Limits...
A model for transcription initiation in human mitochondria.

Morozov YI, Parshin AV, Agaronyan K, Cheung AC, Anikin M, Cramer P, Temiakov D - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, School of Osteopathic Medicine, Rowan University, 2 Medical Center Dr., Stratford, NJ 08084, USA.

Show MeSH
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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4402542&req=5

Figure 2: 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).

Mentions: Using chemical mapping with CNBr under ‘single hit’ (19,20) conditions, we mapped the region in TFB2M that interacts with mtRNAP to the interval flanked by residues 315–352 (Figure 2A and Supplementary Figure S3). This region corresponds to the α8 helix in the model of TFB2M, which was built based on the structure of its homolog, TFB1M (21) (Figure 2B). Despite a relatively low-sequence conservation between TFB1M (which is not a transcription factor) and yeast homolog of TFB2M, Mtf1 (transcription factor) these proteins exhibit high structural conservation (21,22). The very N-terminal domain of TFB2M (res 1–69) is implicated in DNA binding and interaction with the priming ATP (15), however, does not share any sequence homology with TFB1M and is illustrated as a dashed line in this model (Figure 2B).


A model for transcription initiation in human mitochondria.

Morozov YI, Parshin AV, Agaronyan K, Cheung AC, Anikin M, Cramer P, Temiakov D - Nucleic Acids Res. (2015)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402542&req=5

Figure 2: 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).
Mentions: Using chemical mapping with CNBr under ‘single hit’ (19,20) conditions, we mapped the region in TFB2M that interacts with mtRNAP to the interval flanked by residues 315–352 (Figure 2A and Supplementary Figure S3). This region corresponds to the α8 helix in the model of TFB2M, which was built based on the structure of its homolog, TFB1M (21) (Figure 2B). Despite a relatively low-sequence conservation between TFB1M (which is not a transcription factor) and yeast homolog of TFB2M, Mtf1 (transcription factor) these proteins exhibit high structural conservation (21,22). The very N-terminal domain of TFB2M (res 1–69) is implicated in DNA binding and interaction with the priming ATP (15), however, does not share any sequence homology with TFB1M and is illustrated as a dashed line in this model (Figure 2B).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, School of Osteopathic Medicine, Rowan University, 2 Medical Center Dr., Stratford, NJ 08084, USA.

Show MeSH