(Received for publication, March 27, 1995; and in revised form, August 14, 1995)
From the
Footprinting studies with the purine-modifying reagent dimethyl
sulfate and with the single-stranded DNA probing reagent potassium
permanganate were carried out in isolated mitochondria from rat liver.
Dimethyl sulfate footprinting allowed the detection of protein-DNA
interactions within the rat analogues of the human binding sites for
the transcription termination factor mTERF and for the transcription
activating factor mtTFA. Although mTERF contacts were localized only at
the boundary between the 16S rRNA/tRNA genes, multiple mtTFA contacts were detected. Contact sites were
located in the light and the heavy strand promoters and, in agreement
with in vitro footprinting data on human mitochondria, between
the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium
permanganate footprinting allowed detection of a 25-base pair region
entirely contained in CSB-1 in which both strands were
permanganate-reactive. No permanganate reactivity was associated with
the other regions of the D-loop, including CSB-2 and -3, and with the
mTERF contact site. We hypothesize that the single-stranded DNA at
CSB-1 may be due to a profound helix distortion induced by mtTFA
binding or be associated with a RNA polymerase pause site. In any case
the location in CSB-1 of the 3` end of the most abundant replication
primer and of the 5` end of the prominent D-loop DNA suggests
that protein-induced DNA conformational changes play an important role
in directing the transition from transcription to replication in
mammalian mitochondria.
The majority of mammalian mitochondrial (mt) ()DNA
molecules possess a triple helix structure called D-loop due to the
displacement of the parental H-strand by short nascent H-strand chains.
The D-loop region, that is the main noncoding region and the most
variable part of vertebrate mt genomes, contains the origin of H-strand
DNA replication (O
) and the promoters for H and L-strand
transcription (for review see (1, 2, 3) ).
The two promoters (HSP and LSP) hold the binding sites for the
transcription factor mtTFA(4) , which activates H- and L-strand in vitro transcription. The H-strand transcripts initiate in
two points. One, I
, located few bases upstream of the
tRNA
gene, is responsible for the synthesis of the two
rRNAs 16S and 12S and of 2 tRNAs; the other, I
, located
near the 5` end of the 12S rRNA gene, directs the synthesis of 12 mRNAs
and of 12 tRNAs(5, 6, 7) . The L-strand is
entirely transcribed as a single transcription unit, starting from a
point that in humans is located 216 bp upstream of O
. It
has a low informational content, because it directs only the synthesis
of the primer for H-strand replication, of 1 mRNA and of 8 tRNAs. The
contrast between the complete L-strand transcription and its low
informational content is not yet fully understood. Moreover, despite
many studies in different laboratories, the molecular mechanisms that
regulate the production of the mature H- and L-strand transcripts are
not yet completely known. They likely involve protein factors and
nucleolytic enzymes. In particular, the termination of the H-strand
ribosomal transcription unit depends on a mt termination factor, mTERF,
that binds to a sequence located few bases downstream of the 5` end of
the tRNA
gene(8) , whereas the
primary transcript processing seems to be mediated by a RNase P-like
endonuclease, which should recognize the cloverleaf structure of the
tRNA genes that separate most of the rRNA and mRNA genes(9) .
Furthermore, the mapping of the nascent human mt transcripts had
suggested that also transcriptional pausing plays a role in regulating
the expression of mt genes(5) .
As far as L-transcripts are concerned, studies on the biosynthetic
mechanism of human and mouse H-strand replication primers (10, 11) showed the existence of several L-strand
transcripts initiating from the L-strand promoter and terminating in
the D-loop region at three conserved sequence blocks (CSB), CSB-1, CSB-2, and CSB-3. The 3` termini of these three RNA
species were joined with the 5` termini of nascent H-strand DNA chains,
and the prominent replication initiation site was located at the 5` end
of CSB-1. Clayton's group (10) hypothesized that the 3`
ends of the RNA primers were generated by post-transcriptional
processing of the primary transcript. Later it was found that a nuclear
RNA-containing endonuclease (RNase MRP), presumably involved in the
maturation of cytoplasmic rRNA (12, 13) and likely
also present at a low concentration in
mitochondria(14, 15, 16) , was able to cleave in vitro the L-strand primary transcript at some of the sites
found in
vivo(17, 18, 19, 20) .
