| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
2 Graduate School of Engineering Science, Osaka University, Toyonaka 560-8531, Japan
3 Institute of Medical Technology and Tampere University Hospital, University of Tampere, FI-33014, Finland
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Mitochondrial RNA polymerase (POLRMT) is a key molecule of the core complex of the mitochondrial transcription machinery which assembles at promoter sequences on both strands of mtDNA, termed the L-strand promoter (LSP) and H-strand promoter (for reviews, see Gaspari et al. 2004; Asin-Cayuela & Gustafsson 2007). Although POLRMT belongs to the family of Phage T7-like RNA polymerases (reviewed by Shutt & Gray 2006), it cannot interact directly with a promoter sequence and initiate transcription in its own right, in contrast to T7 RNA polymerase. POLRMT requires the assistance of mitochondrial transcription factor A (TFAM) and one of two mitochondrial transcription factor B (TFB1M or TFB2M) to initiate promoter-dependent RNA synthesis (Dairaghi et al. 1995a; Falkenberg et al. 2002). TFB2M has more efficient activity for mitochondrial transcription initiation than TFB1M (Falkenberg et al. 2002). The distinction between the physiological roles of TFB1M and TFB2M is still unclear.
As for mtDNA replication, two models have been proposed, based, respectively, on strand-asynchronous and strand-coupled mtDNA synthesis (Shadel & Clayton 1997; Holt et al. 2000; Falkenberg et al. 2007).
In the strand-asynchronous model, the nascent leading strand initiates at the origin of heavy (H)-strand synthesis (OH) and proceeds unidirectionally, displacing the parental H-strand as single stranded DNA (ssDNA) until the separated origin of light (L)-strand synthesis (OL) is exposed for lagging strand synthesis (Shadel & Clayton 1997). DNA synthesis of each strand occurs unidirectionally and continuously (Clayton 1991). The transcription from LSP is supposed to provide RNA primers for synthesis of the leading strand initiating from OH. Thus, POLRMT is thought to contribute a primer for the initiation of H-strand synthesis (Chang & Clayton 1985), whereas the priming mechanism at the major L-strand origin is largely unknown in this model.
On the other hand, for the strand-coupled mtDNA synthesis model, two mechanisms have been proposed based on electrophoretic analyses of replication intermediates. One mechanism is coupled leading and lagging-strand DNA synthesis, which initiates bidirectionally across a broad zone downstream of the major non-coding region of mtDNA (Holt et al. 2000; Bowmaker et al. 2003). In the other mechanism, initiation is limited to specific sites within the non-coding region, with RNA incorporation throughout the lagging strand (RITOLS) (Yang et al. 2002; Yasukawa et al. 2006). Both strand-asynchronous and RITOLS replication predict delayed second-strand DNA synthesis; however, in the RITOLS model RNA takes the place of mitochondrial single-stranded DNA binding protein (mtSSB). Conventional strand-coupled DNA synthesis requires repeated synthesis of short RNAs on the lagging strand to give rise to Okazaki fragments, which elsewhere is typically mediated by a dedicated primase that forms an integral part of the replisome (Kornberg & Baker 1992). The long tracts of RNA associated with RITOLS replication might also be products of a primase. Although a primase activity in mammalian mitochondrial extracts was reported (Wong & Clayton 1986), the protein responsible has never been identified. Identification and characterization of a mitochondrial primase could reveal important features of one or both models of mtDNA replication.
In previous studies, we and others have reported that mtDNA is organized in a higher order structure termed the nucleoid and that TFAM has an architectural role in nucleoid formation (Garrido et al. 2003; Kanki et al. 2004; Kaufman et al. 2007). It has also been proposed that TFAM affects mitochondrial transcription via a conformational change of mtDNA (Ohgaki et al. 2007).
In order to further elucidate the effect of DNA conformation on human mitochondrial transcription, we reconstituted an in vitro system using minimum protein components (i.e., recombinant POLRMT, TFB2M, and TFAM) plus closed circular DNA as a template. In the course of this study, we found a strong promoter-independent but DNA conformation-dependent activity of POLRMT. Here we characterize this promoter-independent and conformation-dependent RNA synthesis and discuss its potential physiological roles in mtDNA transcription and replication.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
We set up a reconstituted in vitro transcription system in which only purified recombinant proteins were used: POLRMT, TFB2M, and TFAM (Fig. S1, Supporting Information). The molecular mechanisms of mitochondrial transcription have previously been investigated mostly using an in vitro run-off assay used on linear DNA templates (Falkenberg et al. 2002). However, mammalian mtDNA is circular and also adopts different conformations. To determine the effects of DNA conformation on mitochondrial transcription, we designed three kinds of template. The first is a linear DNA fragment including the LSP region and conserved sequence blocks (CSBs) of human mtDNA, used in a run-off assay (Fig. 1A, panel a). Both LSP and CSBs are thought to play important roles for RNA primer formation at the origin of the leading strand (OH) (Shadel & Clayton 1997). The second is a closed circular DNA containing a portion of the non-coding region including LSP (Fig. 1A, panel b). The third is the same as the second but without LSP (Fig. 1A, panel c).
