Báo cáo khoa học: Transcription termination at the mouse mitochondrial H-strand promoter distal site requires an A/T rich sequence motif and sequence specific DNA binding proteins pptx

Eur. J. Biochem. 270, 1128–1140 (2003) Ó FEBS 2003

doi:10.1046/j.1432-1033.2003.03461.x

Transcription termination at the mouse mitochondrial H-strand
promoter distal site requires an A/T rich sequence motif
and sequence specific DNA binding proteins
Vijayasarathy Camasamudram, Ji-Kang Fang and Narayan G. Avadhani
Laboratories of Biochemistry, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania, USA

Termination of mitochondrial (mt) H-strand transcription
in mammalian cells occurs at two distinct sites on the
genome. The first site of termination, referred to as
mt-TERM occurs beyond the 16 S rRNA gene. However,
the second and final site of termination beyond the
tRNAThr gene remains unclear. In this study we have
characterized the site of termination of the polycistronic
distal gene transcript beyond the D-loop region, immediately upstream of the tRNAPhe gene. This region, termed
D-TERM, maps to nucleotides 16274–16295 of the mouse
genome and includes a conserved A/T rich sequence motif
AATAAA as a part of the terminator. Gel-shift analysis
showed that the 22 bp D-TERM DNA forms two major
complexes with mouse liver mt extract in a sequence-specific

manner. Protein purification by DNA-affinity chromatography yielded two major proteins of 45 kDa and 70 kDa.
Finally, the D-TERM DNA can mediate transcription
termination in a unidirectional manner in a HeLa mt
transcription system, only in the presence of purified mouse
liver mt D-TERM DNA binding proteins. We have therefore characterized a novel mt transcription termination
system, similar in some properties to that of sea urchin, as

well as the nuclear RNA Pol I and Pol II transcription
termination systems.

The mitochondrial (mt) genome in mammalian cells is a
double-stranded circular DNA, which encodes two rRNAs,
22 tRNAs, D-loop primer RNAs for DNA replication and
13 polypeptides that are components of the mt electron
transport-coupled oxidative phosphorylation system [1,2].
Both the H- (heavy) and L- (light) strands of the mt DNA in
vertebrate cells are transcribed symmetrically, and nearly
completely [3,4] as polycystronic precursor RNAs starting
from strand-specific promoters, HSP1 and LSP, respectively
[5–10]. Until recently, the H-strand of the D-loop region,
between tRNAPro and tRNAPhe was thought to be a
noncoding region of the genome. The D loop is created by
the displacement of one of the parental strands by 0.5–1 kb
nascent DNA strand needed for the replication of the L
strand. The 0.8–1 kb long D loop is a ubiquitous feature of
the vertebrate mt genomes [4,11]. The D-loop of the

vertebrate mt DNA also houses HSP and LSP organized in
opposite orientations, but within about 100 nucleotides of
each other.
The genes encoded by the H-strand, believed to be the
leading strand, can be classified into two categories: the
promoter-proximal region encoding the tRNAPhe, 12 S
rRNA and the 16 S rRNA genes [1] and the promoter-distal
region which encodes the majority of mRNAs, tRNAs, and
the 0.8 kb D-loop region RNA of unknown function [2].
This region spans a 13.6-kb sequence downstream of the

tRNALeu gene. A majority of the mt RNA species is
processed from larger polycistronic precursors [12]. The
promoter-proximal and -distal regions of the H-strand are
expressed at distinctly different rates: the promoter-proximal region is transcribed at 40- to 80-fold higher rate than
the promoter-distal region [13,14]. A combination of
biochemical and mutational analysis coupled with the
analysis of mt DNA from human patients with mitochondrial diseases led to the identification of a tridecamer
DNA sequence that supports partial transcription termination at the end of the 16 S rRNA gene. The putative
tridecamer terminator sequence has been mapped to the 5¢
end of the tRNALeu gene, which occurs immediately
downstream of the 16 S rRNA gene [15,16]. A 36-kDa
protein, which binds to the tridecamer sequence motif, has
been purified and characterized by cDNA cloning [17]. This
protein, termed the mt transcription termination factor
(mTERF) binds to the promoter proximal terminator
sequence (mt-TERM), and promotes transcription termination, under in vitro conditions, albeit on a partial basis.

Correspondence to V. Camasamudram, Department of Biochemistry,
Faculty of Medicine and Health Sciences, United Arab Emirates
University, PO Box: 17666, Al-Ain, UAE.
Fax: + 971 3 7672033, Tel.: + 971 3 7039502,
E-mail:
Abbreviations: mt, mitochondrial; D-TERM, promoter distal terminator sequence; HSP, H strand promoter; LSP, L strand promoter;
mTERF, mitochondrial transcription termination factor;
mt-TERM, promoter proximal terminator sequence.
(Received 15 October 2002, revised 28 December 2002,
accepted 14 January 2003)

Keywords: mitochondria; transcription termination; mitochondrial H-strand transcription; D-loop binding proteins;
polyadenylation.

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Mitochondrial transcription termination (Eur. J. Biochem. 270) 1129

mTERF, a leucine zipper protein, appears to bind to the
DNA as a monomer, possibly through a novel coiled-coil
structure created by interactions between intramolecular
zipper domains and terminates transcription by a DNA
bending mechanism. Additionally, this terminator sequence
exhibits bi-directional activity as described by Shang and
Clayton [18], thus invoking its possible role in L-strand
transcription termination as well. However, it is not clear as
to how small populations of H-strand transcripts escape
termination at the mt-TERM and continue through the
distal sites of the genome.
Studies from two laboratories showed that the D-loop
regions from the rat and mouse mtDNAs encode a stable
0.8-kb poly(A) containing RNA of as yet unknown function
[19,20]. The 5¢ end of this RNA maps to nucleotide 15 417
of the mouse genome, which marks the start of tRNAPro on
the L-strand. The 3¢ end maps to nucleotide 16295 of the
genome, which is immediately upstream of the tRNAPhe
gene on the H-strand [20]. Our results also showed that in
the mouse mt system, the 3¢ end of this stable RNA is
preceded by a conserved A/T-rich sequence motif,
AAUAAA [20]. This canonical nuclear polyadenylation
signal is believed to have a role in the 3¢ end formation of
nuclear RNA polymerase II transcripts and also yeast mt

mRNAs [21,22]. Similar A/T-rich sequences have been
shown to occur at or close to the 3¢ end of D-loop coded
RNAs from the human and rat systems [1,23]. Based on
this, we proposed that the conserved AAUAAA motif,
along with its flanking sequences, function as the transcription termination site (D-TERM) for the promoter distal Hstrand genes. In the present study we provide evidence that
this A/T-rich D-TERM motif with its flanking sequences
(16274-5¢-ATTACGCAATAAACATTAACAA-3¢-16295¢)
binds to mt-specific proteins, different from the previously
characterized 36-kDa mTERF [17], and terminates transcription in an in vitro reconstituted system in a unidirectional manner.