Here,
in order to elucidate some of the aspects concerning the regulation of
mt transcription and replication, we used an experimental approach
aiming to obtain evidence about the physiological role of some of these
protein factors. In particular we used an in vivo-like
environment, made by rat liver isolated mitochondria, to investigate by
means of in organello footprinting with dimethyl sulfate (DMS)
and potassium permanganate (KMnO) the occurrence of protein
binding sites and of single-stranded DNA structures in the regulatory
region and in the transcription termination region of rat mt DNA. By
these techniques we were able to detect, within the mitochondrion,
multiple protein-DNA interactions and, for the first time, a
single-stranded-DNA region located within CSB-1. The data obtained with
this approach suggest that protein-induced mt DNA conformational
changes play an important role in defining the 5` ends of the prominent
H-DNA nascent chains.
Control samples
of naked DNA (protein-free) were obtained extracting the nucleic acids
from the same amount of mitochondria as above. Then the DNA pellets
were suspended in 100 µl of TE buffer (10 mM Tris-HCl, pH
7.4, 1 mM EDTA), preheated for 2 min at 37 °C, and treated
with 0.1% DMS for 2 min at 37 °C. The reaction was blocked by
adding 25 µl of 1.5 M sodium acetate, pH 7.4, 1 M 2-mercaptoethanol, and then the DNA was ethanol precipitated. The
pellets were lyophilized and treated with 100 µl of 1 M piperidine for 30 min at 90 °C. Sephadex separation was
performed as above, and the final samples were recovered in 35 µl
of water. In most experiments the piperidine treatment of samples and
controls was omitted because preliminary tests showed that the primer
extension effectively terminated in correspondence of modified bases;
in this case the DNA pellets were suspended in 100 µl of TE buffer,
purified by spin column chromatography, and recovered in 35 µl of
water. To set up the footprinting conditions and to check the fidelity
of mt DNA methylation pattern, preliminary control experiments, using
as template a recombinant plasmid containing the DNA region under
investigation, were carried out. 2 µg of the pFF28 plasmid
containing a rat mt DNA insert of 712 bp from position 15719 to
position 132 (21) were combined in a final volume of 100 µl
with 3 mM MgCl, 100 mM KCl, 0.2
mM dithiothreitol, 30 mM Tris-HCl, pH 8.0, and 0.1
mM EDTA. After preheating for 2 min at 37 °C, DMS was
added to a final concentration of 0.1%, and the samples were incubated
for 5 min at 37 °C. The reactions were blocked by adding 200 µl
of cold 3 M ammonium acetate, 1 M 2-mercaptoethanol,
20 mM EDTA, and 250 µg/ml tRNA. The DNA was recovered by
ethanol precipitation and processed as for DMS-treated mt DNA.
Several reports have shown that isolated mitochondria from different mammalian sources are able to synthesize and process mt RNA in a way that closely resembles the in vivo process(21, 22, 23, 24, 27, 28, 29) . Here, in order to detect within the mitochondria protein binding sites and single-stranded DNA regions, we carried out DNA footprinting in an in vivo-like environment made of isolated mitochondria from rat liver. In a typical experiment, freshly purified rat liver mitochondria were treated with the modifying agent, and the mt DNA was isolated. Control samples were obtained by a similar treatment of naked mt DNA (protein-free) purified from the same amount of mitochondria used for in organello footprinting. Mitochondrial DNA, isolated from test and control samples, was subjected to primer extension with Taq DNA polymerase in the presence of a 5` end-labeled oligonucleotide, and the reaction products were resolved on a polyacrylamide sequencing gel. The sites of DMS-altered reactivity or permanganate sensitivity were dependent on the organelle integrity, because they were not detected either in mitochondria stored at -80 °C, or in organelles whose permeability was altered by incubation in hypotonic media (experiments not shown). The results here reported were obtained in at least five different independent experiments on different individuals; each different sample of mitochondria displayed the same in organello footprinting pattern.
