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. 2014 Apr;42(6):4100-12.
doi: 10.1093/nar/gkt1360. Epub 2014 Jan 9.

Organization of the human mitochondrial transcription initiation complex

Elena Yakubovskaya  1 , Kip E Guja, Edward T Eng, Woo Suk Choi, Edison Mejia, Dmitri Beglov, Mark Lukin, Dima Kozakov, Miguel Garcia-Diaz
Affiliations

Organization of the human mitochondrial transcription initiation complex

Elena Yakubovskaya et al. Nucleic Acids Res. 2014 Apr.

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.

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Figures

Figure 1.
Figure 1.
EM reconstruction of the mitochondrial transcription initiation complex. (A) Representative raw micrograph of uranyl-stained grids containing a quaternary POLRMT:TFB2M:TFAM:DNA complex. (B) Representative reference-free class averages (top row) and corresponding re-projections of the final model (bottom row). (C) The final resolution was estimated as 25 Å using the Fourier shell correlation criterion with a cutoff of 0.5. (D) Euler angle distribution plot of POLRMT:TFB2M:TFAM particle projections by the end of the refinement procedure. (E) The final structure of the mitochondrial transcription initiation complex shown in 90° rotations.
Figure 2.
Figure 2.
POLRMT physically interacts with TFAM. (A) Pull-down assay to analyze interactions with purified untagged TFAM or TFAMΔCTE. The proteins were covalently bound to NHS-activated agarose and incubated with POLRMT, TFB2M or POLRMTΔNTE to test for the presence of a direct physical interaction. As a control for non-specific binding to the resin, inactivated resin without immobilized protein was used. Compared with the control, the only clear interaction observed was between full-length POLRMT and full-length TFAM. (B) A reverse experiment performed by covalently immobilizing full-length POLRMT or POLRMTΔNTE onto the resin. We then probed for interactions with TFB2M, TFAM and TFAMΔCTE. The result further suggests the existence of an interaction between POLRMT and TFAM. However, no evidence was found of a direct interaction between POLRMT and TFB2M or between TFB2M and TFAM. (C) Quantification of the results shown in panels A and B. The Precision Plus Protein Dual Color Standard protein marker (BioRad) shown in panels A and B (indicated with the letter M) contains the following bands: 250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa and 25 kDa. A control lane in panels A and B is indicated with the letter C and the predicted molecular masses (based on sequence) for the proteins are as follows: 134.5 kDa for POLRMT, 101 kDa for POLRMTΔNTE, 24.4 kDa for TFAM, 21.9 kDa for TFAMΔCTE and 43.3 kDa for TFB2M.
Figure 3.
Figure 3.
The interaction between POLRMT and TFAM is dependent on the NTE. (A) Isothermal titration calorimetry experiments of TFAM-binding to POLRMT (B) TFAM-binding to POLRMTΔNTE or (C) TFAMΔCTE binding to POLRMT performed at 25°C. In all three cases, the top panel represents the heat signal for injections of TFAM or TFAMΔCTE into the calorimeter cell containing POLRMT or POLRMTΔNTE. The bottom panel displays the integrated heat for each injection after peak integration and subtraction of basal values fitted to a simple single-site binding model (solid line). The stoichiometry (N) and binding constant (KD) for the interaction between TFAM and POLRMT are shown in the bottom panel. No thermal effect was observed for the putative interaction between TFAM and POLRMTΔNTE, or between TFAMΔCTE and POLRMT.
Figure 4.
Figure 4.
POLRMT and TFB2M interact in a DNA-dependent manner. Gel-filtration experiment demonstrating that both proteins could be resolved as separate peaks on a Sephadex 200 10/300 gel filtration column in the absence of promoter DNA (A) and (B). However, when linear promoter DNA was included in the experiment, both POLRMT and POLRMTΔNTE co-eluted with TFB2M, suggesting that a stable POLRMT:TFB2M complex is formed in the presence of promoter DNA (C) and (D). Actual size-exclusion chromatograms for experiments without the promoter DNA (E) and with promoter DNA (F).
Figure 5.
Figure 5.
Localization of the components of the transcription initiation complex. (A) Surface representation of the EM map for POLRMT apoenzyme. The flexibly fitted crystal structure (PDB ID: 3SPA) is shown superimposed (cross-correlation coefficient 0.86) in cartoon representation with a 90° rotation. (B) SAXS/WAXS model of TFB2M. Surface representation (gray) of a filtered average bead model that describes the data (χ2 = 1.59). Shown inside the surface is the X-ray crystallographic structure of TFB1M (PDB: 4GC5). (C) The experimental and theoretical scattering profiles of TFB2M and TFB1M. The observed scattering profile for TFB2M is well fit by the calculated profile derived from the TBF1M crystal structure (χ2 = 1.68). (D) EM and SAXS data reveal the relative positions of POLRMT and TB2M within the complex. The EM envelope for the full complex is shown as a gray mesh. The POLRMT EM map and TFB2M SAXS model have been positioned within the EM envelope and are shown in green and orange, respectively. (E) Relative orientation of the three protein components of the initiation complex (PDB IDs: 3SPA, 4GC5 and 3TQ6). The EM envelope for the full complex is shown as grey mesh, whereas POLRMT, TFAM and TFB2M are shown as ribbons colored green, purple and orange, respectively. The asterisk indicates additional volume corresponding to the N-terminal region of POLRMT that was not resolved in the crystal structure.
Figure 6.
Figure 6.
Model of the intact mitochondrial transcription initiation machinery. The EM envelope for the full complex is shown as a grey mesh, while POLRMT, TFAM, TFB2M and the promoter DNA are shown as ribbons colored green, purple, orange and blue, respectively. The asterisk indicates additional volume corresponding to the N-terminal region of POLRMT that was not resolved in the crystal structure.
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