Helix Unwinding and Base Flipping Enable Human MTERF1 to
Terminate Mitochondrial Transcription
Research by E. Yakubovskaya, E. Mejia, J. Byrnes, E. Hambardjieva, and M. Garcia-Diaz, Cell
2010, 141, 982-993
Summary and commentary prepared by Emi Leonard and Azeen Hadadi for CHEM 4521, Fall
2010, Georgia Institute of Technology
Research Summary
Purpose of Study:
To determine the structure of MTERF1 bound to its recognition sequence, to analyze its binding
mechanism, and to investigate the effect of specific mutations G3249A and G3242A. The
information gathered may provide insight into the role of MTERF proteins and suggest a link
between mitochondrial disease and the regulation of mitochondrial transcription.
Background:
Human mitochondria contain their own genetic material that encodes 13 subunits of the
respiratory chain, 2 rRNAs, and 22 tRNAs (1). This genetic material called mitochondrial DNA
(mtDNA) consists of 2 strands, a heavy strand and a light strand. Transcription from the light
strand promoter (LSP) and heavy strand promoter (HSP) produces lengthy transcripts, which are
processed to release the individual RNA molecules (1).
The two major sites at which mtDNA replication initiates are the origins of light and
heavy strand replication. The mitochondrial RNA polymerase generates the RNA primers
necessary for initiation of DNA synthesis at both of these sites. Initiation depends on the RNA
polymerase and mitochondrial transcription factors A and B2 (TFAM and TFB2M, respectively)
(1). Mitochondrial transcription can be regulated by controlling the expression of the genes
responsible for the process itself.
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Mitochondria contain a family of dedicated transcriptional regulators: the mterf proteins .
Mterf (mitochondrial termination factor) proteins have been implicated in mitochondrial
transcription, the coordination between transcription and replication and the regulation of
mitochondrial protein synthesis. The mterf family contains four members (MTERF1-4), all of
which are homologous to MTERF1 (2). Human MTERF1 is a 342-amino acid protein that was
shown to arrest in vitro mitochondrial RNA polymerase (POLRMT) progression by specifically
binding a 28-nucleotide sequence located immediately downstream of the 3’ end of the 16S
rRNA gene, within the leu-tRNA (3). Asin-Cayuela et al. demonstrated that MTERF1 is able to
promote termination in a bidirectional way, with an even higher efficiency when POLRMT
transcribes in the light-strand direction (4).
It was later shown that MTERF1 is capable of stimulating transcriptional initiation as
well as termination utilizing an artificial rDNA (2,3). MTERF1 causes transcriptional arrest and
activation by simultaneously binding the termination site and a novel site in the H1 region,
causing the looping-out of rDNA that promotes the recycling of transcription machinery (3). This
model accounts for the higher rate of transcription of rDNA compared to mRNA genes,
providing the basic molecular mechanism for the regulation of the rRNA/mRNA ratio (2,3).
These observations are all based on in vitro experiments; no in vivo evidence exists to
support these explanations (2). The lack of structural information on mterf proteins also
contributes to the ambiguity that surrounds their cellular role. Past studies have suggested that
MTERF1 is a modular leucine-zipper containing protein and that was therefore the suggested
method of DNA-binding (2,3). This study looks to determine the structure of MTERF1 and to
confirm the method of binding. This study also investigates two pathogenic mitochondrial
mutations: G3249A that causes a variant of Kearns-Sayre syndrome and G3242A that is
associated with an uncharacterized mitochondrial disorder. These mutations interfere with
protein-DNA interactions that are essential for sequence recognition by MTERF1 and therefore
eliminate its transcriptional termination activity (2).
Researcher’s Approach:
Wild type (WT) and mutant MTERF1 proteins, TFB2M and POLRMT were expressed
and purified for later utilization in structure determination and binding analyses. WT MTERF1,
TFB2M and POLRMT were cloned, allowing expression with his-tagged maltose binding
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protein (MBP). The triple R162A-F243A-Y288A mutant and each of the five arginine mutants
were constructed by QuikChange mutagenesis. His-tagged TFAM was cloned in pET-22. The
proteins were overexpressed in special E. coli cells adapted to low temperature. WT and mutant
MTERF1 proteins, TFB2M, and TFAM were purified by His-tag chromatography, followed by
Heparin and Mono S chromatography. POLRMT was purified by Heparin chromatography and
Mono S chromatography.