Figure 1:
In organello DMS footprinting at the
termination site of ribosomal gene transcription unit. A, DNA
from DMS-treated (D) and untreated (C) mitochondria
was prepared as described in the text. L-strand and H-strand probes
were the oligos ND1 (position 2761-2740) and 16S (position
2542-2563), respectively. Probe sequences were: ND1, 5`
GATTAGGAGTGTTAGGATATTA 3`, and 16S, 5` CCCAGTTACGAAAGGACAAGAG 3`. Rat
mt DNA positions (25) were deduced from control G-ladder and by
sequencing reactions run in preliminary experiments (not shown). Sites
of in organello methylation hypersensitivity are indicated by open triangles, and filled triangles indicate sites
of methylation protection. The size of the triangles is roughly proportional to the amount of modification; larger
triangles indicate at least a 3-fold difference between normalized
values of test and control samples. The bands R, R
, and R
in each panel serve as reference for
normalization. The position of the 16S rRNA/tRNA
boundary is shown. B, sequence positions of the
footprinted bases. The triangles indicate the sites of altered
DMS reactivity as deduced from A. The rat analogue of the
human tridecamer transcription termination site (34) is boxed. The junction between 16S rRNA and
tRNA
genes is indicated. H-strand
transcription proceeds from left to right.
The analyses of protein-DNA contacts in the regulatory region are reported in Fig. 2and Fig. 3. The L-strand and the H-strand probes P-REV1 (position 51-30) and D-rat-viv2 (position 16108-16129) detected two regions of altered DMS reactivity. The first region contained hypermethylated or undermethylated bases from 16197 to 16211; nine of the modified residues were located on the L-strand, and three were on the H-strand. The second region displayed a lower level of altered DMS reactivity; it encompassed 29 bp, from position 16252 to 16280. Also in this case both strands were involved, but the H-strand was slightly more affected than the other. Two more regions of altered DMS reactivity ( Fig. 2and Fig. 4) were detected with the probes D-REV2 (position 16107-15986) and D-rat-viv1 (position 15955-15976). They comprise bases contained between CSB-1 and CSB-2 (from 16042 to 16064) and bases contained within CSB-1 (from 16018 to 16034). DMS-altered activity was not detected in the bases located in CSB-2 and downstream of CSB-1 (results not shown). The region comprised between CSB-1 and CSB-2 showed a level of occupancy of at least 40%, with nucleotides 16050 and 16051 showing a DMS protection of about 90%, whereas CSB-1 was associated with a DMS protection level of about 50%. Although in most of the cases the altered methylation pattern concerned the purines, in some cases pyrimidine residues were also affected. In particular two T residues at positions 16018 and 16019 showed DMS hyper-reactivity (Fig. 4). It has been reported (35, 36) that DMS may methylate cytosine and to a minor extent thymidine when these bases are in a single-stranded DNA. This is the case for T-16018 and T-16019 that, as shown by permanganate footprinting reported below (see Fig. 6), are in a single-stranded DNA.
Figure 2:
Schematic diagram summarizing the results
obtained by DMS and KMnO in organello footprinting in the
regulatory region of rat mt DNA. The positions and the orientation of
the oligonucleotide probes are shown at the top of the figure;
their precise positions are reported in the legends of the following
figures. Hatched boxes (I, II, III and IV) indicate the protein contact sites as deduced
from the DMS footprinting experiments reported in Fig. 3and Fig. 4. Permanganate reactivity at CSB-1 (see Fig. 6) is
indicated by displacing both strands. In the case of DMS footprinting,
probes PREV-1 and D-rat-viv-2 were used to detect regions I and II (see Fig. 3). Regions III and IV (see Fig. 4) were found with
probes D-REV2 and D-rat-viv-1. The permanganate reactive region in
CSB-1 (see Fig. 6) was detected with the probes P-REV1, D-REV2,
and D-rat-viv-1. The bottom part of the diagram shows the
presumptive approximate positions of primer RNAs and of the H-DNAs in
rat as deduced from the mapping of such species in human and
mouse(10, 11) . Wavy and solid lines indicate the primer RNAs and the H-DNAs, respectively. Bold
lines show the most prominent primer and H-DNA species. The
numbers refer to the genomic position of rat mt DNA(25) . H, H-strand; L, L-strand; CSB, conserved
sequence block; CSB-1, 16012-16037; CSB-2,
16068-16084; CSB-3, 16101-16118. O
is the main H-strand replication
origin, which in rat is located at position 16011 (63) . I
and I
are the L and H-strand initiation sites that in rat are
located around position 16178 and 16183,
respectively(63) .(
)
Figure 3:
In organello DMS footprinting
near the H- and L-strand promoters. A, L-strand and H-strand
probes were the oligos P-REV1 (position 51-30) and D-rat-viv-2
(position 16108-16129), respectively. Primer sequences were:
P-REV1, 5` GAATCCATCTAAGCATTTTCAG 3`, and D-rat-viv-2, 5`
CCCCAAAAACATTAAAGCAAGA 3`. The bands R and R
in each panel serve as
reference for normalization. Symbols indicating the site and the extent
of altered methylation reactivity are the same as in Fig. 1. For
each primer a short (S) and a long (L) gel run are
shown. D, DMS treated mitochondria; C, control. B, genomic position of the bases with altered methylation. The triangles indicate the sites of altered DMS reactivity as
deduced from A. I
and I
are the initiation sites of L and H
transcripts. The two boxed regions are the putative binding
sites of rat mtTFA at LSP and HSP.
