Activation and Regulation of
Human Mitochondrial Transcription Factor A
Kelsey McMurtry '14 and Wesley Manz '15
Contents:
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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