|
When RNA synthesis was carried out in the presence of TFAM on the negative supercoiled closed circular DNA template containing LSP, the 132 nt product was clearly observed (Fig. 1B, lane 3), indicating LSP-dependent RNA synthesis. In addition, RNA products with various lengths over 1 kb were observed. These longer RNAs may reflect a natural processivity of POLRMT or may repeated initiation elsewhere in the template. When TFAM was omitted (Fig. 1B, lane 4), the 132 nt product was hardly seen, indicating that LSP-dependent RNA synthesis was suppressed. TFAM is therefore required for LSP-dependent RNA synthesis also on supercoiled DNA. In contrast to the 132 nt product, the longer RNAs were still observed, although signal intensities for these transcripts were only about half of those seen in the presence of TFAM (compare lanes 3 and 4). To test whether they could be derived from LSP-independent RNA synthesis, we performed RNA synthesis on a supercoiled closed circular DNA template lacking LSP, in the presence of TFAM (Fig. 1B, lane 2). The 132 nt product was not seen in the absence of LSP. However, the longer RNA products were observed to a similar extent to those in Fig. 1B, lane 4. We then synthesized RNA on supercoiled closed circular DNA without LSP in the absence of TFAM (Fig. 1B, lane 5). The longer RNA products were still clearly observed although their signal intensities were slightly weaker than the signal seen in the presence of TFAM without LSP (compare lanes 2 and 5 in Fig. 1B). These results support the proposition that POLRMT can synthesize RNA independent of the LSP promoter, if the template is supercoiled circular DNA. To determine whether promoter-independent RNA synthesis is caused by supercoiling of the template or by its circularity, we treated the supercoiled closed circular DNA with DNA topoisomerase I (Topo I). Virtually no RNA synthesis was observed (Fig. 1B, lane 11) using such relaxed circular templates in the absence of TFAM. The only difference between the experiments shown in lanes 5 and 11 is that the templates are either supercoiled or relaxed. These results indicate that POLRMT can thus synthesize RNA in a LSP-independent fashion, using supercoiled DNA as a template.
In lane 3, the longer products should contain both LSP-dependent and independent transcripts, while most of the longer RNAs in lanes 2 and 4 should be LSP-independent. The intensity of signals in lane 3 is almost twice that in lane 2 or 4, which suggests that LSP-independent RNA synthesis by POLRMT is as strong as LSP-independent synthesis under these conditions. When a relaxed DNA template containing LSP was used for RNA synthesis in the presence of TFAM (lane 9), the 132 nt product was observed. The longer RNAs in lane 9 should logically be LSP-dependent, because there should be negligible LSP-independent RNA synthesis on the relaxed templates (see lanes 8, 10, and 11). The presence of very weak signals in lanes 8 and10 as well as a trace amount of 132 nt signal in lane 13 could be due to incomplete relaxation by Topo I. The signals in lanes 2 and 4 (LSP-independent) are rather stronger than those in lane 9 (LSP-dependent). Taken together, it may be concluded that LSP-independent RNA synthesis on supercoiled DNA is at least comparable in amount to LSP-dependent synthesis.
It is known that the C-terminal tail of TFAM is required for promoter-dependent transcription on linear DNA templates (Dairaghi et al. 1995b). However, we were able clearly to see the 132 nt product from the negatively supercoiled DNA template containing LSP even when TFAM devoid of its C-terminal tail was used (Fig. 1B, compare lanes 6 and 7). This suggests that the C-terminal tail of TFAM is not strictly required for LSP-dependent transcription on negatively supercoiled DNA. A trace amount of the 132 nt product was observed when LSP-containing template was relaxed with Topo I, which might be due to incomplete relaxation of the template DNA (Fig. 1B, lane 13). TFAM was not required for LSP-independent RNA synthesis on negatively supercoiled DNA although it enhanced the synthesis (Fig. 1B, lanes 4 and 5).
Promoter-independent RNA synthesis is dependent on DNA supercoiling
To confirm that TFAM-independent mitochondrial transcription requires supercoiling but is independent of the presence of mtDNA promoter sequences, pGEM vector plasmids (i.e. containing no mtDNA sequences) were next tested. Supercoiled pGEM was relaxed in a dose-dependent manner by Topo I (Fig. 2a, lanes 1–3). The vector linearized with PstI was used as a control (Fig. 2a, lane 4). We purified each template and used them for RNA synthesis in vitro, in the absence of TFAM. No RNA synthesis was observed on the linear DNA template (Fig. 2b, lane 4). Promoter-independent RNA synthesis on the circular DNA was inhibited in a Topo I-dependent manner (Fig. 2b, lanes 1–3). There was no RNA synthesis without POLRMT (data not shown).
|
It is well established that TFB2M is required for promoter-dependent RNA synthesis by POLRMT (Falkenberg et al. 2002). TFB2M was always included in the reactions described above. We then examined the effects of TFB2M on promoter-independent RNA synthesis, using two different supercoiled templates, pGEM and pSTV28: the former contains a T7 promoter but the latter does not. In both cases, promoter-independent RNA synthesis by POLRMT was dependent on TFB2M (Fig. 3, center and right). TFB2M did not induce promoter-independent RNA synthesis by T7 RNA polymerase (T7 RNAP) to nearly the same extent (Fig. 3, left). This TFB2M-dependency of the reaction strongly supports the notion that this RNA synthesis is dependent on POLRMT, and not on any contaminating RNA polymerases. Although the elongated promoter-independent RNA products over 1000 nt in length were synthesized strictly dependent on TFB2M, significant level of short RNA product (<150 nt) were observed even if TFB2M was omitted (Fig. 3, lanes 7 and 12).
