Mitochondrion 14 (2014) 34–41

Contents lists available at ScienceDirect

Mitochondrion journal homepage: www.elsevier.com/locate/mito

Characterization of the sea urchin mitochondrial transcription factor A reveals unusual features☆ Stefania Deceglie a, Claudia Lionetti a, James B. Stewart b, Bianca Habermann b, Marina Roberti a, Palmiro Cantatore a, Paola Loguercio Polosa a,⁎ a b

Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro, Via Orabona, 4, 70125 Bari, Italy Max Planck Institute for Biology of Ageing, Robert-Koch-Strasse 21, 50931 Cologne, Germany

a r t i c l e

i n f o

Article history: Received 19 July 2013 Received in revised form 27 September 2013 Accepted 22 October 2013 Available online 1 November 2013 Keywords: mtDNA transcription Mitochondria TFAM Sea urchin DNA binding

a b s t r a c t Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial transcription we have characterized the DNA binding properties of the mitochondrial transcription factor A (TFAM). The protein contains two HMG box domains but, differently from vertebrates, displays a very short C-terminal tail. Phylogenetic analysis showed that the distribution of tail length is mixed in the different lineages, indicating that it is a trait that undergoes rapid changes during evolution. Homology modeling suggests that the protein adopts the same configuration of the human counterpart and possibly a similar mode of binding to DNA. DNase I footprinting showed that TFAM specifically contacts mtDNA at a fixed distance from three AT-rich consensus sequences that were supposed to act as transcriptional initiation sites. Bound sequences are homologous and contain an inverted repeat motif, which resembles that involved in the intercalation of human TFAM in LSP DNA. The here reported data indicate that sea urchin TFAM specifically binds mtDNA. The protein could intercalate residues at the DNA inverted motif and, despite its short tail, might have a role in mitochondrial transcription. © 2013 The Authors. Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction In humans the basal mitochondrial transcription apparatus consists of a phage-like RNA polymerase (POLRMT) and the activating factors TFAM and TFB2M. Modulation of the transcription process requires three additional proteins, MTERF1, MTERF2 and MTERF3, which belong to the MTERF protein family (Park et al., 2007; Terzioglu et al., 2013; Wenz et al., 2009). A novel function for MTERF3 has been recently demonstrated, which points to a close crosstalk between transcription initiation and ribosomal biogenesis (Wredenberg et al., 2013). Human TFAM was the first mitochondrial transcription factor to be identified. It is a member of the high-mobility group box proteins (HMGB) that share the HMG DNA binding domain (known as the HMG box). A hallmark of HMGB proteins is their ability to bend and unwind the DNA helix. HMGB proteins comprise two groups classified by the specificity of their DNA-binding interactions (Malarkey and Churchill, 2012). The nonsequence-specific proteins usually have two HMG domains and are associated with the maintenance and architecture of DNA. The sequence-specific HMGB proteins typically contain

☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author. Tel.: +39 080 5443378; fax: +39 080 5443403. E-mail address: [email protected] (P. Loguercio Polosa).

one HMG domain and are involved in transcription activation of genes through specific binding in promoter regions. Human TFAM displays two tandem HMG boxes, named HMG1 and HMG2 (Fisher et al., 1992; Parisi and Clayton, 1991), separated by a linker and followed by a C-terminal tail that is thought to be necessary for transcription activation (Dairaghi et al., 1995; Parisi et al., 1993). With respect to the classification of HMGB proteins, human TFAM is distinctive in that it shows both sequence-specific and nonsequence-specific DNA binding capacity. TFAM makes specific contacts with the mtDNA upstream of promoters LSP and HSP (Gangelhoff et al., 2009) and activates promoterspecific transcription (Fisher and Clayton, 1988; McCulloch and Shadel, 2003; Sologub et al., 2009). With its non-specific binding activity, TFAM serves a role in maintenance and packaging of mtDNA into nucleoid-like structures (Ekstrand et al., 2004; Kukat and Larsson, 2013). A growing debate has been focused on the core requirement of TFAM for basal transcription initiation in humans. Some groups showed that basal transcription initiation required a three-component apparatus made of POLRMT, TFB2M and TFAM (Litonin et al., 2010); others supported a two-component system consisting of POLRMT and TFB2M (Bestwick and Shadel, 2013; Shutt et al., 2010). Anyhow, there is general agreement that TFAM is required in transcription activation. The protein can also exert a repressive effect on transcription (Lodeiro et al., 2012). Recently the tridimensional structure of the human TFAM-LSP complex has been reported (Ngo et al., 2011; Rubio-Cosials et al., 2011). The

