Part:BBa_K4305002

TFAM (43aa-246aa)

The mature TFAM protein contains two HMG boxes separated by a linker and a charged C-terminal tail. HMG-box domains allow TFAM to bind, wrap, and bend DNA without any sequence specificity. C-terminal tail is required for activation of promoter-specific mtDNA transcription. TFAM gene (43aa-246aa) was cloned into pET28 vector to overexpress TFAM protein in E.coli. This vector allows the TFAM protein to be fused with 6 x Histidine tag at the N-terminal region.

Literature Review & Experimental Results:

In contrast to the chromatin-based packaging of the nuclear genome, the mitochondrial genome is packaged into non-chromatin nucleoids involving proteins specific to mitochondria, such as TFAM [1]. 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) [2]. TFAM functions in determining the abundance of the mitochondrial genome by regulating packaging, stability, and replication [3]. Human TFAM has an ability to bind to DNA in a sequence-independent manner and is abundant enough to cover whole region of mitochondrial DNA, owing to which TFAM stabilizes mitochondrial DNA through formation of the nucleoid and regulates (or titrates) the amount of mitochondrial DNA [4]. TFAM contains two high mobility group (HMG)-box domains (HMG-box A and HMG-box B) that intercalates into the minor groove of a half-site [2].

The mitochondrial genome contains three promoters - the light strand promoter (LSP), the heavy strand promoter 1 (HSP1), and the heavy strand promoter 2 (HSP2) [4]. When TFAM is bound to these promoters, either LSP or HSP, the TFAM protein distorts mitochondrial DNA into a U-turn [5]. TFAM forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. This is shown as each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of DNA, with the positively charged a-helix serving as a platform to facilitate DNA bending [4].

The mitochondrial protein is processed in six steps: (1) The protein containing the signal sequence is synthesized in the cytoplasm. (2) Signal sequence binds to a receptor in the organelle mebrane. (3) Receptor - protein complex diffuses within membrane to a contact site. (4) Protein is unfolded, moved across the membrane, and refolded. These operations are carried out by the protein transporter complex and its associated chaperone proteins. (5) Once inside, the signal sequence is cleaved off by a specific peptidase. (6) Finally, mature protein is formed inside the mitochondrial matrix.

In the experiment, 43-246aa TFAM was cloned into pET28 (N-terminal Histidine tag, E.coli expression vector) to produced His-tagged TFAM protein. The first 42 amino acids of TFAM guides TFAM into the mitochondria. We cloned the TFAM (43aa to 246aa), which does not contain the mitochondrial signal peptide because TFAM recombinant protein does not have to transport to mitochondria in this project. The mature TFAM protein contains two HMG boxes separated by a linker and a charged C-terminal tail. Regarding the amino acid sequence, the 43rd to 50th amino acids act as a linker, the 50th to 122nd amino acids are translated into the HMG Box-A, the 122nd to 152nd amino acids also act as a linker, the 152nd to 223rd amino acids are translated into the HBG Box-B, and the 223rd to 246th amino acids act as a tail. HMG-box domains allow TFAM to bind, wrap, and bend DNA without any sequence specificity. C-terminal tail is required for activation of promoter-specific mtDNA transcription. TFAM gene (43aa-246aa) was cloned into pET28 vector to overexpress TFAM protein in E.coli. This vector allows the TFAM-protein to be fused with 6 x Histidine tag at the N-terminal region.

While TFAM protein distorts mitochondrial DNA into a U-turn, in the TFAM-DNA complex, the DNA was not distorted. This is because TFAM and DNA are not bound at a specific promoter but they combine non-specifically, which therefore leads to a U-turn not being formed. Instead, TFAM and DNA were compactly binded to form the TFAM-DNA complex.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 64
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References:

[1] B. Ngo, Huu et al. The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA. Nature Structural & Molecular Biology. 18, 1290-1296 (2011).

[2] B.Ngo, Huu et al. Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation. Nature Communications. 5, 3077 (2014).

[3] Kang, Inhae et al. The mitochondrial transcription factor TFAM in neurodegeneration: Emerging evidence and mechanisms. FEBS Lett. 5, 793-811 (2018).

[4] Kang, Dongchon et al. Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions. Mitochondrion. 7, 39-44 (2007).

[5] Rubio-Cosials, Anna et al. Protein Flexibility and Synergy of HMG Domains Underlie U-Turn Bending of DNA by TFAM in Solution. Biophysical Journal. 10, 2386-2396 (2018).