Materials and methods
Preparation of mt extract capable of transcription
initiation
Mt extract from HeLa cells rich in RNA polymerase activity
was prepared as described by Shadel and Clayton [24].
HeLa cells were grown in suspension culture in Joklik’s
medium supplemented with 10% new born calf serum (both
from Sigma Chemical Co). The cells (about 1 g wet weight)
in logarithmic phase were harvested and disrupted by
homogenization in a buffer containing 134 mM NaCl, 5 mM
KCl, 1 mM Na2HPO4, and 2.5 mM Tris/HCl pH 7.5.
Mitochondria were isolated from the homogenate by
differential centrifugation, and further purified by sucrose
density banding. Mt particles banding at the interphase of
1.2–1.6 M sucrose was recovered and used for preparing the
mt extract. The RNA polymerase activity from the mt lysate
was enriched by successive chromatography on heparin
agarose and DNA–Sephacel columns [24]. The polymerase
activity was monitored by a filter-binding assay, which
measures the incorporation of [a32P]UTP into nascent

RNA chains programmed on denatured calf thymus DNA
templates [25].

Preparation of mt extract for DNA binding
Mt protein extracts for gel shift analysis were prepared from
freshly isolated mouse liver mitochondria and subjected to
heparin agarose chromatography as described earlier [20].
Briefly, mitochondria were suspended in buffer A (20 mM
Hepes pH 7.9, 50 mM KCl, 10 mM MgCl2, 1 mM EDTA,
1 mM dithiothreitol), lysed by adding 0.6 M KCl and the
soluble fraction was separated by centrifugation at
105 000 g for 60 min at 4 °C. The supernatant fraction
was dialysed against buffer B (20 mM Hepes, 50 mM KCl,
10 mM MgCl2, 1 mM EDTA, 15% glycerol, 2 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride and
1 lgỈmL)1 each of leupeptin, pepstatin and antipain) for
3–4 h, at 4 °C and used for binding to heparin–agarose
resin. About 1 mL heparin–agarose, pre-equilibrated with
buffer B, was added to the extract (100 mg protein) and
mixed with gentle agitation for 1 h at 4 °C. The mixture was
poured into a 1-mL column and the flow-through fraction
was collected. The column was subsequently washed with
15 mL buffer B, followed by a step gradient of 0.1–1.0 M
KCl in buffer B. The fractions eluted between 0.5 and 1.0 M
KCl were pooled and dialysed against buffer B. DNA–
protein binding was assayed by gel mobility shift analysis.
Partial purification of mt transcription termination
factor by DNA affinity chromatography
The putative termination factor was purified by affinity
chromatography [26]. The 22-bp synthetic double-stranded
DNA ) termed D-TERM DNA ) contains the promoterdistal termination sequence of the mouse mt genome

immediately upstream of tRNAPhe (16274-5¢-ATTACG
CAATAAACATTAACAA-3¢-16295). About 1.5 mg 5¢
biotinylated synthetic double-stranded D-TERM DNA of
22 bp was bound to avidin-agarose resin (1 mL swollen
resin, Sigma) in a buffer containing 10 mM Tris/HCl
pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA
and 5% glycerol. The affinity DNA matrix (1 mL) was
mixed with 50 mg heparin–agarose purified mouse liver
mt protein extract by gentle mixing in loading buffer,
containing 10 mM Tris/HCl pH 7.4, 50 mM NaCl, 1 mM
dithiothreitol, 1 mM EDTA, 5% glycerol, 1 mM phenylmethanesulfonyl fluoride, 1 lgỈmL)1 each of leupeptin,
pepstatin and antipain and 25 lg poly dI-dC. The binding
mixture was incubated for 60 min at 4 °C on a rotating
wheel. The contents were then poured into a 0.6 · 5 cm
column and the unbound material was collected as flowthrough. The column was washed twice with 10 mL loading
buffer, and sequentially with a step gradient (4 mL each)
containing 0.2, 0.5 and 1.0 M KCl in loading buffer. Protein
eluted in each step was tested for binding to the putative
termination sequence motif (D-TERM) by gel mobility shift
assays.
Gel mobility shift assays
The 22 bp synthetic double-stranded D-TERM DNA was
5¢ end labelLed using [c-32P]ATP and polynucleotide kinase.
Protein–DNA binding reactions were carried out in 20 lL
volume containing 10 mM Tris/HCl pH 7.4, 50 mM NaCl,
1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 100 ng


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1130 V. Camasamudram et al. (Eur. J. Biochem. 270)

dI:dC, 0.1–0.2 ng 32P-labelled gel purified double-stranded
DNA probe (30 000 c.p.m.) and 2–5 lg mt protein extract
or 1–2 lg affinity purified protein fraction [20]. Synthetic
oligonucleotides with nucleotide replacements targeted to
various positions of D-TERM were used as mutant probes
to assess the specificity of protein–DNA binding. Unlabelled or mutant oligonucleotides were preincubated for
10 min on ice with added proteins under normal assay
conditions before adding the probe. Binding was carried out
for 25 min at room temperature and the DNA–protein
complexes were resolved by electrophoresis on 4% acrylamide gels in Tris/Acetate/EDTA buffer at 4 °C [20].
DNaseI footprint analysis
The putative transcription termination region DNA
(D-TERM DNA) cloned in the BamH1 site of Bluescript
vector (Stratagene) was linearized with either Xba1 or III
and 3¢ end-labelled with Klenow enzyme in the presence of
a-32P dCTP. After a second digestion with either Xba1 or
HindIII the resulting fragments were gel purified and
electroeluted. Gel purified 3¢ end-labelled DNA probe
(190 000 c.p.m. Xba1 end-labelled H strand fragment or
HindIII end-labelled L strand fragment), was incubated
with 25 lg mt protein extract or 25 lg albumin using the
binding reaction conditions as described for the gel mobility
shift assay. The protein-bound DNA complexes and free
DNA probe were resolved by electrophoresis on 4%
acrylamide gels in Tris/acetate/EDTA at 4 °C and were
recovered from the gel following autoradiography and
electroelution. Protein–DNA complex (complex II) and the
free DNA probe, recovered from the gel (20 000 c.p.m.)

