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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.

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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).
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Figure 1: 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).

Mentions: The remnants of the ancient single-subunit RNA polymerases of the Pol A family can be found in modern bacteriophages and DNA-maintaining organelles, such as plastids and mitochondria. In the latter, these RNAPs are charged with responsibility to synthesize mRNA, tRNA and rRNA, and to make RNA primers for replication (1–3). Even though some mitochondrial genomes are small (e.g. human mtDNA), regulation of mitochondrial gene expression is an elaborate process that occurs at various stages and involves many auxiliary factors and DNA and RNA modifying enzymes (4–8). Numerous mitochondrial dysfunctions are associated with defects in expression of mitochondrial genes and contribute to aging and severe pathologies and dysfunctions (9). At the beginning of the gene expression process, transcription of human mitochondrial DNA requires assembly of an initiation complex (IC) composed of mitochondrial RNA polymerase (mtRNAP) and two core transcription factors: TFAM and TFB2M (10–12). Recent studies demonstrated that mtRNAP is recruited to the promoter by formation of direct interactions with the nucleoid protein, TFAM (Figure 1A) (13,14). The resulting complex, called the pre-IC, lacks specificity toward DNA and cannot initiate transcription unless another transcription factor, TFB2M, is bound (13). Upon binding, the N-terminus of TFB2M reaches the active site of mtRNAP where it interacts with the priming substrate and assists in promoter melting (15). However, neither TFB2M nor TFAM binding sites on mtRNAP have been identified and thus the overall architecture of the IC as well as the pre-IC remains obscure. In this work, using biochemical, genetic and structural data we build a comprehensive map of interactions between all components of the IC. These data allowed us to construct a model of transcription initiation which is essential for understanding of molecular mechanisms of promoter binding and recognition and future studies of regulation of gene expression in human mitochondria.


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 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).
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Figure 1: 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).
Mentions: The remnants of the ancient single-subunit RNA polymerases of the Pol A family can be found in modern bacteriophages and DNA-maintaining organelles, such as plastids and mitochondria. In the latter, these RNAPs are charged with responsibility to synthesize mRNA, tRNA and rRNA, and to make RNA primers for replication (1–3). Even though some mitochondrial genomes are small (e.g. human mtDNA), regulation of mitochondrial gene expression is an elaborate process that occurs at various stages and involves many auxiliary factors and DNA and RNA modifying enzymes (4–8). Numerous mitochondrial dysfunctions are associated with defects in expression of mitochondrial genes and contribute to aging and severe pathologies and dysfunctions (9). At the beginning of the gene expression process, transcription of human mitochondrial DNA requires assembly of an initiation complex (IC) composed of mitochondrial RNA polymerase (mtRNAP) and two core transcription factors: TFAM and TFB2M (10–12). Recent studies demonstrated that mtRNAP is recruited to the promoter by formation of direct interactions with the nucleoid protein, TFAM (Figure 1A) (13,14). The resulting complex, called the pre-IC, lacks specificity toward DNA and cannot initiate transcription unless another transcription factor, TFB2M, is bound (13). Upon binding, the N-terminus of TFB2M reaches the active site of mtRNAP where it interacts with the priming substrate and assists in promoter melting (15). However, neither TFB2M nor TFAM binding sites on mtRNAP have been identified and thus the overall architecture of the IC as well as the pre-IC remains obscure. In this work, using biochemical, genetic and structural data we build a comprehensive map of interactions between all components of the IC. These data allowed us to construct a model of transcription initiation which is essential for understanding of molecular mechanisms of promoter binding and recognition and future studies of regulation of gene expression in human mitochondria.

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
Related in: MedlinePlus