Activation and Regulation of Human Mitochondrial Transcription Factor A

Kelsey McMurtry '14 and Wesley Manz '15


I. Introduction

The mitochondrial transcription factor A (TFAM of mtFAM) is a nuclear encoded, mitochondrial-specific, high mobility group (HMG) protein found in humans. Binding of TFAM activates recognition of the promoters on human mitochondrial DNA (mtDNA) by mitochondrial RNA polymerase (MTRNAP). Packaging, replication and maintenance of mtDNA in nucleoprotein entities is dependent upon proteins like TFAM. MtDNA codes for critical factors that form respiratory chain complexes in the mitochondrial oxidative phosphorylation process. This process allows mitochondria to regenerate ATP, making TFAM necessary for efficient generation of energy.

TFAM induces transcription by inserting bends, over regular intervals, in mtDNA. The direction of the DNA helix is reversed by the insertion of TFAMs and into the minor groove of mtDNA at the heavy (HSP1,HSP2) and light strand promoters (LSP.) TFAM forms a with itself and opens DNA allowing for more effective replication of mtDNA by MTRNAP. 

II. General Structure

TFAM is a monomer composed entirely of alpha and is comprised of 204 amino acids with a molecular weight of nearly 26 kDa. TFAM exhibits partial structural disorder by displaying the characteristics of a globular protein, implying folding, as well as the flatter profile of an unfolded protein. TFAM is a high-mobility group (HMG) protein comprised of two HMG-box domains, HMG1 and HMG2, which are separated by a . This linker compensates for the DNA phosphate backbone repulsion by creating a U-turn shape in the minor groove and stabilizing the two kinks in the DNA. Following the two HMG-box domains separated by a linker, TFAM has a that has a specific recognition for LSP.

III. DNA Binding

, composed of two strands, one designated as heavy (H), and the other light (L), interact with TFAM at two promoters, LSP and HSP1. Low TFAM concentrations allow for protein contact of with HMG1 (multi-colored) and HMG2 , respectively. HMG domains involve three helices folding into an L-shaped arrangement (short L-arm: composed of two short antiparallel helices ,HMG1 helix 1 and helix 2 and HMG2 helix 1 and helix 2 , and a long arm (6 to 7 resides from the N-terminus of the domain, packed against the C-terminal alpha helix (HMG1 helix 3 and HMG2 helix 3) , with the inside of the contacting the DNA minor groove. The amino acids interacting with the minor groove of DNA involve Leu58 from HMG1 helix 1 of TFAM (intercalates between DNA bases A3 and C4, which distorts DNA structure). This distortion of DNA structure is stabilized by Tyr57 of helix 1 partially intercalating bases T20 and G19 of chain D, and making a hydrogen bond with G19. The most critical amino acid interactions between TFAM and DNA involve Leu58 of HMG1, Leu182 of HMG2, and Lys137 of the C-terminal tail, all of which comprise the TFAM-LSP complex.

IV. Phosphorylation

Regulation of TFAM is controlled by phosphorylation within its HMG domains by cAMP-dependent protein kinase A (PKA) in mitochondria.The phosphorylation of these residues within the DNA-binding domains of the protein inhibits its ability to bind and activate transcription.

The catalytic subunit of PKA targets serine residues within both the HMG1 and HMG2 domains. Serine reidues on the display water mediatied and direct hydrogen bond interactions with the DNA bases, specifically Ser-55-Thymine 22 , Ser-56-Thymine 21, and Ser-61-Guanine 20. Ser-160, within the subunit, is also targeted by PKA, however it has no direct interactions with DNA. Interactions between HMG1 and are essential to the stability of the protein-DNA complex. Once phosphorylated, electrostatic repulsion between the and the serine residues causes the protein to release from mtDNA. When , the protein exists as a monomer in the mitochondria unless rebound to mtDNA.

V. Implications

Phosphorylation provides a mechanism for rapid control of TFAM concentration, as unbound protein in the mitochondria is degraded by a AAA+ Lon protease. Mitochondrial Lon belongs to a class of ATPase proteins which preform protein catabolism via hydrolysis of peptide bonds. In animal studies, knockouts of the TFAM gene in the mtDNA completely eliminated oxidative phosphorylation and lead to embryonic lethality; a heart-specific knock of TFAM resulted in cardiomyopathy and neonatal death. While TFAM is necessary for cell survival, cells in which the mitochondria were absent the Lon protease responsible for TFAM degradation experienced extensive protein overproduction. This overproduction resulted in an increase in mtDNA, but also cases of cardiac failure, neuro-degeneration, and age dependent deficits in brain function.

VI. References

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Rubio-Cosials, Anna, Sidow, Jasmin F., Jimenez-Menendez, Nereida, Fernandez-Millan, Pablo, Montoya, Julio, Jacobs, Howard T., Coll, Miquel, Bernado, Pau, and Sola, Maria. 2011. Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter. Nature Structural and Molecular Biology 18(11):1281-1289.

Gangelhoff, Todd A., Mungalachetty, Purnima S., Nix, Jay C., and Churchill, Mair E. A. 2009. Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A. Nucleic Acids Research 37: 3153-3164.

Garcia-Nafria, J., Ondrovicova, G., Blagova, E., Levdikov, V.M., Bauer, J.A., Suzuki, C.K., Kutejova, E., Wilkinson, A.J., Wilson, K.S. 2010. Structure of the proteolytic domain of the human mitochondrial Lon Protease. Protein Science. 19(5):987-99

Wu, J., Brown, S.H.J., von Daake, S., Taylor, S.S. 2007. PKA type IIalpha holoenzyme reveals a combinatorial strategy for isoform diversity. Science. 318(5848):274-9.

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