1567-7249/$ – see front matter © 2013 The Authors. Elsevier B.V. and Mitochondria Research Society. All rights reserved. http://dx.doi.org/10.1016/j.mito.2013.10.003

S. Deceglie et al. / Mitochondrion 14 (2014) 34–41

most striking feature of the structure is that the protein forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. This bending is the result of two 90° successive kinks caused by the HMG boxes, which intercalate residues to the DNA. The dramatic bending, which is stabilized by the linker, would position the C-tail in close proximity to the transcription start site. In this way the C-tail would be able to interact with the POLRMT-TFB2M complex, resulting in the activation of transcription (McCulloch and Shadel, 2003; Sologub et al., 2009). The nonsequence-specific HMGB proteins include the budding yeast ortholog of TFAM, named Abf2. Like the human protein, Abf2 consists of two HMG boxes separated by a linker, but it lacks the C-terminal tail. Abf2 does not bind specifically to promoter DNA and is not required for promoter-specific initiation of transcription; it is essential for maintenance and packaging of mtDNA (Dairaghi et al., 1995; Diffley and Stillman, 1992). Sea urchin mtDNA (about 15.7 kbp) has the same coding capacity of mammalian mtDNA, but gene arrangement is remarkably different. Typical features of sea urchin mtDNA gene organization are: i) the separation of the two ribosomal genes, which are adjacent in mammals; ii) the clustering of 15 tRNA genes, which in mammalian mtDNA are scattered along the molecule; and iii) the reduced size (about 130 bp) of the main non-coding region (NCR), which in mammals is larger (about 1000 bp) (Cantatore et al., 1989). Studying mitochondrial transcription in sea urchin is of particular interest because it allows relating changes in mitochondrial gene organization with variations in the mechanism of mitochondrial transcription. Early studies, aiming at mapping mature and precursor mitochondrial RNA species, supported a mechanism based on the existence of multiple and overlapping transcription units, possibly starting from short conserved AT-rich sequences that were proposed to act as bidirectional promoters (Cantatore et al., 1990; Elliott and Jacobs, 1989). In the present study, to gain more information on the mechanism and apparatus of sea urchin mitochondrial transcription, we have

35

produced in insect cells with the baculovirus system the recombinant transcription factor TFAM and have investigated its DNA binding properties. Interestingly, we found that sea urchin TFAM has a very short Cterminal tail compared to human TFAM. The protein specifically recognizes three homologous sequences located at a constant distance from the AT-rich sequences. The bound regions contain an inverted motif that, similarly to what occurs for human TFAM, might be involved in the bending of the contacted DNA.

2. Materials and methods 2.1. Cloning of Paracentrotus lividus TFAM cDNA The cDNA of TFAM was obtained as follows. Egg poly(A)+ RNA was reverse-transcribed with a 36mer oligonucleotide containing (dT)15. The cDNA was amplified by PCR using primer For and Rev bearing the initiator methionine and stop codon, respectively; primers were designed on the sequence of the six P. lividus ESTs retrieved from P. lividus EST database (http://goblet.molgen.mpg.de/cgi-bin/webapps/paracentrotus.cgi) using BLASTP program and human TFAM as query. The baculovirus transfer vector pBacPAK9 containing TFAM cDNA was prepared by standard DNA manipulation techniques as follows. Briefly, TFAM cDNA, without the leader peptide (amino acids 46-237) and bearing a hexa-histidine tag at its N-terminus, was PCR amplified with Phusion High-Fidelity DNA Polymerase (BioLabs), using a primer For containing the BglII site followed by the sequence coding for the initiator methionine and the hexahistidine, and a primer Rev containing the NotI site followed by the stop codon. The amplification product was restriction digested, gel purified and inserted into BglII/NotI sites of the transfer vector pBacPAK9 (Clontech). The recombinant plasmid was transformed into Escherichia coli XL1-Blue Supercompetent (Stratagene) and the correct sequence was verified by the automatic sequencing of both strands.

Leader Peptide Pliv Hum

52 49

HMG1 Pliv Hum

104 101

Linker Pliv Hum

156 153

HMG2 Pliv Hum

208 200

C-tail Pliv Hum

237 246

Fig. 1. Alignment of the amino acid sequences of TFAM from P. lividus (Pliv) and humans (Hum). Alignment was generated with the Swiss-Model Automated Protein Modeling Server (http://swissmodel.expasy.org). Regions with sequence identity or similarity according to the ClustalW program (Thompson et al., 1994) are shaded in black and gray, respectively. Positions of domains and secondary structure elements, indicated above the sequence, are according to the structure of human TFAM (Ngo et al., 2011). Red boxes indicate residues that are identical in all metazoans (Rubio-Cosials et al., 2011). Blue triangles and green dots indicate residues of human TFAM involved in the formation of the hydrophobic core of the L-shaped HMG-boxes, and in contacting DNA, respectively. Green squares denote the two residues of human TFAM, which intercalate at the DNA inverted motif.