|
Thus far, we used a relatively low amount (2 pmol) of TFAM, suitable for promoter-dependent transcription on a linear DNA template (Falkenberg et al. 2002). However, we and other groups have reported data suggesting that TFAM is much more abundant in vivo, and is a major protein component of the mtDNA nucleoid (Shen & Bogenhagen 2001; Alam et al. 2003; Ekstrand et al. 2004), whereas other groups have concluded that TFAM is less abundant (Maniura-Weber et al. 2004; Cotney et al. 2007). It is known that TFAM inhibits promoter-dependent transcription at high concentrations in a run-off assay using a linear template (e.g., 294 molecules of TFAM per template DNA molecule, Falkenberg et al. 2002). We therefore compared the effects of TFAM on promoter-dependent transcription from supercoiled closed circular and linear templates (Fig. 4a). To specifically assay the promoter-dependent transcription, we quantitated the prematurely terminated 132 nt transcripts instead of the longer RNA products, because the latter could contain both LSP-dependent and -independent transcription products (Fig. 4b). The 132 nt product was hardly detected, using the linear template in the presence of 20 pmol of TFAM (i.e., 200 molecules of TFAM per template DNA molecule or about 1 molecule per 20 bases). On the other hand, using the closed circular template, the 132 nt product intensity at 20 pmol of TFAM was still higher than that at 2 pmol of TFAM (Fig. 4c). Thus, promoter-dependent transcription was inhibited by TFAM to a lesser extent on the closed circular template.
|
Mitochondrial single-stranded DNA binding protein (mtSSB) is as abundant as TFAM in mitochondria. In contrast to TFAM, mtSSB did not affect RNA synthesis in the same range of concentrations (Fig. 4d, lanes 8–13). This may also be physiologically important, as will be discussed later.
T7 RNAP does not exhibit promoter-independent RNA synthesis
To test whether promoter-independent RNA synthesis is observed generally under our experimental conditions, i.e. could be an experimental artifact, we examined transcription in vitro by T7 RNAP. The reactions in Fig. 5 were done on supercoiled pSTV28 (no T7 promoter) as a template, with equivalent amount of each RNA polymerase (i.e., UMP incorporation by 500 fmol of POLRMT/TFB2M with 2 pmol of TFAM on a template containing LSP and T7 promoter was almost equal to that by T7 RNAP on the same template; data not shown). Although POLRMT belongs to the same RNA polymerase super-family as T7 RNAP, we did not detect any significant level of promoter-independent RNA synthesis by T7 RNAP, in contrast to the RNA synthesis by POLRMT (Fig. 5a,b). T7 RNAP synthesized RNA more strongly than did POLRMT when the T7 promoter-containing vector, pGEMT, was used (Fig. S2). TFB2M did not induce RNA synthesis on the T7 promoter-less vector (Fig. 3, panel left). These results indicate that our reaction conditions per se do not induce promoter-independent RNA synthesis.
|
Promoter-independent RNA synthesis is not caused by cryptic LSP-like sequences
The observed RNA synthesis is apparently not dependent on LSP. However, there remains the possibility that cryptic LSP-like sequences are present in the templates used. To rule out this possibility, and to confirm the DNA sequence-independence, we determined the 5'-ends of the transcripts synthesized in vitro. To compare with promoter-dependent initiation, T7 RNAP was used as a control. pGEM has a T7 promoter sequence and so we first examined the T7 promoter region. The initiation sites for transcription by T7 RNAP were clustered around the presumptive initiation site for T7 RNAP (Fig. 6a, open circles). In contrast, the 5'-ends of transcripts synthesized by POLRMT were mapped throughout the region without any sequence specificity (Fig. 6a, closed circles). To confirm the random scattering of the initiation sites, we also examined another region using a different primer on the opposite strand. The 5'-ends of POLRMT transcripts were again dispersed over a wide area (Fig. 6b). The numbers of colonies obtained in this assay for the POLRMT transcripts were essentially the same between this region and the T7 promoter region (results not shown), suggesting that the RNA synthesis occurs to a similar extent in these two regions. These results therefore suggest that promoter-independent RNA synthesis is indeed DNA sequence-independent and DNA topology-dependent.
|
In the conventional mechanism of DNA replication, a single priming event for the leading strand but multiple priming events for the lagging strand are required (Kornberg & Baker 1992; Frick & Richardson 2001). In such reactions, primases are characterized as DNA-dependent RNA polymerases that can synthesize a short oligoribonucleotide complementary to a single-stranded DNA template.
Here, we performed in vitro RNA synthesis on single-stranded circular DNA (sscDNA) of a pGEM vector. The products were analyzed by denaturing polyacrylamide gel electrophoresis (Fig. 7). RNA synthesis on the sscDNA template was much stronger than that in the run-off assay (compare Fig. 7, lanes 1 and 2), suggesting that ssDNA is a preferred template for POLRMT. However, the synthesized RNA was much shorter using on the sscDNA template than dsDNA. Interestingly, TFB2M no longer affected RNA synthesis on sscDNA (Fig. 7, lane 3).