Crystallization was performed to determine the structure of MTERF1. Crystals grew at
room temperature and data collection was performed at the National Synchrotron Light Source.
Both datasets were processed using HKL2000. The WT structure was determined by
Multiwavelength Anomalous Diffraction using three datasets. Density modification and phase
extension were performed using DM and the resulting model was refined against the native
dataset. The model included all nucleotides and residues 73-396 of MTERF1. The mutant
structure was solved by molecular replacement.
Binding analyses were conducted to determine the effect of DNA sequence on
MTERF1’s ability to bind the recognition sequence of double stranded DNA. Isothermal titration
calorimetry (ITC) experiments were performed at 4 °C. WT or mutant MTERF1 was titrated
with 22 bp oligonucleotide pairs. Samples were prepared by dialyzing all interacting components
against a buffer and data were analyzed using the ORIGIN software.
Transcriptional termination assays were utilized to analyze the ability of different
proteins to promote transcriptional termination in an in vitro system. Nucleotides 491-790 of the
human mitochondrial DNA were cloned between the Ncol and HindIII sites of pET-22, and the
termination sequence was inserted 100 bp from the initiation site in both orientations by sitedirected mutagenesis. The substrate was linearized using HindIII. WT or mutant MTERF1 was
added. Transcription was initiated by the addition of 400 fmol POLRMT, 500 fmol TFB2M, and
2.5 pmol TFAM, and after 30 min of incubation the reactions were stopped. Products were
phenol extracted, ethanol precipitated, resuspended in loading buffer, and analyzed by PAGE.
Observations:
1. The MTERF1 protein assumes a fold that is unlike previously proposed models. It is an
all-α-helical protein that consists of 19 α helices and 7 310 helices that is built upon a
repeated motif (2 α helices followed by a 310 helix which now will be referred to as the
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mterf motif). The MTERF1 fold consists of 8 mterf motifs and an additional deformed
motif in the C terminus. Within each motif, multiple hydrophobic residues create a
hydrophobic core between the two α helices.
2. MTERF1 binds to the double-stranded DNA containing the termination sequence along
the major groove. MTERF1 covers twenty base pairs and imposes a slight curve (25°) in
the DNA. While most remains in the B-DNA conformation, the DNA structure of the
central part of the recognition sequence is greatly deformed. Binding of MTERF1 results
in significant DNA unwinding and promotes partial melting (eversion of three
nucleotides) (Figure 1).
3. Each nucleotide everted from the double helix is stabilized by MTERF1 via a stacking
interaction and hydrogen bonds to the base and phosphate (Figure 2). The triple R162AF243A-Y288A mutant was studied to observe binding without stacking interactions.
Removal of the stacking interactions modified the conformation of all three everted
nucleotides to a less favorable conformation. Thus stacking interactions are essential to
stabilize the everted nucleotides and MTERF1 actively promotes base-flipping. Arg162, a
stacking residue, is universally conserved in MTERF1 proteins suggesting that
differences must exist in the way MTERF1 associates with DNA in some of these
species.
4. Most of the interactions observed in the structure of MTERF1 are electrostatic in nature
and are established with the phosphate groups of the DNA strands. This type of
interaction does not impart any sequence specificity. The number of sequenceindependent interactions suggested that MTERF1 should be able to bind double stranded
DNA regardless of sequence. ITC experiments determined that MTERF1 is capable of
binding a double stranded DNA of arbitrary sequence. Importantly, the lower DNA to
protein ratio of binding demonstrates that MTERF1 does not preferentially integrate with
the DNA in a particular orientation, consistent with the lack of sequence specificity.
Rather, it can bind to different regions of the DNA and as a result more than one
MTERF1 molecule can bind the same substrate molecule concurrently.
5. Based on the observed binding stoichiometry when titrating the triple mutant (Figure 3),
there are only a limited number of interactions that appear capable of discriminating
against a particular sequence, because most of the protein-DNA interactions seen in the
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structure are in relation to the DNA backbone. Therefore sequence recognition seems to
be mediated by specific hydrogen bonding to the major groove of DNA. Five arginine
residues form base interactions that are likely to determine sequence specificity. Three of
these concurrently hydrogen bond to N7 and O6 of a guanine base (Figure 4). These
arginine residues are conserved in MTERF1, similar to the nucleotides that they
recognize in the mitochondrial DNA. These arginines, however, are not conserved in
other mterf proteins. Individual arginine to alanine substitutions indicated that the
importance of each of these residues for DNA binding and sequence recognition is not
equal, leading to an analysis of their abilities to promote transcriptional termination.