Figure 4:
In organello DMS footprinting
near the replication origin of rat liver mt DNA. A, L-strand
and H-strand probes were the oligos D-REV2 (position 16107-15986)
and D-rat-viv1 (position 15955-15976), respectively. The
sequences were: D-REV2, 5` TTTGGCATTGAAGTTTCAGGTG 3`, and D-rat-viv1,
5` CCTGTGGAACCTTTTAGTTAAG 3`. Symbols used to indicate the sites and
the extent of altered DMS reactivity are as in Fig. 1. The bands R, R
, and R
in each panel serve as reference for
normalization. D, DMS-treated mitochondria; C,
control; Pl, recombinant plasmid pFF28 (it contains a rat mt
DNA insert of 712 bp comprising position 15719-132(21) )
treated in vitro with DMS. B, genomic position of the
bases with altered DMS reactivity as deduced from A. The
positions of the H-strand replication origin (O
) and of CSB-1-2 are
shown.
Figure 6:
In organello permanganate
footprinting near the replication origin of rat mt DNA. A,
L-strand probes were the oligos P-REV1 and D-REV2; H-strand probe was
the oligo D-rat-viv1. Panel I shows the P-REV1 extension
products of undigested mt DNA extracted from KMnO-treated (K) and untreated(C) mitochondria. In lanes 1 and 2 are reported the extension products of the plasmid
pFF28(21) . The plasmid was first treated with DMS (lane
1) and KMnO
(lane 2) as described under
``Materials and Methods,'' digested with BglI, and
then subjected to primer extension with Taq DNA polymerase and
labeled P-REV1 primer. Mitochondrial genomic positions refer to bands
in lanes 1 (underlined) and 2 (not
underlined). In the other two panels mt DNA from test (K)
and control (C) was first digested with BglI and then
subjected to primer extension with D-REV2 and D-rat-viv1, respectively.
Sites of in organello permanganate hyper-reactivity are
indicated by triangles. The positions of
CSB-1(16012-16037), CSB-2(16068-16084), and
CSB-3(16101-16118) are indicated. B, sequence positions
of the permanganate reactive bases. The triangles indicate the
sites of hyper-reactivity as deduced from A. The positions of
CSB-1 to CSB-3) and of H-strand replication origin (O
) are
indicated.
To obtain information relative to the significance of these DNA-protein interactions in rat liver, sequence homology studies were performed. Fig. 5A shows that Rat I, a sequence of 21 bp (from 16196 to 16216) containing the first block of reactive bases, was homologous to the mtTFA binding site of human, mouse, and bovine LSP (4, 32, 33, 37) , whereas (Fig. 5B) the second block of reactive bases contained a region, Rat II (extending from 16247 to 16268), displaying a significative homology with the mtTFA binding site of HSP. Therefore the DMS-modified bases of Rat I and Rat II are probably due to contacts with the rat analogue of human factor mtTFA, which stimulates mt transcription interacting with DNA sequences located upstream of the transcription initiation sites(4) . The similarity of the sequence Rat III, located between CSB-1 and CSB-2 (from 16041 to 16064) with the mtTFA-footprinted regions in LSP and HSP, shown in Fig. 5C, suggests that the altered reactivity of this region is due to contacts with the same protein. This hypothesis is supported by the capacity of mtTFA to bind in vitro an analogous region of human mt DNA(4, 33) . The bases located within CSB-1 (Fig. 2, block IV, and Fig. 3B) do not share significative homology with the other footprinted regions. However footprinting analysis in human mt DNA (33) showed that mtTFA was able to bind nonhomologous sequences in vitro, including those contained in CSB-1. These data, which were confirmed by in organello footprinting(33) , let us to ascribe also the DMS-altered reactivity of CSB-1 to the rat analogue of human mtTFA.