36

S. Deceglie et al. / Mitochondrion 14 (2014) 34–41

2.2. Construction of the baculovirus recombinant expression vectors and protein production in insect cells

equal volume of cold phosphate-buffered saline, frozen in liquid nitrogen and stored at −80 °C.

Linearized baculovirus DNA and recombinant pBacPAK9 plasmid were co-transfected in Spodoptera frugiperda (Sf9) cells according to the manufacturer's protocol (Clontech). Recombinant viruses were plaque purified and evaluated by PCR for the presence of the insert; viral stocks were prepared by two-step growth amplification, as described in the BacPAK manual (Clontech). For protein expression, Sf9 cells were grown in suspension (100 ml, about 1.5 × 108 cells) at 27 °C in SFM-900 II insect cell culture medium with L-glutamine (Invitrogen), supplemented with 2% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin antibiotic mixture (Invitrogen). Cells were infected with TFAM recombinant baculovirus (10 plaque forming units/cell); they were collected 72 h after infection, washed with an

2.3. Protein purification and analysis by SDS–PAGE and Western immunoblot The frozen pellet deriving from 100 ml of culture was resuspended in 10 ml of lysis buffer (50 mM sodium phosphate buffer pH 7.8, 10% glycerol), supplemented with 5 mM β-mercaptoethanol and protease inhibitors (Sigma) 1 mM AEBSF, 2 mM pepstatin A, 0.002 mM leupeptin, 2 mM benzamidine, and incubated on ice for 20 min. Cells were then lysed by 20 strokes in a Dounce homogenizer using a tight-fitting pestle; NaCl was added to a final concentration of 0.8 M. After incubation at 4 °C for 45 min with gentle rotation, the homogenate was centrifuged at 130 000 ×g in the Beckman 70.1 Ti rotor for 45 min at 4 °C. The supernatant was passed 5 times through a 18-gauge needle to shear the

Fig. 2. Nested hierarchy diagram showing the distribution of TFAM in the various lineages with respect to the tail length. Tails were classified on the basis of their length into short (S, b15 residues or absent), medium (M, 15–40 residues) and long (L, N40 residues). No TFAM sequence was available for the taxa in gray.

S. Deceglie et al. / Mitochondrion 14 (2014) 34–41

37

Fig. 3. Homology modeling of sea urchin TFAM. (A) Overall structure of sea urchin TFAM (mature protein, residues 46–237) obtained by using the Swiss-Model Automated Protein Modeling Server (http://swissmodel.expasy.org). It is also shown the structure of human TFAM in complex with 28-bp LSP DNA (accession cod. 3TMM; Ngo et al., 2011), used in the homology modeling. HMG1 and HMG2 are colored in blue and green respectively, the linker in pink and the C-terminal tail in yellow. The side view emphasizes the difference in the tail length. (B) Electrostatic surface potential plot of sea urchin TFAM viewed from the top and flipped 180°. The surface is colored according to the electrostatic surface potential (blue, +4 kT; red, −4 kT).

DNA; it was then supplemented with imidazole solution pH 7.0 to obtain a final concentration of 5 mM and mixed with 1 ml of Ni+2-NTA agarose beads (QIAGEN) equilibrated in buffer A (50 mM sodium phosphate buffer pH 7.8, 10% glycerol, 0.8 M NaCl, 5 mM β-mercaptoethanol, protease inhibitor mix as before) supplemented with 5 mM imidazole. The suspension was incubated at 4 °C for 2 h with gentle rotation. Beads were collected by centrifugation for 10 min at 1500 ×g. After the removal of the supernatant (flow-through), beads were washed with 5 ml of buffer A containing 20 mM imidazole for 10 min with gentle rotation. Beads were then collected by centrifugation and the supernatant (wash) was removed; pelleted beads were gently resuspended in 10 ml of buffer A supplemented with 20 mM imidazole and packed into a Poly-Prep chromatography column (BioRad). Proteins bound to the beads were eluted with 5 ml of buffer A containing 300 mM imidazole. Peak fractions were pooled, diluted to 0.15 M NaCl with buffer B (10 mM Tris–HCl pH 8.0, 10 mM MgCl2, 1 mM EDTA, 20% glycerol, 1 mM DTT, protease inhibitors) and subjected to fast-liquid chromatography onto a 1-ml heparin–sepharose (GE Healthcare) column, previously equilibrated with buffer B containing 0.15 M NaCl. All operations were performed at 0–4 °C. Bound proteins were eluted with a linear NaCl gradient from 0.15 M to 1.2 M in buffer B; TFAM eluted at 0.5 M–0.6 M NaCl. The protein concentration was determined by Bradford protein assay (BioRad), using bovine serum albumin (BSA) as protein standard. Fractions were analyzed by SDS–PAGE using 4–12% precast polyacrylamide gels (BioRad). We estimated a yield of about 0.3 mg of pure TFAM protein from 100 ml of starting cell culture. For Western blot analysis, proteins were subjected to SDS–PAGE and electrotransferred to polyvinylidene fluoride (PVDF) membrane (Immobilon P, Millipore)

