA structure. (D) Scanning cross-linking of pBpa-containing TFAM and mtRNAP. The pre-initiation complexes were assembled using ³²P-labeled mtRNAP (50 nM), 50 nM LSP and 50 nM TFAM having pBpa at the position indicated, UV irradiated and resolved in SDS-PAGE. Note that covalently linked polypeptides may migrate differently depending on the point of attachment. (E) TFAM does not cross-link to the heterologous mtRNAP. Pre-initiation complexes were assembled using MBP-modified Cys217TFAM and mtRNAP (lanes 1–3) or yeast mtRNAP (RPO41) (lanes 4,5) in the absence or presence of DNA (LSP for human mtRNAP and 14S promoter for RPO41), as indicated and UV-irradiated. Molecular weight markers are shown in lane 6. Note that molecular weight of RPO41 (155 kDa) is similar to that of TFAM-mtRNAP cross-link. (F) TFAM does not cross-link to TFB2M. Initiation complexes (150 nM) were assembled using mtRNAP, ³²P-labeled TFB2M, MBP-modified TFAM and the LSP promoter. The grey arrow with an asterisk marks the expected position of the TFB2M-TFAM cross-linking species. (G) TFAM-mtRNAP interactions require DNA long enough to accommodate both proteins. Cross-linking was performed using Cys217MBP-TFAM and WT RNAP and synthetic double-stranded DNA having nonspecific sequence and the lengths indicated.

Figure 2.

TFAM interacts with the N-terminal region of mtRNAP. (A) Mapping of TFAM-mtRNAP cross-link with NTCB. ³²P-labeled Δ119 mtRNAP was treated with NTCB to generate a set of peptide markers (lane 1). The pre-IC (50 nM) was assembled with ³²P-labeled Δ119 mtRNAP and 217MBP-TFAM and UV irradiated. The cross-linked species (lanes 6–9) were separated from the free mtRNAP (lanes 2–5) and treated with NTCB for 5 (lanes 3,7), 10 (lanes 4,8) or 15 (lanes 5,9) min. The residual low molecular bands in lanes 6–9 likely represent de-cross-linking taking place during the electro-elution procedure. (B) Fine mapping of TFAM-mtRNAP cross-link with LysC. The pre-IC was assembled as described above and treated with LysC protease for the time indicated before (lanes 2–4) and after (lanes 6–8) UV-irradiation. The 3.5 kDa peptide visible on Lys C cleavage corresponds to the very N-terminus of mtRNAP (sequence MGHHHHHHRRASVGRWAKILEKDKRTQQMRMQRLK, the PKA site is underlined). (C) Mapping of 217pBpa-TFAM cross-linking region in mtRNAP with hydroxylamine. The cross-linked pre-IC (lane 2) was treated with hydroxylamine for 4 h (lane 3) and the products of the reaction resolved using SDS-PAGE. Radioactive protein markers (lane 1) were generated using CNBr cleavage of ³²P-labeled mtRNAP. (D) Schematics of the cross-link mapping data illustrating regions of mtRNAP–TFAM interactions.

Figure 3.

TFAM interaction region in mtRNAP. (A). Cross-linking of TFAM with mtRNAP deletion mutants. The cross-linking was performed using Cys217MBP-TFAM and the mtRNAP mutants indicated in the presence of the LSP promoter. (B) Importance of 120–134 region of mtRNAP for TFAM interactions. Cross-linking was performed using ³²P-labeled Cys217MBP and the mtRNAP mutants indicated on LSP, HSP1 or nonspecific (NS) DNA template. (C) Sequence conservation in the TFAM-binding region of mtRNAP of different mammalian and avian species. Black arrows and letters indicate point mutations made in this region of mtRNAP. The star indicates substitutions to pBpa. Lysine residues cleaved by LysC are marked by blue arrows. (D and E) Relative transcription activity of mtRNAP mutants having deletions or substitutions in the region of TFAM binding. In vitro transcription initiation assay was performed as described in ‘Materials and Methods’ section using the LSP template.

Figure 4.

Functional activity of mtRNAP mutants. (A). Substitutions within mtRNAP TFAM-binding region result in a loss of cross-linking efficiency. The pre-ICs (50 nM) were assembled using ³²P-labeled 217MBP-TFAM and Δ119 (lanes 1,2) or mutant mtRNAP (lanes 3–6) as indicated and UV-irradiated. The cross-linking species were separated using 10% SDS-PAGE. (B). MtRNAP having pBpa in the TFAM-binding region cross-links to TFAM. The pre-ICs were assembled using ³²P-labeled TFAM (50 nM) and T132pBpa-mtRNAP (50 nM, lane 2, and 100 nM, lane 3), UV-irradiated for 5 min and analyzed as described above.

Figure 5.

The upstream promoter DNA region contours mtRNAP molecule in the pre-IC. (A) DNA cross-linking at −49 base is TFAM-dependent. Pre-initiation complexes (150 nM of ³²P-labeled template containing 4-thio UMP at position −49, 150 nM mtRNAP and 0–400 nM TFAM) were UV-irradiated for 15 min. (B) Mutant mtRNAP lacking N-terminal TFAM-binding site does not cross-link to the upstream promoter DNA. The cross-link was performed using WT or Δ150 mtRNAP in the presence (lanes 2,4) or absence (lanes 1,3) of TFAM by UV-irradiation for 15 min. (C) Promoter sequence is required for DNA-TFAM cross-linking. The reaction contained WT mtRNAP, TFAM (where indicated) and templates with (lanes 1,2) or without (lanes 3,4) LSP promoter sequence. (D) The far upstream promoter region (−60 to −40) is important for efficient transcription. Transcription activity was measured using synthetic template having LSP promoter from −40 (‘−40’, lane 1), nonspecific sequence from −40 to −60 (‘−60NS’, lane 2) or LSP promoter from −60 (‘−60’, lane 3).

Figure 6.

Assembly of transcription initiation complexes in human mitochondria. Binding and bending of DNA allows for TFAM and mtRNAP interaction and recruitment of the latter to the promoter where it forms a pre-initiation complex. The pre-IC, in turn, recruits TFB2M, which is required for promoter melting and initiation of RNA synthesis.