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

Transcription termination at the mouse mitochondrial H-strandpromoter distal site requires an A/T rich sequence motif and sequence specific DNA binding proteins Vijayasarathy Camasamudra

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,

immedi-ately upstream of the tRNAPhegene 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 chromato-graphy 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 there-fore 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

Keywords: mitochondria; transcription termination; mito-chondrial H-strand transcription; D-loop binding proteins; polyadenylation

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, HSP1and 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 0.5–1kb

nascent DNA strand needed for the replication of the L

strand The 0.8–1kb 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, 1 2 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-pro-ximal 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 mito-chondrial diseases led to the identification of a tridecamer DNA sequence that supports partial transcription termin-ation 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 termin-ation, 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: + 9713 7672033, Tel.: + 9713 7039502,

E-mail: vijayc@uaeu.ac.ae

Abbreviations: mt, mitochondrial; D-TERM, promoter distal

termi-nator 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)

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 tRNAProon

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

transcrip-tion terminatranscrip-tion site (D-TERM) for the promoter distal

H-strand 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

tran-scription in an in vitro reconstituted system in a

unidirec-tional 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 1g wet weight)

in logarithmic phase were harvested and disrupted by

homogenization in a buffer containing 134 mMNaCl, 5 mM

KCl, 1mM 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

1.2–1.6Msucrose 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 mMKCl, 10 mM MgCl2,1 mMEDTA,

1 mM dithiothreitol), lysed by adding 0.6M KCl and the soluble fraction was separated by centrifugation at

105 000 g for 60 min at 4C The supernatant fraction was dialysed against buffer B (20 mMHepes, 50 mMKCl,

1 0 mMMgCl2,1 mM EDTA, 1 5% glycerol, 2 mM dithio-threitol, 1mM phenylmethanesulfonyl fluoride and

1 lgÆmL)1 each of leupeptin, pepstatin and antipain) for 3–4 h, at 4C and used for binding to heparin–agarose resin About 1mL heparin–agarose, pre-equilibrated with buffer B, was added to the extract (100 mg protein) and mixed with gentle agitation for 1h at 4C 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 0.1–1.0M KCl in buffer B The fractions eluted between 0.5 and 1.0M 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 promoter-distal 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 (1mL swollen resin, Sigma) in a buffer containing 10 mM Tris/HCl

pH 7.4, 50 mM NaCl, 1mM dithiothreitol, 1mM EDTA and 5% glycerol The affinity DNA matrix (1mL) 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, 1mM dithiothreitol, 1mM EDTA, 5% glycerol, 1mM phenyl-methanesulfonyl fluoride, 1 lgÆmL)1 each of leupeptin, pepstatin and antipain and 25 lg poly dI-dC The binding mixture was incubated for 60 min at 4C on a rotating wheel The contents were then poured into a 0.6· 5 cm column and the unbound material was collected as flow-through 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 1.0MKCl 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 mMTris/HCl pH 7.4, 50 mMNaCl,

1 m dithiothreitol, 1m EDTA, 5% glycerol, 100 ng

dI:dC, 0.1–0.2 ng32P-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

Unla-belled 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%

acryl-amide gels in Tris/Acetate/EDTA buffer at 4C [20]

DNaseI footprint analysis

The putative transcription termination region DNA

(D-TERM DNA) cloned in the BamH1site of Bluescript

vector (Stratagene) was linearized with either Xba1or III

and 3¢ end-labelled with Klenow enzyme in the presence of

a-32P dCTP After a second digestion with either Xba1or

HindIII the resulting fragments were gel purified and

electroeluted Gel purified 3¢ end-labelled DNA probe

(190 000 c.p.m Xba1end-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 4C 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)

treat-ment 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

MgCl2for 2 min at 25C as described by Henninghausen

and Lubon [27] Samples were phenol extracted,

concen-trated 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

dimethyl-sulphate 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

electrophor-esis 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 tRNAPhegene

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 l TTP and 100 l 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 denatur-ation at 95C 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 cross-linked 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 mMTris and 20% methanol buffer The proteins

on the blot were denatured with 6M guanidine/HCl, in binding buffer (10 mM Tris HCl pH 7.5, 50 mM NaCl,

1 mM dithiothreitol, 0.1mM EDTA, 0.01% NP40, 5% glycerol), for 5 min and renatured by stepwise dilution of guanidine/HCl solution from 6M to 0.375M 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.ÆmL)1) in binding buffer containing

2 lgÆmL)1 poly(dI/dC) for 12 h at 4C 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 termin-ation (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 EcoR1site 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 Mfe1site 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

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 Mfe1site 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

run-through 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 0.5Mor 1MKCl 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,

1 0 mM MgCl2, 1 mM dithiothreitol, 100 lgÆmL)1 BSA,

400 lM ATP, 200 lM GTP, 200 lM CTP, 40 lM UTP,

20 lgÆmL)1 linearized transcription template, 4 lg HeLa

mt RNA polymerase and 0.25 lM[a-32P]UTP (5 lCi from

a stock of 400 CiÆmmol)1 specific activity), at 28C 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 mgÆmL)1 Proteinase K) and incubation continued at