for 2 h. Membranes were blocked for 1 h in PBS containing 0.1% Tween 20, 5% non-fat milk (BioRad), and incubated for 1 h in the same buffer composition with rabbit antibodies against human TFAM 1:10,000. Blots were then incubated in the same buffer composition for 1 h with anti-rabbit IgG HRP conjugate (GE Healthcare, 1:10,000). Signals were visualized using the ECL Plus Detection System (GE Healthcare). Quantitative analysis was performed with ChemiDoc using Quantity-One software (BioRad).

2.4. DNase I footprinting assay DNase I footprinting probes were obtained by PCR on P. lividus mtDNA (see legend of Fig. 4A for the position of probe ends). Products were fluorescently end-labeled by PCR using one Cy5 labeled primer and loaded on a native 6% polyacrylamide gel run in 0.5X TBE at 300 V at 4 °C. Gel slices containing the labeled probe were excised and DNA was eluted overnight according to standard conditions. Probe concentration was determined with ChemiDoc using Quantity-One software (BioRad). Probes (45 fmol) were incubated with the indicated amounts of TFAM in a 50-μl volume containing 20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 mM KCl, 1 mM DTT, 0.01 mM EDTA, 0.1 mg/ml BSA. After incubation at room temperature for 20 min at 25 °C, reactions were added with an equal volume of 5 mM CaCl2, 10 mM MgCl2, and with 0.5 U/ml of DNase I (GE Healthcare). After 1 min of incubation at room temperature, reactions were stopped by adding an equal volume of 1% SDS, 200 mM NaCl, 20 mM EDTA, 0.25 mg/ml tRNA. Samples were phenol extracted, ethanol precipitated and loaded onto a 6% sequencing gel,

38

S. Deceglie et al. / Mitochondrion 14 (2014) 34–41

alongside with sequencing reactions performed on identical labeled fragments. Wet gels were analyzed by phosphor imaging. 3. Results and discussion 3.1. Cloning, expression and purification of sea urchin TFAM To clone the sea urchin transcription factor TFAM we searched the P. lividus EST database (http://goblet.molgen.mpg.de/cgi-bin/webapps/ paracentrotus.cgi) using the BLASTP program and the human TFAM as query. We retrieved six ESTs that were completely sequenced to confirm the position of the initiator methionine and stop codon. The cDNA encoding P. lividus TFAM is 714 bp long (GenBank accession no. JN412094) and predicts a 237 amino acid protein (Fig. 1). Sequence analysis with programs for subcellular localization prediction, such as MitoProt II and WoLF PSORT (Claros and Vincens, 1996; Horton et al., 2007), indicates a mitochondrial localization; the potential cleavage site was placed using as reference human TFAM, whose cleavage site had been determined by sequencing the mitochondrial purified protein (Parisi and Clayton, 1991). TFAM cDNA, deprived of the leader peptide and with an exahistidine tag at its N-terminus, was expressed in Sf9 insect cells by using the baculovirus expression system. The soluble fraction of the cell lysate was subjected to Ni+2 affinity chromatography and analyzed by western blotting (Supplementary Fig. 1A). The immunoreactive fractions were pooled and further purified by heparin– sepharose chromatography in FPLC. The eluted proteins were separated by SDS–PAGE and Coomassie blue stained. As shown in Supplementary Fig. 1B, TFAM eluted around 0.5 M NaCl and was highly pure, as judged on the basis of the quantitative analysis of the gel. 3.2. Characterization of TFAM protein structure Alignment of sea urchin TFAM with the human counterpart revealed a 25% amino acid identity and 40% amino acid similarity rather uniformly distributed, with a higher conservation in correspondence of HMG1 and HMG2 (Fig. 1). A linker of 30 amino acids connects the two HMG boxes; HMG2 is followed by a carboxyl-tail that is 9 residues long and contains 4 glutamate residues. To gain more information on the distribution of TFAM tail length in the different lineages we collected full-length sequences of TFAM from several species using NCBI–BLAST (Altschul et al., 1997) and aligned them with each other (Katoh et al., 2002). Sequences were compared with respect to the length of their tail beyond the conserved HMG2 (position 229 in P. lividus TFAM). The results of the analysis were mapped onto the nested hierarchy diagram, which is presented in Fig. 2. This investigation showed that the reduced size of the C-tail is shared by other Echinoderms such as Strongylocentrotus purpuratus, as well as by Nematoda, Cnidaria and some Saccharomycetaceae. In human TFAM, the conserved HMG2 is followed by a sequence of 26 amino acids, which can be considered a medium-length tail. Vertebrates like fish, birds or amphibians have an extremely long tail up to 80 amino acids. The longest observed tail was found in the Hymenoptera red-fire ant (Solenopsis invicta) with 131 amino acids. It appears that the distribution of the tail length is mixed in the different lineages; in Hexapoda, for example, there are organisms with long and short tails. This suggests that the tail length is a trait that undergoes rapid changes during evolution.