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
We report here that POLRMT can synthesize RNA independent of a promoter sequence on supercoiled DNA (Fig. 1B). Based on the following arguments, we conclude that this activity is mediated by POLRMT and not by an unknown contaminating RNA polymerase in the protein samples. First, promoter-independent activity was comparable to LSP-dependent activity, whereas no contaminating proteins of a comparable amount were seen in the samples of POLRMT or TFB2M (Fig. S1). Secondly, the activity required TFB2M, which is considered specifically to associate with POLRMT (Falkenberg et al. 2002). It is unlikely that the TFB2M sample was contaminated by a highly active RNA polymerase because the TFB2M sample alone had no RNA synthesis activity (data not shown) and TFB2M did not induce promoter-independent RNA synthesis by T7RNAP (Fig. 3).
The simplest explanation for the effect of negative DNA supercoiling is that it reduces the thermodynamic energy required for the formation of single-stranded regions in double-stranded DNA (dsDNA) (Ohyama 2005). Consistent with this explanation, transcription initiation by RNA polymerase is generally enhanced by negative DNA supercoiling (Ohyama 2005). Negative DNA supercoiling favors the induction of unpaired regions at some sequence motifs on dsDNA. For example, mirror repeats may adopt intra-molecular triplex with ssDNA, and AT-rich sequences can experience DNA unwinding (Ohyama 2005). RNA synthesis on supercoiled dsDNA might be explained in part by the strong activity of ssDNA-directed RNA synthesis by POLRMT (Fig. 7). ssDNA-directed RNA synthesis by an RNA polymerase fraction prepared from HeLa cell mitochondria was already reported some 20 years ago (Buzan & Low 1988). Recently, the ssDNA-directed activity of POLRMT which synthesize short RNA species, under 100 nt in length, has been reported using other sscDNA templates (Wanrooij et al. 2008).
TFB2M was not required for RNA synthesis on an ssDNA template. Considering its strong homology to a family of RNA-binding rRNA methyltransferases (Falkenberg et al. 2002; McCulloch et al. 2002), TFB2M might have a capacity to bind ssDNA. Therefore, one possibility to explain the requirement for TFB2M in transcription of dsDNA is that it could stabilize an incompletely single-stranded template established by negative supercoiling. Transcriptional inhibition by a high amount (20 pmol) of TFAM (Fig. 4) might indicate that DNA packaging by TFAM makes the DNA more resistant to unwinding. This inhibition was observed at the putative in vivo level concentration of TFAM that was extrapolated previously (Takamatsu et al. 2002). This may be physiologically important for suppressing unnecessary and random RNA synthesis in vivo, except when ssDNA is exposed, for example, during DNA replication.
Considerable amount of short RNA products (<150 nt) were synthesized on negatively supercoiled DNA templates (Figs 2b, 3 and 4d). The length of short RNA products was similar to that of RNA products synthesized on sscDNA (Fig. 7 in this article and Wanrooij et al. 2008). The synthesis of short RNA products seemed to be enhanced by negative supercoiling of DNA templates (Fig. 2b, lane 3) and independent of the presence of TFB2M (Fig. 3, lanes 7 and 12), in contrast to strict TFB2M-dependence of the synthesis of long RNA products (Fig. 3, lanes 8–11 and 13–16). These observations raise the possibility that POLRMT may synthesize short RNAs in the absence of TFB2M on both ssDNA and negatively supercoiled dsDNA but TFB2M may enhance processivity of POLRMT and/or re-initiation at the 3'-end of RNA products during promoter-independent RNA syntheses only on the supercoiled DNA templates.
Promoter-independent RNA synthesis was strongly dependent on DNA supercoiling in the reconstituted system when using dsDNA (Fig. 2). However, the mechanism that regulates mtDNA conformation in vivo is unclear. One possibility is promoter-dependent RNA synthesis itself. Stable RNA–DNA hybrid formation by transcription from LSP is thought to serve as a primer for leading strand (H-strand) DNA synthesis (Shadel & Clayton 1997; Ohsato et al. 1999; Pham et al. 2006). This RNA–DNA hybrid may also introduce positive supercoiling into the region downstream of it, which can change DNA conformation. Another possibility is an effect of the triple-stranded D-loop structure found in many molecules of mammalian mtDNA (Arnberg et al. 1971; Kasamatsu et al. 1971), which can introduce positive DNA supercoiling into mtDNA. Conversely, resolution of the D-loop structure introduces negative superhelicity into closed circular mtDNA. The D-loop structure can be destabilized by binding of TFAM in vitro and in vivo (Takamatsu et al. 2002; Ohgaki et al. 2007). The mtDNA helicase Twinkle might also affect D-loop structures (Korhonen et al. 2003). Moreover, a D-loop-stabilizing protein has also been reported (He et al. 2007). These observations suggest that the D-loop could be important for transcription because it can effect a rapid transition in DNA conformation.
Possible physiological implications
Although the observed promoter-independent activity could be considered as a nonspecific and artificial activity in vitro, it should be noted that this activity of POLRMT is as strong as its promoter-dependent RNA synthesis activity (Fig. 1B). This suggests that it may also operate, or at least, be regulated, in vivo. Unregulated RNA synthesis would otherwise be a potential hazard to mtDNA maintenance.