Figure 5:
Sequence alignment of DMS footprinted
regions. A, alignment of rat footprinted region near I with the mtTFA binding sites at the LSP
of human, mouse, and bovine mt DNA(33, 35) . B, alignment of rat footprinted region near I
with mtTFA binding sites at the HSP of
human, mouse, and bovine mt DNAs. C, alignment of DMS
footprinted bases in the noncoding region of rat mt DNA. The numbers in parentheses (human, bovine, and mouse genomic
positions are according to Anderson et al.(70, 71) and Bibb et al.(72) )
refer to the position of the first nucleotide of the
sequence.
To test the presence of
single-stranded DNA regions in other locations and in particular at a
site where protein-dependent transcription termination takes place, we
tested the permanganate hypersensitivity of the region containing the
16S rRNA/tRNA boundary. This is the site
where the ribosomal transcription unit ends due to the interaction with
the termination factor mTERF(8) . Fig. 7shows the
absence of any significative signal due to permanganate hyper-reactive
bases. These data were quite surprising in the light of a recent report (49) showing that mTERF binding induced DNA bending. The lack
of permanganate sensitivity in the presence of DNA bending might be
explained by hypothesizing that the mt DNA complexed with mTERF assumes
a stacked configuration that prevents the thymidine oxidation by
permanganate(50) . Moreover, this result implies that the
single-stranded DNA regions functioning as pause sites are not required
for termination, so that mTERF alone is able to generate, probably by a
physical blockage mechanism, the end of transcription.
Figure 7:
In organello permanganate
footprinting at the boundary between 16S rRNA/tRNAUUR
genes. Probes used were the oligos ND1 and 16S (see Fig. 1). The
position of the gene boundary is indicated. C, control; K, permanganate-treated mitochondria. The numbers at
the left of each lane indicate the genomic positions
of some bands, as deduced by control T+C ladder (not
shown).
The mtTFA binding to the different portions of the D-loop is related to the multiple roles of this factor in mt transcription and replication. The binding that takes place at LSP and HSP serves for stimulating mt transcription(4, 51) . It occurs with different efficiencies ranging from 82% in LSP (Fig. 2, block I) to 45-50% in HSP (Fig. 2, block II); this is in agreement with both in vitro binding experiments with human mtTFA (4) and with the higher rate of in vivo mt transcription of the L-strand compared with that of H(3, 5) . It is likely that variations in the level of mtTFA within the mitochondria may regulate promoter selection such that at low concentrations L-strand transcription, which is linked to H-DNA replication, would predominate, whereas at higher mtTFA levels HSP transcription would also take place. The recent finding of a NRF-1 binding site in the human mtTFA gene (52) is a first evidence of the modulation of mtTFA gene expression. The mtTFA contacts with LSP and HSP are quite asymmetric; at LSP most of the purines located on the L-strand contact mtTFA, whereas on the H-strand only 3 purine residues exhibit DMS hyper-reactivity. Although both H- and L-strand promoters may function bidirectionally in vitro and in vivo(53) , the transcription of the respective main coding strand largely predominates. The substantial asymmetric binding of mtTFA to LSP and HSP found in organello and in vitro(4) may represent the mechanism used for the substantial unidirectionality of the two promoters.
Recent in organello and in vitro footprinting experiments of Ghivizzani et al.(33) showed that human mtTFA, in addition to binding LSP and HSP, is able to bind the entire regulatory region of mt DNA in a phased arrangement, contributing to specific packaging of mt DNA control region in vivo. It has been suggested that phased mtTFA binding might be required for facilitating mt transcription (33, 54) or RNA processing(17) . The data reported in this paper confirm this view because DMS-altered reactivity, presumably due to the rat analogue of human mtTFA, was found in the region between CSB-1 and CSB-2 and within CSB-1.