were subjected to DNAse 1 (Boehringer Mannheim) treatment at a concentration ranging from 0.025 to 0.100 U per
50 lL reaction, in the presence of 0.5 mM CaCl2, 1 mM
MgCl2 for 2 min at 25 °C as described by Henninghausen
and Lubon [27]. Samples were phenol extracted, concentrated by ethanol precipitation and resolved on 8%
sequencing gels as described before.
Methylation interference analysis
The 3¢ end-labelled D-TERM DNA probe prepared as
described above was partially methylated using dimethylsulphate essentially as described by Maxam and Gilbert [28].
Binding reactions contained 500 000 c.p.m. methylated
DNA and 30 lg mt extract. The resolution of the protein
bound (complex II) and free DNA probes by electrophoresis on 4% acrylamide gels under gel shift conditions,
treatment of gel recovered DNA with piperidine and
analysis of cleaved DNA strands on a sequencing gel, were
carried out as described before [26,27].
UV induced protein-DNA cross-linking
A photoreactive 32P-labelled D-TERM DNA probe was
prepared from a template that encompasses the mouse mt
genome sequences 16274–16295 upstream of tRNAPhe gene.
The 22-bp template was annealed to a 8-base primer oligo
5¢-TTGTTAAT-3¢ and filled using Klenow fragment in the
presence of 10 lCi [a-32P]dATP/dCTP, 200 lM dGTP,
100 lM TTP and 100 lM Brd UTP as described [26,29].

Protein–DNA binding reactions containing photoreactive
probe were carried out in a 500-lL eppenderof tube as
described in gel mobility shift assays. The reaction mixtures
were then placed on a bed of ice and irradiated with UV
light (wavelength 256 nm) for 10 min at a distance of 5 cm.
After the addition of SDS sample buffer and heat denaturation at 95 °C for 5 min, the reaction mixtures were
subjected to electrophoresis on a SDS/12% polyacrylamide

gel and the DNA–protein complexes were visualized by
autoradiography. For competition experiments, unlabelled
competitor oligonucleotides or mutants were added at
20–100-fold molar excess and incubated for 10 min on ice
prior to the addition of the photoreactive probe. To identify
the proteins in complex II, gel shift reactions containing the
photoreactive probe were run on gels as described in
previous sections. The protein–DNA complexes were crosslinked by UV irradiation of the gel for 30 min on a
transilluminator (Fotodyne, Hartland, USA), vacuum dried
and autoradiographed. The individual complexes were
excised, and subjected to SDS/PAGE (12% acrylamide)
followed by autoradiography.
South-western blotting
The South-western protocol was modified from Mangalam
et al. [30]. The proteins resolved on 12% SDS/PAGE were
electroblotted onto nitrocellulose membrane using 40 mM
glycine, 50 mM Tris and 20% methanol buffer. The proteins
on the blot were denatured with 6 M guanidine/HCl, in
binding buffer (10 mM Tris HCl pH 7.5, 50 mM NaCl,
1 mM dithiothreitol, 0.1 mM EDTA, 0.01% NP40, 5%
glycerol), for 5 min and renatured by stepwise dilution of
guanidine/HCl solution from 6 M to 0.375 M in binding
buffer. Washed membranes were equilibrated with 3% BSA
in binding buffer for 5–15 min. The excess BSA was rinsed
off with binding buffer and the membranes were incubated
with c-32P-labelled D-TERM or mutant DNA probes
(8 · 105 c.p.m.ỈmL)1) in binding buffer containing
2 lgỈmL)1 poly(dI/dC) for 12 h at 4 °C on a rotating
wheel. The filters were removed, washed with binding buffer
and exposed to X-ray film for autoradiography.

Construction of transcription vectors and assay
of transcription termination
Because of the known specificity of the human mt RNA
polymerase for human mt promoters LSP and HSP [31], we
constructed chimeric templates containing the human LSP
(pLSP) for transcription initiation and wild-type or mutated
mouse D-TERM motifs for assaying transcription termination (Fig. 7A). A 526-bp human mt D-loop DNA
fragment (nucleotides 530–534) was amplified by PCR
using the pKB741SP (a kind gift from D. A. Clayton) DNA
template and cloned in the EcoR1 site of pGEM 7Z plasmid
DNA. This region of DNA is selected for the presence of
LSP segment as well as the L-strand transcription start site
(nucleotide 407). The chimeric templates were constructed
by introducing the mouse D-TERM sequences (listed in
Fig. 2B) at Mfe1 site of pLSP (at nucleotide 242; 165 bases
downstream of L-strand start site) either in the forward
pD-TERM (F) or in reverse orientation pD-TERM (R).
The mutant versions containing the nucleotide replacements


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Mitochondrial transcription termination (Eur. J. Biochem. 270) 1131

in the D-TERM sequence (see Fig. 2B; pD-TERM Mut1,
pD-TERM Mut2 and pD-TERM Mut3) were cloned in a
similar way at the Mfe1 site of pLSP in the forward
orientation.
Prior to use in in vitro transcription reactions, the
affinity-purified fractions were tested for DNAse and

RNAse contamination. The mt DNA template and runthrough RNA product were incubated with increasing
concentrations of affinity-purified fractions and tested by
agarose gel electrophoresis. Treatment of DNA or RNA
with 4 and 10 lg of 0.5 M or 1 M KCl eluted fractions did
not cause any DNA or RNA degradation thus indicating
the absence of nuclease activity in the affinity purified
protein fractions.
Run-off transcription assays were carried out in 25 lL
reaction mixture containing, 10 mM Tris/HCl pH 8.0,
10 mM MgCl2, 1 mM dithiothreitol, 100 lgỈmL)1 BSA,
400 lM ATP, 200 lM GTP, 200 lM CTP, 40 lM UTP,
20 lgỈmL)1 linearized transcription template, 4 lg HeLa
mt RNA polymerase and 0.25 lM [a-32P]UTP (5 lCi from
a stock of 400 CiỈmmol)1 specific activity), at 28 °C for
30 min [24,25]. In some experiments, the reaction mixture
was supplemented with 4 lg affinity-purified moue liver mt
protein fraction. After a 15-min chase with cold 200 lM
UTP, the reactions were terminated by the addition of
equal volume of stop buffer (20 mM EDTA, 1% SDS,
0.5 mgỈmL)1 Proteinase K) and incubation continued at
37 °C for 30 min. The in vitro transcription products were
recovered by phenol extraction followed by ethanol
precipitation and resolved on 6% polyacrylamide, 8 M
urea gels.

