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

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.

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.

Figure 2. TFAM binds HSP1 in a reverse orientation

(A) The labeled DNA template used to determine the orientation of TFAM on HSP1. Thymine 550 was replaced with bromo-uracil to label the proximal half-site. (B) A presentation of the 22 bp HSP1 structure, showing its U-turn shape and the location of intercalating residues. (C) The TFAM/HSP1 complex showing location of the anomalous signal of bromo-uracil (orange) relative to the domains of TFAM. The anomalous signal is adjacent to HMG-box B. (D) Close-up view of the anomalous electronic density in a Friedel-pair difference map revealing a > 5σ peak (orange).

Figure 3. Conversion of HSP1 into a promoter dependent on DNA bending

(A) Schematic of HSP1, LSP, and two engineered promoters (EP1 and EP2) derived from HSP1. EP1 and EP2 were designed to reverse the orientation of TFAM on HSP1. (B) Representative transcription reactions with wild-type TFAM and the L6 mutant that is deficient in DNA bending. (C) Quantification of transcription reactions, with error bars representing standard deviations from three independent experiments. Values are normalized to that of wild-type TFAM.

Figure 4. Dimerization interface

(A) Overview of two molecules of TFAM forming a dimer in the TFAM/HSP1crystal structure. Each TFAM molecule is bound to its own DNA fragment. Helix 3 from one HMG-box A domain forms an antiparallel interface with the corresponding helix 3 from another molecule. The locations of the cysteines used for protein labeling are indicated in red. (B) Close-up of the antiparallel dimerization interface. Residues involved in hydrogen bonds and salt bridges are labeled. (C) Superimposition of the dimerization interfaces from all four TFAM/DNA structures: TFAM/LSP-28 bp (green, pdb:3TMM), TFAM/LSP-22 bp (cyan, pdb:3TQ6), TFAM/HSP1-22 bp (purple), and TFAM/nonspecific DNA-22 bp (grey). RMSD values relative to TFAM/LSP-22bp are as follows: TFAM/LSP-28 bp, 0.887; TFAM/HSP1-22 bp, 1.056; TFAM/nonspecific DNA-22 bp, 0.951.

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.

Figure 6. Structural determinants of DNA compaction

(A) A schematic of the TPM assay. The bead is attached by a single DNA molecule to the glass surface. Upon addition of TFAM, the contour length of the DNA molecule is reduced, causing a decrease in the bead’s radius of motion. (B) Effect of increasing concentrations of TFAM on the DNA contour length. A 1910 bp DNA fragment was used. Error bars indicate standard error of the mean from three independent experiments. (C) DNA compaction by TFAM on DNA fragments of varying lengths. Error bars represent standard error of the mean from three independent experiments. (D) Fractional shortening of DNA by TFAM as a function of DNA length. (E) Maximal DNA compaction by wild-type TFAM and mutants. A 1910 bp DNA fragment was used. Error bars illustrate standard error of the mean from three independent experiments.

Figure 7. Models of transcription activation by TFAM and mtDNA packaging

(A), (B) Comparison of TFAM function on LSP versus HSP1. When bound on LSP (A), TFAM is oriented with the HMB-box B domain binding the distal half-site. As a result, the U-turn in DNA is necessary to position the C-terminal tail (small yellow region) towards the transcriptional machinery. The C-terminal tail is essential for transcriptional activation and physically interacts with TFB2M, but additional interactions with mtRNA polymerase are also possible. When bound to HSP1 (B), TFAM is oriented with the HMG-box B domain binding the proximal half-site. The C-terminal tail is positioned close to the transcriptional machinery, and DNA bending is dispensable for transcriptional activation. (C) Model of mtDNA compaction. Upon binding to mtDNA, each TFAM molecule imposes a local U-turn. When TFAM coats mtDNA, the formation of multiple U-turns results in mtDNA compaction. In addition, TFAM monomers can dimerize through the HMG-box A domain. This interaction forms DNA loops, which further compact mtDNA in the nucleoid.