6. According to the transcriptional termination assay, TFAM, TFB2M, and POLRMT
prompt a unique runoff transcription product on the transcriptional termination substrate;
however, upon addition of MTERF1, a specific termination product appears. When the
termination sequence is inverted (as to mimic termination of LSP-initiated transcription),
termination is greater, possibly in accord with the stronger affinity of MTERF1 for the
light strand. Termination was neither observed with the triple mutant nor with the
arginine mutants.
7. The G3242A mitochondrial DNA mutation interacts in the MTERF1 structure with
Arg251, while G3249A interacts with Arg387. Each of these mutations would destroy a
guanine-arginine interaction. The G3242A mutant led to weak or no effect on binding.
The G3249A mutation, however, seems to fully eliminate specific DNA binding. Both
resulted in a significant decrease in termination.
What Researchers Accomplished:
Yakubovskaya et al. determined the structure of human MTERF1. Their results suggest a
specific mechanism of sequence recognition and binding in which sequence recognition follows
sequence-independent binding, ultimately resulting in a conformational change that unwinds the
DNA double helix leading to base-flipping. Base-flipping is a pivotal feature of the interaction of
MTERF1 with DNA. It is often employed by a variety of DNA repair and replication proteins
that demand access to a specific base in the DNA. The everted base is typically stabilized by πstacking interactions due to the fact that nucleotide eversion is not energetically favorable in
these proteins. Base-flipping is normally used either to help recognize specific features of the
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DNA or to aid in the catalytic mechanism; this is typically done to gain access to a substrate that
would otherwise be concealed within the DNA double helix. Here base-flipping’s sole purpose is
to stabilize MTERF1 onto DNA and to prevent its displacement.
MTERF1 appears to be much more effective at promoting termination from the LSP than
from the HSP. This could be due to the protein’s stronger affinity for the light strand as well as
the large number of interactions associated with this strand. Despite the fact that the projected
structure of MTERF1 does not explain how the protein can form the mitochondrial DNA loop
implicated in rRNA synthesis, the protein-DNA interaction surface seen in the structure
illustrates that MTERF1 would not be able to associate with two DNA molecules at the same
time. This ultimately indicates that it is unlikely for the loop to be mediated by a single MTERF1
molecule. However, the partial melting seen with the structure, together with the preferential
binding to the light strand and the small number of interactions with the heavy strand, propose a
mechanism by which MTERF1 (or other mterf proteins) could aid transcriptional initiation by
adding to the initial melting. Also, parallels between MTERF1 and families of RNA binding
proteins suggest that the mterf fold may not be limited to binding double stranded DNA.
After determining the crystal structure it can be concluded that two pathogenic mutations,
G3242A and G3249A, that interfere with arginine-guanine interactions that are involved in
sequence recognition by MTERF1 compromise the ability of MTERF1 to terminate
transcription. G3249A seems to completely eliminate specific binding by MTERF1 and
ultimately hinders termination, while G3242A does not significantly affect binding, but seriously
reduces the ability of MTERF1 to terminate transcription. This intense effect on termination
proposes that the pathogenic effects of these two mitochondrial DNA mutations could be
partially mediated by inhibiting the activity of MTERF1 at the leu-tRNA site.
Commentary on the Research
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is
a disease that affects several of the body’s systems, specifically the brain, muscles, and nervous
system. Child or adult onset, this neurological disease is caused by mutations in mitochondrial
DNA genes (5). The A3243G mitochondrial DNA mutation is commonly identified in MELAS
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patients and has been shown to reduce MTERF1 binding and interfere in vitro with
transcriptional termination. The A3243 mutation is one of the three nucleotides that have been
everted, and a transition mutation would substitute an A:T base pair with a stronger G:C base
pair. This nucleotide substitution can explain the moderate decrease in binding (2). The results
indicate that the A3243G mutation exhibits slightly decreased binding, similar to that of A3243T
mutation. With the A3243 mutation, the A:T base pair is maintained but the larger purine ring
has been located in the heavy strand (which would lead to a steric clash). The remaining
mutations should not be expected to significantly conflict with MTERF1 sequence recognition,
with the exception of G3242A and G3249A (2).