The binding of mtTFA to CSB-1 probably underlies a further function of this factor. In fact, contrary to the rest of the other binding sites whose sequence among mammalian species is highly diverging(37, 55) , CSB-1 is universally conserved among vertebrates(55, 56, 57) . Moreover, it contains the transition site between the most abundant replication primer and the prominent D-loop DNA(55, 58, 59) . Therefore, it is likely that the binding of mtTFA to CSB-1 might have a rather different role with respect to that hypothesized for the other regions of the D-loop. The most likely possibility is that the mtTFA-CSB-1 complex may be part of a recognition signal for the transition from L-RNA to H-DNA chains. The presence of a single-stranded DNA in this region (see below) reinforces the hypothesis of a peculiar role for the mtTFA-CSB-1 complex.
Few cases of DNA distortions induced by DNA-binding proteins have been reported. They include the RAP-1 protein, whose binding to Saccharomyces cerevisiae telomers induces an unusual permanganate reactivity of the C-rich strand(60) , the Epstein-Barr virus DNA replication origin binding protein EBNA1(61) , and the TATA box-binding protein, whose binding in vivo to the coding strand of the promoter of the phosphoenolpyruvate carboxykinase gene produces an altered permanganate activity on the opposite strand(62) . It is interesting to observe that although the distortions induced by these proteins (60, 61, 62) concern a limited stretch of DNA, the extended permanganate reactivity of CSB-1 (13 reactive bases on the L-strand and 7 on the H-strand in a stretch of 25 bp) would create a profound structural change that should involve more than one helical turn.
An alternative explanation of the permanganate reactivity at CSB-1 is based on the presence among nascent human transcripts of many paused RNA molecules (5) and based on the location within CSB-1 of the multiple 3` ends of RNAs originating from the L-strand initiation site (10, 11, 21, 63) . These data suggest that the single-stranded DNA at CSB-1 may be associated with a RNA-polymerase pause site. The existence of a transcription pause site in CSB-1 might have implications for the mechanism of replication primer formation. It could be hypothesized that the formation of the 3` end of the longest and presumably more active replication primer (7S RNA), which terminates in CSB-1, might take place by transcriptional pausing and would not necessarily require the action of a nuclease activity. On the contrary, the lack of permanganate-reactive bases at CSB-2 and CSB-3 would require the presence of one or more nuclease activities needed to create the 3` ends of the primers terminating at these sites. The existence of a transcription pause site at CSB-1 might also help to explain the regulation mechanism of L-strand transcription. Although the L-strand is transcribed at a higher rate than the H-strand(5) , its products are not more abundant than the H-strand coded RNAs(64) . This could depend either on a lower stability of the L-strand polycistronic transcript (65) or on the existence of a pause site at CSB-1. According to the latter hypothesis the L-strand transcription would start at a high rate, producing the replication primer; then the RNA-polymerase should meet the pause site from where only a minority of the polymerase molecules should escape to synthesize the L-strand coded products. Transcriptional regulation at steps following the initiation is a well established mode of controlling gene expression, not only in prokaryotes (66) but also in eukaryotes. Recently, Giardina et al.(44) showed by in vivo permanganate footprinting that pausing occurred after few bases from the transcription initiation site of two Drosophyla heat shock genes. Krumm et al.(45) reported that the block to transcriptional elongation of the human c-myc gene, which occurs near the end of the first exon, was determined by promoter proximal pausing.
The two explanations of permanganate reactivity at CSB-1 are not necessarily in contrast with each other because both envisage a role for single-stranded DNA at CSB-1 in the transition from transcription to replication. The overall data suggest that the mtTFA binding at CSB-1 and the strong and specific permanganate sensitivity of this region are probably both part of the same signal that regulates the transition from RNA primers to nascent H-DNA chains. Moreover the results reported in this paper emphasize the potential of in organello footprinting with chemical reagents in analyzing at a molecular level the changes in mt DNA structure that are linked to transcriptional or replicative activity of the organelle. This technique will represent a useful approach for studying the changes in mt structure and expression observed in different physiological and pathological conditions in an in vivo-like environment, such as during aging or in mitochondrial diseases(67, 68, 69) .