Journal: Nucleic Acids Res / Year: 2014 Title: Organization of the human mitochondrial transcription initiation complex. Authors: Elena Yakubovskaya / Kip E Guja / Edward T Eng / Woo Suk Choi / Edison Mejia / Dmitri Beglov / Mark Lukin / Dima Kozakov / Miguel Garcia-Diaz / 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 ...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.
History
Deposition
Dec 6, 2013
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Header (metadata) release
Jan 15, 2014
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Map release
Jan 29, 2014
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Update
Apr 16, 2014
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Current status
Apr 16, 2014
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_2529.map.gz / Format: CCP4 / Size: 284.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Reconstruction of the human mitochondrial transcription initiation complex
Voxel size
X=Y=Z: 3.4 Å
Density
Contour Level
By EMDB: 4.77 / Movie #1: 4.61
Minimum - Maximum
-2.27669072 - 6.27684975
Average (Standard dev.)
0.0 (±0.99999994)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
-6
-6
-6
Dimensions
42
42
42
Spacing
42
42
42
Cell
A=B=C: 142.8 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
3.4
3.4
3.4
M x/y/z
42
42
42
origin x/y/z
0.000
0.000
0.000
length x/y/z
142.800
142.800
142.800
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-207
-207
-206
NX/NY/NZ
414
414
414
MAP C/R/S
1
2
3
start NC/NR/NS
-6
-6
-6
NC/NR/NS
42
42
42
D min/max/mean
-2.277
6.277
-0.000
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Supplemental data
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Sample components
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Entire : Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA
Entire
Name: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA
Components
Sample: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA
Protein or peptide: Dimethyladenosine transferase 2, mitochondrial
Protein or peptide: Dimethyladenosine transferase 2, mitochondrial
Protein or peptide: Transcription factor A, mitochondrial
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Supramolecule #1000: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA
Supramolecule
Name: Quaternary complex of POLRMT, TFAM, TFB2M and LSP DNA / type: sample / ID: 1000 Details: The complex also has a DNA, that has a mitochondrial light strand promoter sequence Oligomeric state: one POLRMT one TFB2M and one TFAM TFAM) / Number unique components: 3
Reference free classes were divided into two subsets, based on particle size, and two initial models were generated from each subset. One model had a visibly elongated shape, whereas the other was more compact. We utilized both models as starting points for parallel refinement, as implemented in EMAN MULTIREFINE routine
CTF correction
Details: EMAN2 protocol each particle
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 12453
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