Biased accumulation of RNA in the nascent L-strand appears to be a unique feature of replicating mtDNA (Yang et al. 2002). However, its molecular mechanism remains to be elucidated. The strong ssDNA-directed RNA synthesis activity of POLRMT (Fig. 7) and its potential for priming DNA synthesis (Wanrooij et al. 2008) might explain this unique character of mtDNA. Given the ability of POLRMT to synthesize RNA on ssDNA coated with mtSSB (Fig. 4d), it is possible that strand-asynchronous and RITOLS replication could be sequential steps in the mitochondrial replication cycle. Thus, the first two-thirds of the genome could be coated with mtSSB while leading strand synthesis traverses the 11 kb between OH and OL, as envisaged by proponents of the strand-asynchronous models (Clayton 1991). POLRMT could then synthesize RNA, dislodging the mtSSB in the process. However, there is no obvious reason why POLRMT should delay synthesizing RNA on the displaced strand template. This problem is avoided in the simplest version of the RITOLS model (Yasukawa et al. 2006), in which RNA incorporation on the lagging strand is coupled to leading-strand DNA synthesis, with the repeated formation of short ‘RNA Okazaki fragments’. Just how tightly these two processes are coupled remains to be established; the case for involvement of POLRMT in synthesizing lagging-strand RNA would become more compelling if it could be shown that the enzyme was an integral component of the mitochondrial replisome.
TFAM is tightly associated with mtDNA (Takamatsu et al. 2002; Alam et al. 2003). Promoter-independent RNA synthesis may therefore only occur immediately after TFAM is released from mtDNA during replication. The strong promoter-independent activity of POLRMT may explain the presence of a large amount of RNA in replication intermediates without assuming any other primases or RNA polymerases. However, further in vitro and in vivo studies are required to verify the involvement of POLRMT. Elucidation of how promoter-independent RNA synthesis is regulated in vivo may give a new insight into the understanding of mtDNA metabolism, including its replication.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
T7 RNA polymerase, Topo I, DNaseI, RNase H, RNase A, and PrimeScriptTM Reverse Transcriptase were purchased from Takara (Shiga, Japan). KOD-plus-TM (Toyobo, Shiga, Japan) was used for polymerase chain reaction (PCR) and TaKaRa ExTaqTM (Takara) was used for colony PCR and SLIC-PCR. A TA-cloning vector, pGEM-T Easy, was supplied by Promega (Madison, WI) and pSTV28 vector was from Takara. All reagents were of analytical grade.
DNA templates.
We PCR-amplified DNA fragments corresponding to nucleotide positions (np) 193–504 of human mtDNA for use in run-off transcription. For RNA synthesis on closed circular DNA templates, human mtDNAs from np 15888 to 504 containing LSP, CSBs and TAS (+LSP) and from np 15888 to 395 containing CSBs and TAS (–LSP) were amplified by PCR. The PCR products were cloned into pGEM-T Easy. Clones which contained the desired fragment with the same direction to T7 promoter were picked up by colony PCR. Clones with the correct DNA sequences were selected and named pGEMLSPhmD (+LSP) and pGEMhmD (–LSP) (Fig. 1A, b and c). To prepare negatively supercoiled DNA, Escherichia coli strain DH5
was transformed by each plasmid and grown to middle-log phase (A660 of 0.6) in Luria-Bertani medium. The plasmid was extracted and purified using a QIAGEN plasmid Midi kit (Qiagen, Valencia, CA). Over 90% of the purified plasmid DNAs were covalently closed circular molecules (Fig. 2a, lane 5). For the preparation of relaxed DNA, supercoiled DNA was incubated with DNA Topo I at 37 °C for 30 min. To prepare single-stranded circular DNA, E. coli strain JM109 was transformed by pGEM-T Easy. Single-stranded molecules of pGEM-T Easy DNA were induced with R408 helper phage and purified according to the manufacturer’s instructions (Promega).
Preparation of recombinant proteins
To express N-terminally His6-tagged human TFAM (full and 
C) and N-terminally His6-tagged human mitochondrial single-stranded DNA binding protein (His-mtSSB), DNA fragments encoding TFAM (amino acid residues 43–246 and 43–211) and mature human mtSSB (amino acid residues 17–148) were inserted between BamHI and EcoRI sites of pPRO-EX-HTb (Invitrogen, Carlsbad, CA). Recombinant TFAM and mtSSB were expressed in E. coli BL21 cells and purified as described previously (Takamatsu et al. 2002; Kanki et al. 2004; Ohgaki et al. 2007). Human RNA polymerase II holoenzyme was purified from human HeLa cell extracts as described previously (Kuraoka et al. 2007).