Results
Locations and sequence properties of the proximal
and distal transcription terminators
Fig. 1A shows the locations of the promoter proximal
transcription terminator (mt-TERM) downstream of the

16 S rRNA gene, and at the 5¢ end of the tRNALeu gene on
mouse mt genome [15,16]. Studies on S1 nuclease analysis,
coupled with the 5¢- and 3¢-end mapping of D-loop
H-strand coded RNA suggested that the distal gene
transcription proceeds until the end of the D-loop, overlapping with the HSP [19,20]. In the mouse system, the 3¢
end of the D-loop H-strand RNA maps to the CAA
sequence ending at nucleotide 16 295 of the genome ([20]
see Fig. 1). Since the RNA is polyadenylated, it is uncertain if the termination occurs at the C residue at 16 293
or the A residue at 16 295 of the genome. The 22-bp
nucleotide sequence (16 274-5¢-ATTACGCAATAAAC
ATTAACAA-3¢-16 295) containing the mouse mt H strand
transcription start site and also the putative termination site
will be referred to as promoter distal transcription termination sequence, D-TERM (see Fig. 1B).
Protein binding property of the distal terminator
sequence
In a previous study we showed that the 22-bp D-TERM
DNA forms two differently migrating complexes with
protein from mt extract as tested by gel mobility shift

analysis [20]. In the present study, the sequence specificity of
D-TERM DNA binding to mt proteins was tested in gel
mobility shift assays by competition with wild-type and
synthetic mutant D-TERM DNA and mt-TERM DNAs.
The sequences of the normal and mutant versions of
the promoter distal putative transcription terminator
D-TERM, as well as the promoter proximal terminator
motif mt-TERM (mouse H strand sequence 2681–2660) are
shown in Fig. 2B.
As shown in Fig. 2A, both complexes I and II were
effectively competed with 10–20-fold molar excess of

unlabelled D-TERM DNA (lane 3–5), while even a 50-fold
molar excess of mt-TERM DNA did not compete significantly with either of the complexes (lanes 12–14). Thus, the
mt-TERM DNA containing A/T rich sequence seems to
have binding specificity different from that of the D-TERM
DNA. The D-TERM DNA contains the cononical polyadenylation signal sequence AATAAA. Further, 20 and 50
molar excesses of Mut1 DNA, with nucleotide replacements
targeted to the AATAAA sequence region failed to compete
for protein binding to D-TERM DNA (Fig. 2A, lanes 6
and 7). Similar concentrations of Mut2 and Mut3 DNAs
with nucleotide replacements targeted upstream or downstream of the AATAAA sequence, respectively, competed
slightly differently (Fig. 2A, lanes 8–11) from Mut1 with
both complexes. These results show that the putative
polyadenylation signal AATAAA, and also the sequences
upstream and downstream of the canonical polyadenylation
signal are important for protein binding to D-TERM DNA.
The binding specificity of D-TERM DNA and also the
possible protein–DNA contact sites were further investigated using DNAse1 footprinting and methylation interference analysis. Fig. 3A shows the DNAse1 footprint of
complex II (see gel shift Fig. 2A) using the H-strand labelled
135-bp DNA probe. It is seen that relatively low level of
protection against DNAase1 is afforded in the absence of
added mt protein (lane 7). Reactions with added mt protein
(lanes 3–5) however, showed a window of protection
spanning the entire D-TERM region, against 0.025–0.1 U
DNAse1 per 50 lL reaction. Although not shown, the
L-strand labelled probe showed a similar generalized
protection of the entire D-TERM sequence region. Additionally, analysis of complex I from the gel shift in Fig. 2A
showed a comparable footprint pattern. These results
suggest a surprising possibility that complexes II and I
may be closely related. The methylation interference analysis of complex II was carried out using the L-strand
labelled 135-bp D-TERM DNA probe. Fig. 3B shows that

a G (nucleotide 16 279) and two A (nucleotides 16 282 and
16 291) residues, as indicated by arrows, are protected
suggesting possible protein–DNA contact sites (lanes 3–4).
While one of the protected residues is localized in the
polyadenylation signal sequence (A at 16 282) the remaining two residues lie in the flanking sequences. Additionally,
two A residues (nucleotides 16 294 and 16 295, respectively), immediately 3¢ to the protected A residue of
D-TERM DNA become hypermeyhlated. Hypermethylation is thought to be the result of conformational changes in
the DNA leading to enhanced sensitivity to dimethyl
sulfoxide. These results together suggest that the protein
complex probably spans the entire D-TERM region with
three purine residues in contact with the protein(s).


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1132 V. Camasamudram et al. (Eur. J. Biochem. 270)

Fig. 1. Location of the putative D-TERM
element for H-strand transcription termination
on the mouse mt genome. (A) Location of the
D-TERM element on the mouse mt genome.
The triangles represent the tRNA genes designated by the single-letter amino acid code.
The polarity of triangle indicates the direction
of the transcription. OL and OH represent the
origin of L and H strand replication, respectively. LSS, L strand start site; HSS, H strand
start site. The map positions of the rRNA,
tRNAs and mRNAs (ND1 to ND6, A6 and
A8 and COX I to III) are based on Bibb et al.
[2]. The continuous outer arrow indicates the
promoter proximal transcript terminating at

mt-TERM [15,16], while the discontinuous
outer arrow indicates the promoter distal
transcript terminating at D-TERM sites.
(B) Schematic representation of the mouse mt
D-loop region encompassing the conserved
sequence boxes I-III (CSB), tRNA genes, L
and H strand promoters (LSP and HSP), L
and H strand transcription start sites (LSS and
HSS). The L-strand sequence and position of
the putative promoter distal transcription termination element (D-TERM) is also shown.

Nature of mt proteins binding to the D-TERM DNA
sequence
UV-induced DNA cross-linking was carried out to investigate the size and complexity of mt proteins binding to the
D-TERM DNA. 32P-labelled photo-sensitive DNA probe
was bound to mouse liver mt protein extract subjected to
UV irradiation in solution and the cross-linked products
were analysed by SDS/PAGE following denaturation at
95 °C for 5 min. As expected, bound complexes without
UV irradiation did not yield any radioactive bands on the
gel (see Fig. 4, lane 1). UV cross-linked protein complexes
with mouse liver mt extract yielded three distinct bands of
about 77, 75 and 55 kDa (lane 2). Assuming a molecular

mass of  7 kDa for single-stranded DNA probe, the
apparent size of the bound proteins correspond to  70,
 68, and  48 kDa, respectively. Results also show that
protein cross-linking with labelled DNA probe was effectively competed by a 10-fold molar excess of unlabelled
D-TERM DNA (lane 3), while a 20-fold molar excess of
mt-TERM DNA did not affect the level of protein crossliking (lane 4). These results further indicate the sequence

specificity of protein cross-linking.
Purification of D-TERM binding proteins
With a view to understand the nature of D-TERM DNA
binding proteins and their possible role in transcription


