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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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Biochemical analysis of TFAM dimerization(A) Emission spectra in a FRET assay measuring the physical interaction between TFAM molecules. Reactions contained Alexa Fluor 488 (donor)-labeled and/or Alexa Fluor 594 (acceptor)-labeled TFAM. Fluorescence emission spectra showed FRET signal only in the presence of plasmid DNA (magenta trace). Note that this signal was abolished in the dimer mutant (blue trace). (B) Emission spectra of wild-type TFAM incubated with linear DNA of varying lengths. (C) DNA bending by the dimer mutant on three templates. Data points are the average of three independent experiments, with error bars representing standard deviations. (D) Representative transcription assay using wild-type TFAM or the dimer mutant. The LSP template generates a 420 nt full-length (run-off) transcript and a truncated 120 nt transcript. (E) Quantification of transcription reactions with error bars representing standard deviations from three independent experiments.
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Figure 5: Biochemical analysis of TFAM dimerization(A) Emission spectra in a FRET assay measuring the physical interaction between TFAM molecules. Reactions contained Alexa Fluor 488 (donor)-labeled and/or Alexa Fluor 594 (acceptor)-labeled TFAM. Fluorescence emission spectra showed FRET signal only in the presence of plasmid DNA (magenta trace). Note that this signal was abolished in the dimer mutant (blue trace). (B) Emission spectra of wild-type TFAM incubated with linear DNA of varying lengths. (C) DNA bending by the dimer mutant on three templates. Data points are the average of three independent experiments, with error bars representing standard deviations. (D) Representative transcription assay using wild-type TFAM or the dimer mutant. The LSP template generates a 420 nt full-length (run-off) transcript and a truncated 120 nt transcript. (E) Quantification of transcription reactions with error bars representing standard deviations from three independent experiments.

Mentions: To test the physiological function of dimerization, we generated a TFAM mutant with five substitutions (K95A, Y99F, E106A, E112A and R116A; hereafter termed “dimer mutant”) designed to disrupt polar and electrostatic interactions at the interface. These 5 residues in the dimerization interface are conserved in mammals (Supplementary Fig. 3). To assess TFAM dimerization, we developed a FRET-based assay to measure TFAM/TFAM contact. We covalently labeled TFAM molecules with either a donor fluorophore (Alexa Fluor 488) or an acceptor fluorophore (Alexa Fluor 595) using cysteine-maleimide3 chemistry at the single cysteine residue at position 49 (Fig. 4A), which is close to the dimer interface. In the absence of DNA, no FRET was detected between the two labeled populations (red trace, Fig. 5A). However, in the presence of DNA, we found a decrease in donor emission and an increase in acceptor emission (magenta trace, Fig. 5A). This FRET signal could be competed off by excess unlabeled TFAM (Supplementary Fig. 4A). This FRET signal was abolished in the dimer mutant (blue trace, Fig. 5A, Supplementary Fig. 4B), suggesting loss of dimerization. Interestingly, the dimerization of wild-type TFAM was not only dependent on DNA, but also on the length of the DNA. Testing a range of DNA lengths (100, 150, 200, 300, to 400 bp), we found robust TFAM dimerization only with DNA fragments ≥ 200 base pairs (Fig. 5B).


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

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

Biochemical analysis of TFAM dimerization(A) Emission spectra in a FRET assay measuring the physical interaction between TFAM molecules. Reactions contained Alexa Fluor 488 (donor)-labeled and/or Alexa Fluor 594 (acceptor)-labeled TFAM. Fluorescence emission spectra showed FRET signal only in the presence of plasmid DNA (magenta trace). Note that this signal was abolished in the dimer mutant (blue trace). (B) Emission spectra of wild-type TFAM incubated with linear DNA of varying lengths. (C) DNA bending by the dimer mutant on three templates. Data points are the average of three independent experiments, with error bars representing standard deviations. (D) Representative transcription assay using wild-type TFAM or the dimer mutant. The LSP template generates a 420 nt full-length (run-off) transcript and a truncated 120 nt transcript. (E) Quantification of transcription reactions with error bars representing standard deviations from three independent experiments.
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Figure 5: Biochemical analysis of TFAM dimerization(A) Emission spectra in a FRET assay measuring the physical interaction between TFAM molecules. Reactions contained Alexa Fluor 488 (donor)-labeled and/or Alexa Fluor 594 (acceptor)-labeled TFAM. Fluorescence emission spectra showed FRET signal only in the presence of plasmid DNA (magenta trace). Note that this signal was abolished in the dimer mutant (blue trace). (B) Emission spectra of wild-type TFAM incubated with linear DNA of varying lengths. (C) DNA bending by the dimer mutant on three templates. Data points are the average of three independent experiments, with error bars representing standard deviations. (D) Representative transcription assay using wild-type TFAM or the dimer mutant. The LSP template generates a 420 nt full-length (run-off) transcript and a truncated 120 nt transcript. (E) Quantification of transcription reactions with error bars representing standard deviations from three independent experiments.
Mentions: To test the physiological function of dimerization, we generated a TFAM mutant with five substitutions (K95A, Y99F, E106A, E112A and R116A; hereafter termed “dimer mutant”) designed to disrupt polar and electrostatic interactions at the interface. These 5 residues in the dimerization interface are conserved in mammals (Supplementary Fig. 3). To assess TFAM dimerization, we developed a FRET-based assay to measure TFAM/TFAM contact. We covalently labeled TFAM molecules with either a donor fluorophore (Alexa Fluor 488) or an acceptor fluorophore (Alexa Fluor 595) using cysteine-maleimide3 chemistry at the single cysteine residue at position 49 (Fig. 4A), which is close to the dimer interface. In the absence of DNA, no FRET was detected between the two labeled populations (red trace, Fig. 5A). However, in the presence of DNA, we found a decrease in donor emission and an increase in acceptor emission (magenta trace, Fig. 5A). This FRET signal could be competed off by excess unlabeled TFAM (Supplementary Fig. 4A). This FRET signal was abolished in the dimer mutant (blue trace, Fig. 5A, Supplementary Fig. 4B), suggesting loss of dimerization. Interestingly, the dimerization of wild-type TFAM was not only dependent on DNA, but also on the length of the DNA. Testing a range of DNA lengths (100, 150, 200, 300, to 400 bp), we found robust TFAM dimerization only with DNA fragments ≥ 200 base pairs (Fig. 5B).

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