The G3249A mutation leads to the development of a form of Kearns-Sayre syndrome, a
mitochondrial myopathy specifically involving muscles controlling eyelid movement (6). The
G3242A mutation has been linked with an uncharacterized mitochondrial disorder potentially
caused by transcriptional deregulation. Yakubovskaya et al. determined that these mutations both
hinder MTERF1’s ability to terminate transcription, resulting in these disorders. However
because this research did not test the affects of these mutations in vivo, future experiments need
to be conducted in order determine whether the G3249A and G3242A mutations result in
transcriptional alterations in vivo.
The researchers describe the binding nature of MTERF1 as being sequence-independent
and therefore unspecific and utilize ITC experiments to support this claim. However, within the
same paragraph the researchers support the specific binding of MTERF1. Although the authors
attempt to explain this discrepancy, the divide between specific and unspecific binding and their
relation to one another is still unclear, resulting in confusion. Initially the authors discuss the
unspecific binding of MTERF1 related to the electrostatic interactions of the phosphate groups of
the DNA strands. Later it is observed that nucleotide eversion is essential for stable sequencespecific binding. Without explicitly stating the extent to which each of these interactions
influences the binding of MTERF1 to DNA, the authors leave the reader to interpret this
interaction. The researchers should have better developed each concept separately and then
brought them together to explain the overall binding mechanism.
The researchers do not fully explain the transcriptional termination assay methods, but
reference a past study (4) which in turn references another past study (7). The search for the
original methods continues on to at least two other studies, which was unnecessary and
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frustrating for the reader. The researchers should have located the study with the original
methods rather than ultimately referencing a series of studies that did not explicitly describe the
methods. The results indicated the critical importance of base flipping for transcriptional
termination and the varying roles of some residues; therefore the lack of understanding of the
original procedures utilized to draw these conclusions resulted in reduced credibility of the
researchers.
Overall, the research conducted by Yakubovskaya et al. was thorough and well done. A
few more details in the methods and further explanation in some of the results would have helped
the reader better understand the decisions made and conclusions reached. The greatest error
occurred in the contradictory information when explaining the binding mechanism; however, this
was most likely a failure to fully explain the concepts and the relationship of specific and
unspecific binding. Further description would have better supported the conclusions drawn in
this area. Discovering more about the binding of MTERF1 to DNA strands would greatly
enhance the current knowledge of the mutations leading to Kearns-Sayre syndrome and MELAS.
This research, in addition to in vivo experiments, could lead to determination of potential
treatments for these mitochondrial myopathys.
Bibliography
1. Wanrooij, P., Uhler, J., Simonsson, T., Falkenberg, M., Gustafsson, C. (2010) Proc. Natl.
Acad. Sci. 107, 16072-6077.
2. Yakubovskaya, E., Mejia, E., Byrnes, J., Hambardjieva, E., Garcia-Diaz, M. (2010) Cell
141, 982-993.
3. Roberti, M., Polosa, P., Bruni, F., et al. (2009) Biochim. Biophys. Acta 1787, 303-311.
4. Asin-Cayuela, J., Schwend, T., Farge, G., Gustafsson, C. (2005) J. Biol. Chem. 280,
25499-25505.
5. Rahman, S., Poulton, J., Marchington, D., Suomalainen A. (2001) Am. J. Hum. Genet. 68,
238-240.
6. Spector, R. and Johanson, C. (2010) Cerebrospinal Fluid Research 7, 14-17.
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7. Falkenberg, M., Gaspari, M., Rantanen, A., Trifunovic, A., Larsson, N., Gustafsson, C.
(2002) Nature Genet. 31, 289-294.
Figures
Figure 1. Three nucleotides (yellow in the figure; corresponding to A3243 of the light strand and
T3243 and C3242 of the heavy strand) are everted from the double-helix in the central part of the
structure. A simulated annealing fo-fc election density map is shown contoured at 3α (2).
Figure 2. The three everted nucleotides are stabilized by π-stacking interactions. R162 (orange),
F234 (magenta) and Y288 (red) stack against each of the everted nucleotides. The light strand is
green and the heavy strand is yellow (2).
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Figure 3. Summary of the observed binding constants. The sequence of each substrate (one of
the strands) is indicated at the top of the table (2).
Figure 4. Sequence recognition by arginine residues. Five arginine residues determine sequence
recognition by MTERF1. A representative example showing how R169 and R202 interact with
their partner guanine residues. The R202 interaction is atypical in that only one hydrogen bond is
established with the guanine base. Hydrogen-bonding distances are shown in orange. A
simulated annealing fo-fc electron density map is shown contoured at 4α (2).
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