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Mitochondrial transcription termination (Eur. J. Biochem. 270) 1133

Fig. 2. Protein binding property of the D-TERM DNA. (A) DNAprotein binding by gel mobility shift analysis was carried out using
32
P-labelled D-TERM probe (0.2 ng, 30 000 c.p.m.) encompassing the
mouse mt genome sequence 16 274–16 295 as described in Materials
and methods. Lane 1 represents control with no added protein. In
lanes 2–14, 5 lg mouse liver mt extract was used. A 10–50 molar excess
of unlabelled DNAs, D-TERM (lanes 3–5) and mt-TERM (lanes 12–
14) as well as mutant versions of D-TERM (Mut) were also used for
competition: Mut1 (lanes 6–7), Mut 2 (lanes 8–9), Mut 3 (lanes 10–11).
(B) Nucleotide sequences of the L-strand D-TERM DNA and
H-strand mt-TERM DNA probes as well as their positions on the
mouse mt genome are shown. Mutant probes are the synthetic
D-TERM DNA probes with nucleotide replacements in polyadenylation signal sequence (Mut1) or upstream (Mut2) or downstream
(Mut3) of the signal sequence. The nucleotide replacements are highlighted in bold letters.

termination, we have partially purified the proteins by DNA
affinity chromatography. To this end heparin agarosebound mouse mt protein fraction was subjected to DNA
affinity chromatography. The ability of the affinity columnpurified protein fractions to bind to D-TERM DNA was
tested by gel mobility shift analysis. The results presented in
Fig. 5A show that the input protein fraction yielded both

complex I and II, as described in Fig. 2A. The flow-through

fraction yielded both of the complexes, though at reduced
intensities (lane 3). Further, the two wash fractions (lanes 4
and 5) and also the fraction eluted with 0.2 M KCl (lane 6)
did not form complexes with the DNA probe. The 0.5 M
and 1 M KCl eluates, on the other hand, formed complexes
with D-TERM DNA (Fig. 5A, lanes 7 and 8, respectively).
The latter fraction (1 M KCl eluted), however, formed
negligible complex I suggesting possible loss of proteins or a
change in the protein composition.
The D-TERM DNA binding activity was calculated
based on gel shift analysis using excess probe followed by gel
quantification. The combined intensity of complexes I and
II extrapolated for 1 mg of HA fraction was considered as
1. Quantitative results of protein recovery at different steps
of purification and relative DNA binding properties of
purified proteins show that the flow through fraction and
the wash fraction-1, which together represent < 90% of the
input protein, show reduced DNA binding efficiency of 0.3.
The wash fraction-2 and 0.2 M KCl eluted fractions which
represent  5–6% of the input protein also show very low
DNA binding activity in the range of 0.05–0.1. The 0.5 M
KCl eluted fraction, which represents < 1% of input
protein showed  20-fold higher DNA binding activity as
compared with control input protein. The 1 M KCl eluted
fraction representing > 0.5% of the input protein showed
DNA binding activity of  4.7-fold that of control input
protein.
The binding specificity of the affinity purified termination

factor(s) was tested by gel mobility shift using radiolabelled
mt-TERM DNA probe. As shown in Fig. 5A (lane 11) the
mtTERM DNA bound to the partially purified factor
(0.5 M KCl elute) with  20-fold lower efficiency as
compared with the D-TERM DNA probe. Additionally,
the input mouse liver mt extract also did not form the
characteristic complexes I and II, with the mt-TERM DNA
probe (lane 10). These results further support the view that
the D-TERM DNA has a distinct protein binding property
and suggest that protein factors binding to the D-TERM
DNA are distinctly different from the 36-kDa mt-TERM
protein factor mTERF [17].
The SDS/PAGE patterns of affinity purified proteins is
presented in Fig. 5B. It is seen that the heparin–agarose
binding step selectively enriched proteins in the range of
29 kDa to > 95 kDa with prominent bands of 70 kDa
and 45 kDa. The 0.5 M KCl fraction eluted from affinity
column contained predominantly the 70-kDa and 45-kDa
components, while the 1 M KCl eluted fraction contained
only the 45-kDa species. Further, the 45-kDa species in
both the 0.5 M and 1.0 M KCl eluted fractions resolve as
doublets. Currently it remains unknown if these doublets
represent post-translationally modified versions of the
same protein. The difference in the electrophoretic
patterns of the 0.5 M and 1.0 M KCl eluted fractions
(Fig. 5B) is consistent with the observed differences in
D-TERM DNA binding properties of these purified
fractions (Fig. 5A).
The DNA binding ability of the purified 70-kDa and
45-kDa proteins was tested by South-western blotting, in

which the heparin–agarose bound fraction (20 lg) and the
proteins eluted from the affinity column (1–10 lg) were
probed with 32P-labelled wild-type and mutant D-TERM
DNAs. As shown in Figs 6 D-TERM DNA specifically


1134 V. Camasamudram et al. (Eur. J. Biochem. 270)

Ó FEBS 2003

Fig. 3. Protein binding specificity and protein contact sites of the D-Term DNA. (A) DNAse 1 footprinting using mt protein extract. Binding
reactions contained 3¢ end-labelled H-strand D-TERM DNA probe (190 000 c.p.m.) and 25 lg BSA or mt extract. Following gel mobility shift
assay, the wet gel was autoradiographed and the free and protein bound complex II or complex I were excised, electro eluted and digested with
DNAse1 as described in Materials and methods. Maxam and Gilbert sequencing reactions of the D-TERM probe (GA and CT) were used as
markers in lanes 1 and 2. A vertical bar shows the protected sequence: lanes 3–6, reactions containing protein–DNA complexes (complex II, 20 000
c.p.m.) were treated with 0.025 U, 0.050, 0.075 and 0.1 U DNAse1 per 50 lL of reaction, respectively. The free probe treated with 0.075 U of
DNAse1 per 50 lL reaction is shown in lane 7. (B) Methylation interference analysis of the protein-bound D-TERM DNA. The 3¢ end-labelled
L-strand D-TERM DNA fragment was partially methylated and subjected to gel mobility shift analysis using mt extract as described in Materials
and methods. F and B indicate piperidine cleavage products of DNA recovered from free and protein-bound DNA (complex II), respectively. The
arrows on the right and asterisks on the left hand sequence bar denote the protected nucleotides. The Maxam and Gilbert sequence reactions were
loaded in lanes marked as GA and TC.