37C for 30 min The in vitro transcription products were

recovered by phenol extraction followed by ethanol

precipitation and resolved on 6% polyacrylamide, 8M

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 tRNALeugene 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,

over-lapping 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

uncer-tain 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

termin-ation 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 signifi-cantly 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 poly-adenylation signal sequence AATAAA Further, 20 and 50 molar excesses of Mut1DNA, 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 down-stream 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 investi-gated using DNAse1footprinting and methylation interfer-ence analysis Fig 3A shows the DNAse1footprint 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 DNAase1is 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 0.025–0.1U DNAse1per 50 lL reaction Although not shown, the L-strand labelled probe showed a similar generalized protection of the entire D-TERM sequence region Addi-tionally, 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 ana-lysis 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 remain-ing two residues lie in the flankremain-ing sequences Additionally, two A residues (nucleotides 16 294 and 16 295, respect-ively), immediately 3¢ to the protected A residue of D-TERM DNA become hypermeyhlated Hypermethyla-tion is thought to be the result of conformaHypermethyla-tional 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)

Nature of mt proteins binding to the D-TERM DNA

sequence

UV-induced DNA cross-linking was carried out to

investi-gate 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

95C 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 effect-ively 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 cross-liking (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

Fig 1 Location of the putative D-TERM element for H-strandtranscription termination

on the mouse mt genome (A) Location of the D-TERM element on the mouse mt genome The triangles represent the tRNA genes desig-nated by the single-letter amino acid code The polarity of triangle indicates the direction

of the transcription O L and O H represent the origin of L and H strand replication, respect-ively LSS, L strand start site; HSS, H strand start site The map positions of the rRNA, tRNAs and mRNAs (ND1to 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 ter-mination element (D-TERM) is also shown.

termination, we have partially purified the proteins by DNA

affinity chromatography To this end heparin

agarose-bound mouse mt protein fraction was subjected to DNA

affinity chromatography The ability of the affinity

column-purified 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 0.2MKCl (lane 6) did not form complexes with the DNA probe The 0.5M and 1MKCl eluates, on the other hand, formed complexes with D-TERM DNA (Fig 5A, lanes 7 and 8, respectively) The latter fraction (1M 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 1mg 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 0.2MKCl 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 0.5M KCl eluted fraction, which represents < 1% of input protein showed 20-fold higher DNA binding activity as compared with control input protein The 1MKCl 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 (0.5M 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 0.5MKCl fraction eluted from affinity column contained predominantly the 70-kDa and 45-kDa components, while the 1MKCl eluted fraction contained only the 45-kDa species Further, the 45-kDa species in both the 0.5Mand 1.0M 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 0.5M and 1.0M 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 with32P-labelled wild-type and mutant D-TERM DNAs As shown in Figs 6 D-TERM DNA specifically

Fig 2 Protein binding property of the D-TERM DNA (A)

DNA-protein 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 1represents 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

polyadeny-lation signal sequence (Mut1) or upstream (Mut2) or downstream

(Mut3) of the signal sequence The nucleotide replacements are

high-lighted in bold letters.

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 0.5MKCl eluted fraction

However, the 1MKCl 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 0.5MKCl 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

Mut1D-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 cross-linking 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 pro-tein clearly suggest the need for much higher propro-tein purification levels

Fig 3 Protein binding specificity and protein contact sites of the D-Term DNA (A) DNAse 1footprinting 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 DNAse1as described in Materials and methods Maxam and Gilbert sequencing reactions of the D-TERM probe (GA and CT) were used as markers in lanes 1and 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 0.1U DNAse1per 50 lL of reaction, respectively The free probe treated with 0.075 U of DNAse1per 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.

Factor-dependent transcription termination by D-TERM

DNA underin 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 construc-ted 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 Mfe1site 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 Ssp1were 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 Mfe1and Ssp1, respect-ively 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 Ssp1digested 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 Ssp1linearized pD-TERM DNA templates

As shown in Fig 7C, HeLa polymerase fraction alone or the reaction mixture supplemented with the wash fraction yielded transcripts terminating at the Ssp1site with no significant termination at the D-TERM site (lanes 2 and 3) Addition of 0.5MKCl fraction (4 lgÆreaction)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 import-ance 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

premature temination sites in Fig 7C Finally, the protein fraction eluted with 1.0M 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 transcrip-tion terminatranscrip-tion 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

Fig 4 Nature of protein binding to D-TERM DNA by UV

cross-linking Brd UTP-substituted and32P end-labelled D-TERM DNA

was incubated with mouse liver mt extract under conditions of

pro-tein–DNA binding reactions as described in Fig 2 Following the

binding reaction at room temperature for 30 min, the reaction

mix-ture 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

unla-belled 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.

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 medi-ated 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 transcrip-tion terminatranscrip-tion came from studies on the characterizatranscrip-tion

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 polyadenyla-tion in the nuclear RNA Pol II transcrippolyadenyla-tion terminapolyadenyla-tion is

a well established entity In view of these facts, we postulated

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 32 P 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.

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]

In the present study, we demonstrate the ability of the 22-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 vitromt transcription system In a human mt

transcrip-tion system driven by HeLa mt RNA polymerase,

tran-scription 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 vitrosystem

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

mt-TERM sequences appear to be linked to polyadenylation

However, we do not have experimental evidence to

indicate whether the polyadenylation is a

termination-linked 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 [1 8] 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

1M 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 0.5M 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 Further-more, the affinity purified 45-kDa species migrated as a doublet on SDS/PAGE, although the South-western ana-lysis showed a single protein band interacting with the DNA

Fig 6 DNA binding properties of affinity

purifiedproteins 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 · 10 5

c.p.m.ÆmL)1each), as described in

Materials and methods Figures represent

autoradiograms of blots.