To express N-terminally FLAG-tagged human POLRMT and C-terminally His6-tagged human TFB2M, we amplified each gene without leader peptides (amino acid residues 42–1230 for POLRMT, amino acid residues 20–396 for TFB2M) by PCR. Each gene was inserted into the pFastBacDual vector (Invitrogen) and each recombinant baculovirus was produced according to the instructions for the Bac-to-Bac system (Invitrogen). The recombinant proteins were expressed in Spodoptera frugiperda (Sf9) cells in suspension in SFM 900II medium (Invitrogen). For protein expression, Sf9 cells were grown at 27 °C and collected 48–72 h after infection. The cells were suspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, and 1X Complete Mini (Protease Inhibitor Cocktail Tablet EDTA free; Roche, Mannheim, Germany). After adding 1 M NaCl (final concentration), the cells were disrupted by sonication for 5 s five times at 1-min intervals on ice. Extracts were cleared by centrifugation. For purification of FLAG-POLRMT, the cell extracts were made by co-expression of POLRMT and TFB2M. After adding an equal volume of buffer A containing 50 mM Tris-HCl (pH 8.0), 1 M NaCl, 10% glycerol and the protease inhibitor cocktail, the extracts were purified using 1 mL of Anti-FLAG M2 agarose (Sigma, St Louis, MO) pre-equilibrated with buffer A. After washing with at least 30 column volumes of the same buffer, FLAG-POLRMT was eluted by the buffer A with 100 mg/mL of FLAG peptide (Sigma).
TFB2M-His6 alone was expressed in Sf9 cells and purified using Ni-NTA agarose (Qiagen) according to the manufacturer’s instructions and the eluted fractions were pooled. The pooled fractions were diluted with buffer B containing 50 mM Tris-HCl (pH 8.0) and 10% glycerol to a final concentration of 0.2 M NaCl and poured over a HiTrap Heparin column (GE Healthcare, Piscataway, NJ) pre-equilibrated with buffer B containing 0.2 M NaCl. After washing with 10 column volumes of the same buffer, TFB2M-His6 was eluted at 0.4 M NaCl in a stepwise gradient of buffer B (0.2–1.0 M NaCl). All recombinant proteins were dialyzed in phosphate-buffered saline containing 20% glycerol, frozen in liquid nitrogen, and stored at –80 °C. After separation by SDS–PAGE, protein was stained with Coomassie brilliant blue. The concentrations were determined using bovine serum albumin (BSA) as a standard with an LAS-1000 CCD camera and Image Gauge image analysis software (Fuji, Tokyo, Japan).
In vitro RNA synthesis reactions
In the standard reaction, 100 fmol of template DNA was incubated at 32 °C for 20 min with proteins (500 fmol POLRMT/TEB2M, and 2 pmol TFAM) in a reaction mixture (30 µL) containing 10 mM Tris-HCl (pH 8.0), 1 mM DTT, 10 mM MgCl2, 100 µg/mL BSA, 5 U RNasin (Promega), 0.4 mM ATP, 0.15 mM each CTP, GTP and UTP, and 0.2 µM [-32P] UTP (3000 Ci/mmol). The reactions were stopped by the addition of 3 µL of stop buffer (containing 10% sodium dodecylsulfate and 5 mg/mL proteinase K) followed by incubation at 55 °C for 10 min. For gel electrophoretic analysis, samples were precipitated by adding 0.15 mL of ice-cooled ethanol, 1.5 µL of 1 mg/mL glycogen, and 10 µL of 10 M ammonium acetate. The precipitates were solubilized in 5 µL of loading buffer (99% formamide, 1 mM EDTA, 0.1% bromphenol blue), incubated at 70 °C for 3 min, and analyzed on an 8 M urea–4% polyacrylamide gel in TBE buffer (50 mM Tris, 48.5 mM borate, and 2 mM EDTA). DNA size makers (100 bp DNA ladder and 1 kb DNA ladder; New England BioLabs, Ipswich, MA) were labeled with [
-32P] ATP using Ready-To-Go T4 polynucleotide kinase (GE Healthcare). DEAE filter-binding assay used 5 µL of the individual samples, spotted on DE81 filter paper (GE Healthcare). After washing three times with 5% Na2HPO4 (1 mL per cm2 of filter paper), the filter paper was rinsed with diethyl-pyrocarbonate-treated water and dried up with 100% ethanol. AMP incorporation was analyzed with a FUJIFILM BAS-2500 bio-image analyzer.