bound to two protein components of 70 kDa and 45 kDa
from heparin–agarose purified protein fraction (HA Fr).
The DNA probe also bound to two similarly migrating
proteins from the affinity purified 0.5 M KCl eluted fraction.
However, the 1 M KCl eluted fraction yielded a prominent
band at 45 kDa. Because the SDS gel profile in Fig. 5B
shows a 4- to 5-fold higher abundance of the 45-kDa species
in the 0.5 M KCl eluted fraction, these results suggest that

the 45-kDa protein is a weak DNA binding protein while
the 70-kDa species is a high affinity DNA binding
protein. Under identical experimental conditions, the
Mut1 D-TERM DNA probe with nucleotide replacements
targeted to the AATAAA sequence region did not bind to
these protein fractions thus indicating sequence specificity.
These results collectively show that the 70- and 45-kDa
proteins purified by DNA affinity chromatography bind to

D-TERM DNA in a sequence-specific manner, but with
different affinities.
Although not shown, UV-mediated DNA–protein crosslinking with gel eluted complex II, showed a 68-kDa protein
in addition to a major 70- and a minor 48-kDa component,
a pattern similar to that obtained with mt extracts (see
Fig. 4). It is likely, that the -48 kDa species identified by
UV cross-linking is probably an overestimate of 45-kDa
protein. The closely migrating 70- and 68-kDa cross-linked
products may represent the same protein bound to the H
and L-strands of the probe. Some of the discrepancies
between the South-western and UV cross-link analyses as
well as the failure to obtain the sequence of the
poly(vinylidine difluoride) (PVDF) membrane-bound protein clearly suggest the need for much higher protein
purification levels.


Ó FEBS 2003

Mitochondrial transcription termination (Eur. J. Biochem. 270) 1135

Fig. 4. Nature of protein binding to D-TERM DNA by UV crosslinking. Brd UTP-substituted and 32P end-labelled D-TERM DNA

was incubated with mouse liver mt extract under conditions of protein–DNA binding reactions as described in Fig. 2. Following the
binding reaction at room temperature for 30 min, the reaction mixture was irradiated at 254 nm of a UV lamp for 10 min at a distance
of 5 cm. The cross-linked products were incubated in Laemmli’s
sample buffer at 95 °C for 5 min and subjected to electrophoresis on
a 10% SDS polyacrylamide gel. DNA–protein complexes were
visualized by autoradiography. For competition experiments unlabelled competitor DNAs were added at 50-fold molar excess and
incubated for 10 min prior to the addition of labelled photoreactive
DNA probe. Lanes 1–4: mouse liver mt extract without (lane 1) and
with UV exposure (lanes 2–4). Lanes 3 and 4 contained 50-fold molar
excess of unlabelled D-TERM(DT) and mt-TERM (MT) DNAs,
respectively.

Factor-dependent transcription termination by D-TERM
DNA under in vitro conditions
We used the well-established HeLa mt lysate system for
in vitro transcription initiation and termination assays
because our attempts to develop an active in vitro system
from the mouse liver/heart mt extracts were unsuccessful.
To test the ability of the putative termination signal

D-TERM DNA to terminate transcription, we constructed chimeric DNA templates containing human LSP and
mouse D-TERM sequences. As shown in Fig. 7A, we
placed mouse D-TERM DNA sequence downstream of
the human LSP (pLSP) at an Mfe1 site in forward
orientation [pD-TERM (F)]. In addition, the D-TERM
DNA sequence was also placed in the reverse orientation
[pD-TERM (R)] to see if the termination is bi-directional.
Plasmid DNAs linearized with Ssp1 were used as
templates in run-off transcription reactions using HeLa
mt RNA polymerase.

Fig. 7B shows the read-through transcription with pLSP
plasmid templates linearized with Mfe1 and Ssp1, respectively. As expected, pLSP template DNAs yielded run-off
transcripts of 165 and 230 nt in length, respectively (lanes 1
and 2). Introduction of the putative terminator DNA in
pD-TERM (F) resulted in a longer read-through transcript
of 265 nt with Ssp1 digested DNA, consistent with the 35-nt
D-TERM added to the template (lane 3). However, no
detectable transcription termination was observed at the site
of inserted D-TERM sequence. We therefore decided to test
the effects of affinity purified mouse mt protein fractions
that were devoid of any contaminating DNAse or RNAse
activity on transcription termination with Ssp1 linearized
pD-TERM DNA templates.
As shown in Fig. 7C, HeLa polymerase fraction alone or
the reaction mixture supplemented with the Ôwash fractionÕ
yielded transcripts terminating at the Ssp1 site with no
significant termination at the D-TERM site (lanes 2 and 3).
Addition of 0.5 M KCl fraction (4 lgỈreaction)1), however,
yielded a major termination downstream of AAUAAA
signal at the end of CAA*, A being the terminal nucleotide
16 295 of the mouse mt genome (189 nt transcript; Fig. 7A
and C, lane 4). An additional major termination was also
observed at the downstream vector site corresponding to a
CAA sequence motif (196 nt transcript). The significance of
the termination at the latter downstream site remains
unclear, although it corresponds to a CAA sequence motif
similar to the upstream D-TERM site. Transcription
termination at the second CAA site, indicates the importance of this sequence motif in addition to AAUAAA
sequence, in the termination process. Quantitation of the gel
by radiometric imaging showed that nearly 60% of the

RNA chains were terminated at the two sites marked
Ôpremature temination sitesÕ in Fig. 7C. Finally, the protein
fraction eluted with 1.0 M KCl also caused transcription
termination at the same two sites, though at vastly reduced
rates (lane 5). The D-TERM sequence cloned in reverse
orientation [D-TERM (R)] was unable to induce transcription termination in the presence of added affinity-purified
factor suggesting that the termination is unidirectional or
polar (lane 6). Results also show that pD-TERM-Mut1,
Mut2 and Mut3 sequences with negligible to marginal
ability to compete for protein binding with D-TERM DNA
yielded vastly reduced transcription termination (lanes 7–9).
These results provide direct evidence that D-TERM DNA
functions as a transcription terminator under in vitro
conditions and that its activity is dependent on the presence
of a novel mt protein factor. Termination was concentration
dependent and was also inhibited by more than 60% by
50 ng D-TERM DNA added to the transcription reaction
mixture.


