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Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation.

Ngo HB, Lovely GA, Phillips R, Chan DC - Nat Commun (2014)

Bottom Line: Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not.This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction.We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA [2] Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA.

ABSTRACT
TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)--the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

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Overview of the TFAM-mtDNA complexes(A) The domain structure of mature TFAM. Residues 1–42 constitute the mitochondrial targeting sequence that is cleaved upon import of TFAM into the mitochondrial matrix. (B) Schematic of DNA sequences bound within TFAM crystals. Note the different orientations of TFAM on LSP versus HSP1. The nonspecific sequence is from the ATPase6 gene. The half-sites of LSP and HSP1 are indicated. (C), (D), (E) Side view of the TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA complexes, respectively. The major intercalating residues, Leu58 and Leu182, are highlighted. The DNA fragments are color-coded as in (B). (F) Superimposition of TFAM crystal structures, color-coded as in (B). (G) Comparison of roll angle values for TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA. Note that there are two peaks of DNA distortion, at the positions where Leu58 and Leu182 intercalate. (H) FRET assay for DNA bending with three different DNA templates: LSP, HSP1, and nonspecific DNA. Data points are the average of three independent experiments, with error bars representing standard deviations.
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Figure 1: Overview of the TFAM-mtDNA complexes(A) The domain structure of mature TFAM. Residues 1–42 constitute the mitochondrial targeting sequence that is cleaved upon import of TFAM into the mitochondrial matrix. (B) Schematic of DNA sequences bound within TFAM crystals. Note the different orientations of TFAM on LSP versus HSP1. The nonspecific sequence is from the ATPase6 gene. The half-sites of LSP and HSP1 are indicated. (C), (D), (E) Side view of the TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA complexes, respectively. The major intercalating residues, Leu58 and Leu182, are highlighted. The DNA fragments are color-coded as in (B). (F) Superimposition of TFAM crystal structures, color-coded as in (B). (G) Comparison of roll angle values for TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA. Note that there are two peaks of DNA distortion, at the positions where Leu58 and Leu182 intercalate. (H) FRET assay for DNA bending with three different DNA templates: LSP, HSP1, and nonspecific DNA. Data points are the average of three independent experiments, with error bars representing standard deviations.

Mentions: We and others have previously solved the crystal structure of TFAM bound to LSP8, 9. The TFAM binding site at LSP is 22 base pairs long and is composed of two half-sites (Fig. 1A, B). TFAM contains two HMG (high mobility group)-box domains (HMG-box A and HMG-box B) that each intercalates into the minor groove of a half-site. Each intercalation contributes to distortion of the DNA, resulting in a dramatic U-turn of the LSP sequence (Fig. 1C). Between the two HMG-box domains is a helical linker with a positively charged surface that interacts with the negatively charged backbone of the DNA. The C-terminal tail of TFAM is required for activation of the transcriptional machinery. In the TFAM/LSP structure, the carboxyl terminal HMG-box B domain binds to the half-site distal from the transcriptional start site. Because the C-terminal tail is adjacent to HMG-box B, the U-turn in the LSP DNA enables the C-terminal tail to contact the transcriptional machinery. Consistent with this idea, TFAM mutants that are deficient in DNA bending are inactive for transcriptional activation at LSP8. In contrast, the same mutants are fully active at HSP1. Based on sequence comparisons, TFAM has been suggested to bind to HSP1 in the reverse orientation compared to LSP10, 11. We proposed that in this reverse orientation, the C-terminal tail would be located near the half-site adjacent to the transcriptional machinery, rendering DNA bending unnecessary8. However, this model hinges on the expectation that the TFAM is indeed reversed on HSP1 compared to LSP, an idea that lacks experimental evidence.


Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation.

Ngo HB, Lovely GA, Phillips R, Chan DC - Nat Commun (2014)

Overview of the TFAM-mtDNA complexes(A) The domain structure of mature TFAM. Residues 1–42 constitute the mitochondrial targeting sequence that is cleaved upon import of TFAM into the mitochondrial matrix. (B) Schematic of DNA sequences bound within TFAM crystals. Note the different orientations of TFAM on LSP versus HSP1. The nonspecific sequence is from the ATPase6 gene. The half-sites of LSP and HSP1 are indicated. (C), (D), (E) Side view of the TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA complexes, respectively. The major intercalating residues, Leu58 and Leu182, are highlighted. The DNA fragments are color-coded as in (B). (F) Superimposition of TFAM crystal structures, color-coded as in (B). (G) Comparison of roll angle values for TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA. Note that there are two peaks of DNA distortion, at the positions where Leu58 and Leu182 intercalate. (H) FRET assay for DNA bending with three different DNA templates: LSP, HSP1, and nonspecific DNA. Data points are the average of three independent experiments, with error bars representing standard deviations.
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Related In: Results  -  Collection

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Figure 1: Overview of the TFAM-mtDNA complexes(A) The domain structure of mature TFAM. Residues 1–42 constitute the mitochondrial targeting sequence that is cleaved upon import of TFAM into the mitochondrial matrix. (B) Schematic of DNA sequences bound within TFAM crystals. Note the different orientations of TFAM on LSP versus HSP1. The nonspecific sequence is from the ATPase6 gene. The half-sites of LSP and HSP1 are indicated. (C), (D), (E) Side view of the TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA complexes, respectively. The major intercalating residues, Leu58 and Leu182, are highlighted. The DNA fragments are color-coded as in (B). (F) Superimposition of TFAM crystal structures, color-coded as in (B). (G) Comparison of roll angle values for TFAM/LSP, TFAM/HSP1, and TFAM/nonspecific DNA. Note that there are two peaks of DNA distortion, at the positions where Leu58 and Leu182 intercalate. (H) FRET assay for DNA bending with three different DNA templates: LSP, HSP1, and nonspecific DNA. Data points are the average of three independent experiments, with error bars representing standard deviations.
Mentions: We and others have previously solved the crystal structure of TFAM bound to LSP8, 9. The TFAM binding site at LSP is 22 base pairs long and is composed of two half-sites (Fig. 1A, B). TFAM contains two HMG (high mobility group)-box domains (HMG-box A and HMG-box B) that each intercalates into the minor groove of a half-site. Each intercalation contributes to distortion of the DNA, resulting in a dramatic U-turn of the LSP sequence (Fig. 1C). Between the two HMG-box domains is a helical linker with a positively charged surface that interacts with the negatively charged backbone of the DNA. The C-terminal tail of TFAM is required for activation of the transcriptional machinery. In the TFAM/LSP structure, the carboxyl terminal HMG-box B domain binds to the half-site distal from the transcriptional start site. Because the C-terminal tail is adjacent to HMG-box B, the U-turn in the LSP DNA enables the C-terminal tail to contact the transcriptional machinery. Consistent with this idea, TFAM mutants that are deficient in DNA bending are inactive for transcriptional activation at LSP8. In contrast, the same mutants are fully active at HSP1. Based on sequence comparisons, TFAM has been suggested to bind to HSP1 in the reverse orientation compared to LSP10, 11. We proposed that in this reverse orientation, the C-terminal tail would be located near the half-site adjacent to the transcriptional machinery, rendering DNA bending unnecessary8. However, this model hinges on the expectation that the TFAM is indeed reversed on HSP1 compared to LSP, an idea that lacks experimental evidence.

Bottom Line: Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not.This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction.We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA [2] Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA.

ABSTRACT
TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)--the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

Show MeSH
Related in: MedlinePlus