Mapping of transcript 5'-ends by SLIC-PCR
SLIC-PCR was performed as described by Baptiste et al. (1993) with the following modifications. In vitro synthesized RNA products were purified using an RNeasy Mini kit (Qiagen) with DNaseI treatment according to the instructions (Qiagen). Single-stranded cDNA (ss-cDNA) was then synthesized by PrimeScriptTM RTase. To perform the reverse transcription reaction, the purified transcribed products (37 ng) were mixed with 13 pmol of random 6-mer in 20 µL of reaction mixture (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 15 mM MgCl2, 0.5 mM dNTPs, 20 U RNase inhibitor, and 100 U PrimeScriptTM RTase). After pre-incubation at 30 °C for 10 min, the mixture was incubated at 42 °C for 1 h and then at 75 °C for 15 min. The reaction sample was adjusted to 100 µL with nuclease-free water and the remaining primer was removed using a Micro Spin S-400R column (GE Healthcare). The purified ss-cDNA was mixed with 15 µL of 0.1 M NaOH for RNA hydrolysis and incubated at 65 °C for 1 h. After neutralization with 10 µL of 2 M Tris-HCl, pH 7.0, the sample was precipitated by the addition of 10 µL of glycogen (1 mg/mL), 50 µL of 10 M ammonium acetate, and 500 µL of ethanol. The precipitate was resolubilized in 5 µL of nuclease-free water. Thus obtained ss-cDNA (1 µL) was incubated with 5 ng of the anchor oligomer (5-p-GCACTTGACTATGACTGACTGAATTCTTTAGTGAGGGTTAATTGCC-NH2-) in RNA ligase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 10 mM DTT) containing 1 mM ATP, 0.1 mg/mL BSA, 50% polyethylene glycol, and 20 U of T4 RNA ligase (Takara) at 22 °C for 48 h. The single-strand ligation product (1 µL) was mixed with 0.5 U of KOD-plus- (Toyobo) in the reaction mixture containing 0.2 mM dNTPs, 5 pmol each of primer 1 (5'-CAATTAACCCTCACTAAAGA-3'), and template specific primer 3-pGEM#176-192 (5'-CAGGAAACAGCTATGAC-3') or 5-pGEM#996-1015 (5'-CCTAACTACGGCTACACTAG-3'). The initial denaturation was done at 94 °C for 1 min and then amplification was performed by 25 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, and extension at 68 °C for 30 s. Following the first PCR, 1 µL of the reaction mixture was added to the second PCR reaction mixture. Cycles of the second PCR were run in the same manner except that primer 3 (5'-CTAAAGAATTCAGTCAGTCA-3') was used in place of primer 1. The PCR products were analyzed by 2% agarose gel electrophoresis and cloned into pBluescript II SK+ vector (Stratagene, La Jolla, CA). Initiation points of the RNA synthesis were determined by sequencing using SK primer and KS primer (Stratagene).
|
Acknowledgements |
|---|
|
Footnotes |
|---|
aPresent address: Institute of Medical Technology, University of Tampere, FI-33014, Finland.
* kang{at}mailserver.med.kyushu-u.ac.jp |
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Arnberg, A., van Bruggen, E.F. & Borst, P. (1971) The presence of DNA molecules with a displacement loop in standard mitochondrial DNA preparations. Biochem. Biophys. Acta 246, 353–357.[Medline]
Asin-Cayuela, J. & Gustafsson, C.M. (2007) Mitochondrial transcription and its regulation in mammalian cells. Trends Biochem. Sci. 32, 111–117.[CrossRef][Medline]
Baptiste, J., Edwards, D.M., Delort, J. & Mallet, J. (1993) PCR Protocols; Current Methods and Applications. Totowa, NJ: Humana Press.
Bowmaker, M., Yang, M.Y., Yasukawa, T., Reyes, A., Jacobs, H.T., Huberman, J.A. & Holt, I.J. (2003) Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J. Biol. Chem. 278, 50961–50969.
Buzan, J.M. & Low, R.L. (1988) Preference of human mitochondrial RNA polymerase for superhelical templates with mitochondrial promoters. Biochem. Biophys. Res. Commun. 152, 22–29.[CrossRef][Medline]
Chang, D.D. & Clayton, D.A. (1985) Priming of human mitochondrial DNA replication occurs at the light-strand promoter. Proc. Natl Acad. Sci. USA 82, 351–355.
Clayton, D.A. (1991) Replication and transcription of vertebrate mitochondrial DNA. Annu. Rev. Cell Biol. 7, 453–478.[CrossRef][Medline]
Cotney, J., Wang, Z. & Shadel, G.S. (2007) Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression. Nucleic Acids Res. 35, 4042–4054.
Dairaghi, D.J., Shadel, G.S. & Clayton, D.A. (1995a) Human mitochondrial transcription factor A and promoter spacing integrity are required for transcription initiation. Biochem. Biophys. Acta 1271, 127–134.[Medline]
Dairaghi, D.J., Shadel, G.S. & Clayton, D.A. (1995b) Addition of a 29 residue carboxyl-terminal tail converts a simple HMG box-containing protein into a transcriptional activator. J. Mol. Biol. 249, 11–28.[CrossRef][Medline]
Ekstrand, M.I., Falkenberg, M., Rantanen, A., Park, C.B., Gaspari, M., Hultenby, K., Rustin, P., Gustafsson, C.M. & Larsson, N.G. (2004) Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum. Mol. Genet. 13, 935–944.
Falkenberg, M., Gaspari, M., Rantanen, A., Trifunovic, A., Larsson, N.G. & Gustafsson, C.M. (2002) Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA. Nat. Genet. 31, 289–294.[CrossRef][Medline]
Falkenberg, M., Larsson, N.G. & Gustafsson, C.M. (2007) DNA replication and transcription in mammalian mitochondria. Annu. Rev. Biochem. 76, 679–699.[CrossRef][Medline]
Frick, D.N. & Richardson, C.C. (2001) DNA primases. Annu. Rev. Biochem. 70, 39–80.[CrossRef][Medline]
Garrido, N., Griparic, L., Jokitalo, E., Wartiovaara, J., van der Bliek, A.M. & Spelbrink, J.N. (2003) Composition and dynamics of human mitochondrial nucleoids. Mol. Biol. Cell 14, 1583–1596.