1136 V. Camasamudram et al. (Eur. J. Biochem. 270)

Ó FEBS 2003
Fig. 5. Purification of D-TERM binding protein by DNA affinity
chromatography. The preparation of D-TERM DNA affinity column
and chromatography using heparin–agarose purified mouse liver mt
extract were as described in Materials and methods. After washing the
column with 10 vols loading buffer, the DNA–Resin-bound proteins
were eluted sequentially with a step gradient of 4 column vols buffers,
each containing 0.2, 0.5, and 1 M KCl as indicated. The fractions were

tested for the presence of D-TERM DNA binding proteins by gel
mobility shift analysis. (A) Lanes 1–8, gel mobility shift patterns of the
D-TERM DNA binding to proteins from various column fractions.
Reactions were run using 4 lL of input protein heparin–agarose
fraction (HA fraction) and 8–9 lL each of flow-through, wash-1 (W1),
wash-2 (W2) and 0.2 M KCl eluted fractions. In lane 7, 0.5 lL of 0.5 M
KCl eluted fraction and in lane 8 1 lL of 1 M KCl eluted fractions
were used for binding. Lanes 9–11, gel mobility shift assays were
carried out using 32P labelled mt-TERM DNA (0.2 ng, 30 000 c.p.m.)
and 5 lL each of HA and 0.5 M KCl eluted fractions. (B) SDS/PAGE
of various column fractions indicated in (A). Proteins were resolved by
electrophoresis through SDS/12% polyacrylamide gels and visualized
by silver staining. Lane 1, crude mouse liver mt extract (30 lg); lane 2,
input heparin–agarose column purified extract (HA Fr, 20 lg); lane 3,
0.5 M KCl eluted (4 lg); and lane 4, 1.0 M KCl eluted (2 lg) fractions.

Discussion
It is well established that £ 60-fold higher steady-state levels
of rRNAs as compared to distal gene coded mRNAs in the
vertebrate mitochondria, result from a partial termination
of transcription at the end of the 16 S rRNA gene [13].
Several mechanisms, including transcription attenuation at

the putative hairpin structure of precursor RNA (at the 5¢
end of the tRNALeu gene) have been proposed for the
partial termination at the end of the rRNA genes [32,33].
Use of in vitro transcription systems, coupled with extensive
mutagenesis at the putative termination region, led to the
identification of a tridecamer sequence termed mt-TERM.
A 36-kDa protein termed mTERF has been to shown to

bind to the mt-TERM DNA in a sequence-specific manner
and promote partial termination of transcription [15–17].
Despite extensive characterization of the mt-TERM mediated partial termination past the rRNA genes, details as to
the specific site and the mechanism of termination of the
H-strand distal gene transcription remain to be elucidated.
Insight into the possible site of distal H-strand transcription termination came from studies on the characterization
of novel H-strand coded polyadenylated RNAs, mapping to
the D-loop regions of the rat and mouse mtDNAs [19,20].
Occurrence of such RNAs of relatively high abundance in
the mouse and rat tissues was surprising since the D-loop
region of the vertebrate mtDNA was believed to be the only
nontranscribed region of the genome. We identified a 0.8-kb
poly(A)-containing RNA, whose 3¢ end maps to the CAA
sequence at nucleotide 16 295 of the mouse mt genome [20].
The 3¢ terminus of the 0.8-kb RNA is preceded by a putative
polyadenylation signal AAUAAA [21]. Analysis of the 3¢
end polyadenylation sites of the H-strand encoded RNAs
by cDNA sequencing, has revealed that the putative
polyadenylation signal, AAUAAA, is conserved in human,
mouse and rat mt genomes [20]. A dodecamer sequence
AAUAA(U/C)AUUCUU was also shown to be the site of
pre-mRNA processing and 3¢ end formation in yeast mt
mRNAs [34]. The occurrence of polyadenylated and
oligoadenylated rRNAs in animal cell mitochondria is well
documented and known to be coupled to mt RNA
processing in the vertebrates [35]. The role of polyadenylation in the nuclear RNA Pol II transcription termination is
a well established entity. In view of these facts, we postulated


Ó FEBS 2003

Mitochondrial transcription termination (Eur. J. Biochem. 270) 1137

Fig. 6. DNA binding properties of affinity
purified proteins by South-western analysis. HA
fraction (25 lg protein), 0.5 M KCl eluted
fraction (2 lg protein) or 1 M KCl eluted
fraction (15 lg) were resolved, on SDS/12%
acrylamide gels, the proteins were transferred
to nitrocellulose membranes and probed with
32
P labelled D-TERM or Mut1 DNA probes
(8 · 105 c.p.m.ỈmL)1 each), as described in
Materials and methods. Figures represent
autoradiograms of blots.

that the conserved sequence motif, AAUAAA might be the
site of termination of distal region H-strand transcription,
and that the termination may be linked to polyadenylation
[20].
Inthepresentstudy,wedemonstratetheabilityofthe22-bp
putative D-TERM sequence (nucleotide 16 274–16 295 of
the mouse genome), containing the polyadenylation signal
and the flanking sequences to terminate transcription in an
in vitro mt transcription system. In a human mt transcription system driven by HeLa mt RNA polymerase, transcription termination was dependent on the addition of
DNA-affinity purified mouse liver mt protein fraction.
Further confirmation of the need for affinity purified
protein(s) comes from experiments showing that nucleotide
replacements targeted to the D-TERM sequence, which
affect protein binding also yield reduced factor-dependent

transcription termination (Fig. 7C). Finally, D-TERM
sequence cloned in the reverse orientation failed to induce
significant termination suggesting the specificity of the
in vitro system.
The D-TERM-mediated transcription termination
exhibits some similar, yet a number of distinct features
as compared to the mt-TERM dependent transcription
termination. Although both of these DNA motifs contain
A/T rich sequences, they show distinct protein binding
properties. The D-TERM DNA probe formed two
complexes (complex I and II), both of which were not
competed by even 50-fold molar excess of mt-TERM
DNA (Fig. 2A). Termination by both D-TERM and mtTERM sequences appear to be linked to polyadenylation.
However, we do not have experimental evidence to
indicate whether the polyadenylation is a terminationlinked or a processing event. While D-TERM mediated
transcription termination characterized in the present
study is unidirectional or polar, the mt-TERM dependent
termination is reported to be bidirectional [18]. In keeping

with their different binding specificities, the two terminator
sequences seem to bind to distinctly different proteins. The
D-TERM DNA binding proteins purified by affinity
chromatography contain two major protein components
of 70 and 45 kDa, while the mt-TERM DNA binding
protein is of 36 kDa [17]. It should also be noted that the
1 M KCl eluted fraction containing predominantly the
45-kDa protein, shows a weak DNA binding and only a
marginal termination activity under in vitro conditions,
while the 0.5 M KCl eluted fraction, containing both the
45-kDa and 70-kDa, proteins exhibits full activity

