Fluorescent Polarization Assays Quantify Human Mitochondrial Transcription Factor A DNA Binding
Human mitochondrial transcription factor A (TFAM) is a DNA binding protein re- sponsible for transcription activation in mitochondria. It is also required for DNA compaction into nucleoids and DNA maintenance. TFAM belongs to the high mobil- ity group (HMG) superfamily, containing two HMG boxes and an additional carboxy- terminal domain. To date, quantitative studies addressing DNA binding specificity have not been performed. A detailed understanding of basic events underlying the specific DNA binding of TFAM as a transcription factor and its role in nucleoid formation has yet to be achieved. The purpose of this study is to develop an assay to further understand what these determinants are, in regards to specific DNA binding. We have used, in the past, fluorescence polarization to study DNA binding. In these assays, increasing concentrations of TFAM are incubated with fluorescently labeled DNA, and the change in polarization is utilized to infer DNA binding affinity. Here, we discuss results obtained with mutant TFAM carrying deletions in the carboxy terminal domain. Our results suggest a key role of this domain in DNA binding. We also describe the development of an alternative approach in which the fluorescent label is located on the protein. This approach may permit the characterization of TFAM binding to various DNA sequences and gain insight into the specificity of DNA binding.
Mitochondria are the powerhouses of the
cell. Their main role is the production of
energy for cellular function through oxidative phosphorylation. Mitochondria also
play a role in fatty acid oxidation, metabolism of calcium and iron, biosynthesis
of amino acids and heme, and apoptosis
. Mitochondria have their own genome
that encodes information essential for
the expression of components of oxidative phosphorylation including, mRNA,
tRNA and rRNA, which are necessary
for protein synthesis in organello -. It
is very well established that mutations in
the genes that encode components of the
oxidative phosphorylative system can lead
to mitochondrial diseases, typically with
manifestations that affect the main energy-consuming organs, such as the heart, 47
brain and muscles. In recent years, it has
been recognized that several human diseases, such as cancer, diabetes, Parkinson's
disease, dementia, and diabetes mellitus,
all have a correlation with defective mitochondrial function -. One hypothesis explaining this phenomenon is that
mitochondrial DNA (mtDNA) can accumulate mutations over the life span of the
mitochondria. An underlying reason why
mtDNA is highly susceptible to mutation
is because it lacks efficient DNA repair
mechanisms and it functions in a highly
Figure 1: Model of TFAM-DNA binding and mitochondrial transcription initiation. First, TFAM binds the LSP promoter, forming a complex. The complex changes the DNA conformation, allowing the recruitment of POLRMT and TFB2M to initiate transcription .
Transcription is the process of converting information stored in the form of
DNA into RNA. This then allows that information to be converted into proteins
for a wide array of functions. Only a single
regulatory region, called a displacement
loop (D-loop), is contained in mtDNA.
Transcription in mitochondria starts from
three promoters: the light strand promoter
(LSP) and heavy strand promoters 1 and
2 (HSP1 and HSP2). Transcription from
the most studied promoter, LSP, requires
three protein components: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM)
and mitochondrial transcription factor B
(TFB2M) (Figure 1). We hypothesize that
mutations within the mtDNA regulatory
region, particularly within the promoters,
may affect TFAM binding and/or POLRMT/TFB2M recruitment to the promoter. Thus transcription initiation, and therefore gene expression, may ultimately lead
to impairment of mitochondrial function.
Materials and Methods
Materials: Purified TFAM, TFAMdCT,
TFAMd10CT, and TC-TFAM were previously prepared in the Cameron laboratory. DNA oligonucleotides were obtained
from Integrated DNA Technologies Inc.
and purified by PAGE gel electrophoresis. BSA was obtained from New England
Biolabs. The Biarsenical Fluorescein derivative FlAsH was obtained from Invitrogen. All other reagents were received
from VWR and Fisher.
Figure 2: Experimental design to study protein-DNA interaction by FP. a- A fluorescent labeled DNA is incubated with increasing concentrations of TFAM. This approach permits the study of determinants for specific binding in the protein. b- A fluorescently labeled TFAM derivative is incubated with increasing concentrations of DNA. This approach permits the study of different DNA sequences and dissection of determinants for specific binding within the DNA.
TFAM-DNA Binding Characterization using fluorescence polarization
(FP): To study TFAM target site we used
fluorescence polarization (FP). The advantages of fluorescence polarization include that it is fast, reproducible, reports
on the equilibrium in solution and does
not require radioactivity materials. FP permits the study of molecular interactions
by tracking size changes of fluorescent
molecules. The polarization depends on
the apparent size of the fluorescent molecule. As the fluorescent molecule is incubated with its binding partner, binding
occurs. The complex formed has a higher
apparent size, which polarizes the emitted light. For protein-DNA interactions,
the fluorophor can be incorporated in the
protein or in the DNA (Figure 2).
Figure 3: TFAM-DNA Binding Fluoresce- in-labeled DNA was used to titrate TFAM concentrations ranging from 0.03125 nM to 500 nM. The data points were plotted using Eq 1 to determine the Kd.
TFAM carboxy terminal domain is
important for DNA binding: As mentioned above TFAM contains two HMG
boxes and a carboxy-terminal domain
(CTD) that have been implicated in transcription initiation. In order to determine
the function of the carboxy-terminal domain we used a protein variant in which
this domain was deleted (TFAMdCT).
We performed the FP assay as described
above, titrating TFAMdCT. We found that
the deletion of the CT domain greatly decreased the binding affinity (30 fold). The
Kd value is 31 +/- 4 nM (Figure 4). This result explains the observation that the CT
domain fails to activate transcription and
suggests that this domain is essential for
high-affinity DNA binding .
Figure 4: The carboxy terminal domain of TFAM is essential for specific DNA binding. a - Schematic of TFAM domains. b - TFAMdCT-DNA Binding. Fluorescein-labeled DNA was incubated with increasing concentrations of TFAMdCT, ranging from 1 nM to 1000 nM. The data points were plotted using Eq 1 to measure the Kd.
Figure 5: The carboxy terminal domain of TFAM is essential for specific DNA binding. a - Sequence alignment of TFAM from human, mouse and bovine species. b - TFAMd10CT-DNA Binding. Fluorescein-labeled DNA was incubated with increasing concentrations of TFAMdCT, ranging from 1 nM to 1000 nM. The data points were plotted using Eq 1 to measure the Kd.
TC-TFAM-DNA Binding Characterization: As depicted in Figure 2b, to study
the determinants of binding specificity
within the DNA, it is useful to incorporate
the fluorescent label into the protein. To
do so, we introduced a tetra cystein (TC)
motif in the N-terminal region of TFAM.
The TC motif can then conjugate the
fluorescent dye, FlAsH. It was important
to confirm that TFAM function has not
been affected by this modification. This
was also determined using FP in which
TC-TFAM was titrated.
Figure 6: TC-TFAM-DNA Binding. Fluorescein-labeled DNA was used to titrate TC-TFAM concentrations ranging from 0.0625 nM to 200 nM. The data points were plotted using Eq 1 to measure the Kd. The Kd was found to be 3 +/- 0.3 nM.
The present work illustrates the use of
FP to study TFAM binding to its DNA
cognate. Here we showed that the carboxy-terminal domain has a critical role in
DNA binding, explaining previous observations that TFAMdCT does not support transcription.
 N. D. Bonawitz et al., "Initiation and beyond: multiple
functions of the human mitochondrial transcription machinery," Mol Cell, vol. 24, no. 6, pp. 813-825, 2006.