Gaspari, M., Larsson, N.G. & Gustafsson, C.M. (2004) The transcription machinery in mammalian mitochondria. Biochim. Biophys. Acta 1659, 148–152.[Medline]
He, J., Mao, C.C., Reyes, A., Sembongi, H., Di Re, M., Granycome, C., Clippingdale, A.B., Fearnley, I.M., Harbour, M., Robinson, A.J., Reichelt, S., Spelbrink, J.N., Walker, J.E. & Holt, I.J. (2007) The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. J. Cell Biol. 176, 141–146.
Holt, I.J., Lorimer, H.E. & Jacobs, H.T. (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100, 515–524.[CrossRef][Medline]
Kanki, T., Ohgaki, K., Gaspari, M., Gustafsson, C.M., Fukuoh, A., Sasaki, N., Hamasaki, N. & Kang, D. (2004) Architectural role of mitochondrial transcription factor A in maintenance of human mitochondrial DNA. Mol. Cell. Biol. 24, 9823–9834.
Kasamatsu, H., Robberson, D.L. & Vinogard, J. (1971) A novel closed-circular mitochondrial DNA with properties of a replicating intermediate. Proc. Natl Acad. Sci. USA 68, 2252–2257.
Kaufman, B.A., Durisic, N., Mativetsky, J.M., Costantino, S., Hancock, M.A., Grutter, P. & Shoubridge, E.A. (2007) The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures. Mol. Biol. Cell 18, 3225–3236.
Korhonen, J.A., Gaspari, M. & Falkenberg, M. (2003) TWINKLE Has 5'
3' DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J. Biol. Chem. 278, 48627–48632.
Kornberg, A. & Baker, T.A. (1992) DNA Replication, 2nd edn. W. H. Freeman & Co., New York.
Kuraoka, I., Suzuki, K., Ito, S., Hayashida, M., Kwei, J.S., Ikegami, T., Handa, H., Nakabeppu, Y. & Tanaka, K. (2007) RNA polymerase II bypasses 8-oxoguanine in the presence of transcription elongation factor TFIIS. DNA Repair 6, 841–851.[CrossRef][Medline]
Maniura-Weber, K., Goffart, S., Garstka, H.L., Montoya, J. & Wiesner, R.J. (2004) Transient overexpression of mitochondrial transcription factor A (TFAM) is sufficient to stimulate mitochondrial DNA transcription, but not sufficient to increase mtDNA copy number in cultured cells. Nucleic Acids Res. 32, 6015–6027.
McCulloch, V., Seidel-Rogol, B.L. & Shadel, G.S. (2002) A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine. Mol. Cell. Biol. 22, 1116–1125.
Ohgaki, K., Kanki, T., Fukuoh, A., Kurisaki, H., Aoki, Y., Ikeuchi, M., Kim, S.H., Hamasaki, N. & Kang, D. (2007) The C-terminal tail of mitochondrial transcription factor A markedly strengthens its general binding to DNA. J. Biochem. 141, 201–211.
Ohsato, T., Muta, T., Fukuoh, A., Shinagawa, H., Hamasaki, N. & Kang, D. (1999) R-loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction specific helicase. Biochem. Biophys. Res. Commun. 255, 1–5.[CrossRef][Medline]
Ohyama, T., ed. (2005) DNA Conformation and Transcription. New York, NY: Springer Science + Business Media, Inc.
Pham, X.H., Farge, G., Shi, Y., Gaspari, M., Gustafsson, C.M. & Falkenberg, M. (2006) Conserved sequence box II directs transcription termination and primer formation in mitochondria. J. Biol. Chem. 281, 24647–24652.
Shadel, G.S. & Clayton, D.A. (1997) Mitochondrial DNA maintenance in vertebrates. Annu. Rev. Biochem. 66, 409–435.[CrossRef][Medline]
Shen, E.L. & Bogenhagen, D.F. (2001) Developmentally-regulated packaging of mitochondrial DNA by the HMG-box protein mtTFA during Xenopus oogenesis. Nucleic Acids Res. 29, 2822–2828.
Shutt, T.E. & Gray, M.W. (2006) Bacteriophage origins of mitochondrial replication and transcription proteins. Trends Genet. 22, 90–95.[CrossRef][Medline]
Takamatsu, C., Umeda, S., Ohsato, T., Ohno, T., Abe, Y., Fukuoh, A., Shinagawa, H., Hamasaki, N. & Kang, D. (2002) Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein. EMBO Rep. 3, 451–456.[CrossRef][Medline]
Wanrooij, S., Fuste, J.M., Farge, G., Shi, Y., Gustafsson, C.M. & Falkenberg, M. (2008) Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro. Proc. Natl Acad. Sci. USA 105, 11122–11127.
Wong, T.W. & Clayton, D.A. (1986) DNA primase of human mitochondria is associated with structural RNA that is essential for enzymatic activity. Cell 45, 817–825.[CrossRef][Medline]
Yang, M.Y., Bowmaker, M., Reyes, A., Vergani, L., Angeli, P., Gringeri, E., Jacobs, H.T. & Holt, I.J. (2002) Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication. Cell 111, 495–505.[CrossRef][Medline]
Yasukawa, T., Reyes, A., Cluett, T.J., Yang, M.Y., Bowmaker, M., Jacobs, H.T. & Holt, I.J. (2006) Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J. 25, 5358–5371.[CrossRef][Medline]
Received: 20 April 2009
Accepted: 27 May 2009
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