(Fig. 7C). The precise roles of the two proteins in
transcription termination remain to be elucidated. In the
case of mt-TERM mediated termination, the DNA affinity
purified mTERF factor was fully active in promoting
termination in an in vitro system [16]. However, the
bacterially expressed, purified 36-kDa factor showed no
significant termination activity [17]. These results suggest
the possibility that mt-TERM dependent transcription
termination requires multiple protein factors including the
well-characterized 36-kDa protein. It is possible that the
H-strand transcription termination system also requires
multiple protein factors.
The DNA binding properties of the proteins were studied
by multiple approaches. Initially, use of DNA-affinity
chromatography resulted in the purification of two major
proteins of 45 and 70 kDa (Fig. 5B). UV-mediated DNA–
protein cross-linking with crude extracts as well as with
complex II, however, showed a 68-kDa protein in addition
to a major 70- and a minor 48-kDa component. We believe
that the  48-kDa species identified by UV cross-linking is
probably an overestimate and may be the same as that
purified as a 45-kDa protein by affinity binding. Furthermore, the affinity purified 45-kDa species migrated as a
doublet on SDS/PAGE, although the South-western analysis showed a single protein band interacting with the DNA


1138 V. Camasamudram et al. (Eur. J. Biochem. 270)

Ó FEBS 2003

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Mitochondrial transcription termination (Eur. J. Biochem. 270) 1139

Fig. 7. Factor-dependent termination of human LSP initiated transcripts, in an in vitro HeLa mt transcription system. (A) Schematic representation of
the DNA templates tested for transcription termination (mt: mitochondria; the numbers in light print represent human and the numbers in bold
italics represent the mouse mt genome sequences). The template DNAs consisted of human LSP DNA [pLSP: human mt DNA sequences
(nucleotides 5¢-530–4-3¢) without or with mouse mt putative terminator sequence containing D-TERM DNA (5¢)16 274–16 295-3¢). In all the
chimeric transcription vectors used in termination reactions, mouse D-TERM DNA was inserted at the single Mfe1 site (nt 242), which is 165 bp
downstream of L strand start site at nt 407 [5,7]. pD-TERM (F) contained mouse D-TERM sequences cloned in forward orientation (5¢-16 274–
16 295-3¢) while pD-TERM (R) contained the mouse terminator sequences cloned in reverse orientation (5¢-16 295–16 274-3¢). Although, not
shown pD-TERM Mut1, Mut2, and Mut 3 transcription vectors contained mutant forms of D-TERM cloned at the same Mfe1 site in forward
orientation (see Fig. 2B). The transcription vectors were linearized with Ssp1, which results in run-off (RO) transcripts of 230 and 265 nucleotides
from pLSP and pD-TERM templates, respectively. Also the two possible D-TERM dependent termination transcripts of 189 and 196 nucleotides
arising from pD-TERM templates are also shown. (B) Patterns of in vitro run-off transcripts using the control pLSP and pD-TERM (F) DNA that
were digested with either Mfe1 or Ssp1 to generate 3¢ tailed templates. Run-off transcription was carried out using HeLa cell mt RNA Pol (4 lg) and
a32P UTP as described in Materials and methods. Lanes 1 and 2 show the run-off (RO) transcription with pLSP plasmid templates (20 lgỈmL)1)
linearized with Mfe1 and Ssp1 restriction enzymes, respectively. Lane3, run-off transcript of the pD-TERM (F) linearized with Ssp1. (C) Effects
of affinity purified protein fractions (4 lg) on in vitro transcription termination using Ssp1 linearized pD-TERM (F) or pD-TERM (R) or mutant
(pD-TERM Mut1, pD-TERM Mut2 and pD-TERM Mut3) templates. Run-off transcription reactions were carried out with HeLa mt RNA Pol
fraction alone (lane 2) or that supplemented with the various DNA-affinity column fractions (4 lg protein each lanes 3–9). Lane 3, wash fraction,
lanes 4 and 5, 0.5 and 1.0 M KCl eluted fractions, respectively. Lanes 6, same as lane 4, except that 20 lgỈmL)1 pD-TERM (R) DNA was used as a
template. Lanes 7–9, same as in lane 4, except that pD-TERM (F) DNA carrying various nucleotide replacements (Mut1, Mut2, and Mut3 as
shown in Fig. 2B) were used as templates. Although not shown, sequence ladders with primers starting from the transcription initiation nucleotide
at +1 were run alongside for determining the exact positions of in vitro terminated RNA.

probe. The precise nature of these two proteins remains
unclear, though they may represent post-translationally
modified forms. Based on the relative DNA binding
affinities we postulate that the 70-kDa protein is the major

DNA binding component and the 45-kDa protein may
associate with the DNA bound protein complex through
protein–protein interaction.
Most interestingly, another protein that binds to
D-loop region of mt DNA with a role in transcription
termination has recently been identified and cloned [36].
Studies by Fernandez-Silva [36] have established that the
binding of sea urchin mt displacement (D)-loop binding
protein (mtDBP) to the noncoding region (D-loop: 133 bp
region between tRNAThr and tRNAPro) of sea urchin mt
genome leads to transcription termination. Interestingly,
the 40-kDa sea urchin mtDBP shows a significant
sequence homology with the mammalian mTERF [37]
and functions as a bipolar transcription termination
factor. They propose a regulatory role for mtDBP even
in mt DNA replication [38].
In summary, we describe a novel sequence-specific
termination of the mouse mt H-strand distal transcripts,
which is reminiscent of the sea urchin mt transcription
termination system as well as the nuclear Pol I-dependent
rRNA [39] and Pol II-dependent mRNA transcription
termination systems [21]. Similar to the recently identified
sea urchin mtDBP, the D-TERM binding proteins bind in a
sequence specific manner to terminator motif localized in the
noncoding region (D-loop) of mouse mt DNA. However,
while both sea urchin mtDBP and mTERF function as
bipolar transcription termination factors, D-TERM proteins show polarity, similar to the TTF1/Reb-1 mediated
termination of rRNA [39]. Whether this is due to a
structurally asymmetric protein–DNA complex is not
known. Further details of the termination mechanism and

the nature of the D-TERM binding proteins are currently
under investigation.

Acknowledgements
We thank D. A. Clayton, Howard Hughes Medical Institute, Chevy
Chase, MD, USA, for providing the human LSP DNA used in this
study. We also thank M.-A. Robin, G. Biswas and C. Chandran
(FMHS, UAE University) for helping with the illustrations and M.
Higgins for editorial assistance. This research was supported in part by
NIH grant GM49683 and Common wealth and General Assembly of
Pennsylvania grant awarded to N. G. Avadhani.

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