The knowledge of the tridimensional structure of human TFAM-LSP complex prompted us to perform a comparative study with the sea urchin protein. Homology modeling of sea urchin TFAM resulted in the structure reported in Fig. 3A. It contains two L-shaped HMG domains, each formed by 3 alpha helices, which are separated by an alpha helical linker. Fig. 3B displays the molecular surface of P. lividus TFAM with computation of the electrostatic surface potential. The positions of regions with a positive surface charge considerably match those found in the human protein. We analyzed the conservation of some critical amino acids between the human and sea urchin TFAM and found that, at the positions equivalent to the intercalating residues Leu58 and Leu182 (green squares in Fig. 1), the sea urchin protein displays two Phe residues. This is in line with the observations by Rubio-Cosials et al. (2011), who found that the two intercalating Leu can be replaced by a hydrophobic residue in some animal species. Regarding the residues involved in specific contacts with bases of the minor groove, alignment of sea urchin and human TFAM showed that these residues, denoted by green dots in Fig. 1, are partly conserved, suggesting that also the sea urchin protein could make specific contacts with the DNA. Alignment of TFAM sequences among metazoans indicated 10 universally identical amino acids that are spread along the protein sequence (Rubio-Cosials et al., 2011). Of these residues, denoted with red boxes in Fig. 1, seven are also conserved in sea urchin and two of them, Trp88 and Trp189, are involved in contacting the oxygen atoms of the DNA sugar-phosphate backbone, as can be inferred from the crystal structure of human TFAM-DNA complex. Furthermore, we checked the conservation of the eight residues that in the human protein are involved in the formation of the hydrophobic core that stabilizes the L-shaped configuration of each HMG box (Ngo et al., 2011). All these residues (indicated by blue triangles in Fig. 1) are either identical or similar in sea urchin TFAM, suggesting a comparable arrangement of the two DNA binding domains. Given the key role of the alpha helical linker in facilitating the DNA bending, we also inspected the conservation of the residues of the linker involved in contacting the DNA. We found that sea urchin TFAM keeps the same residues except for His137, Met143 and Lys147, which are replaced by Arg, Arg and Ala, respectively. All together, these observations indicate that the HMG boxes and the linker are highly conserved in the sea urchin protein and suggest that the protein may adopt the same configuration of the human counterpart and possibly a similar mode of binding to the DNA. Recently it was shown that human TFAM is phosphorylated at Ser 55 and Ser 56 within its HMG1 box, as part of a mechanism for rapidly regulating protein DNA-binding and abundance (Lu et al., 2013). We found that in sea urchin TFAM Ser 56 is conserved and Ser 55 is replaced by Thr. This suggests that phosphorylation may occur also in sea urchin, possibly at those residues. 3.3. Detection of specific binding sites on mtDNA In order to investigate the DNA binding specificity of the sea urchin TFAM, we selected six regions of P. lividus mtDNA for the presence of short AT-rich consensus sequences that were previously supposed by us and others to be involved in mitochondrial RNA synthesis as possible transcriptional start sites (Cantatore et al., 1990; Elliott and Jacobs, 1989) (Fig. 4A). Of the six AT-rich sequences, four are not coding and are found in the main NCR (nt 1203–1220) and between three pairs of oppositely transcribed genes (tRNAVal and tRNAMet, nt 1799–1811;

Fig. 4. DNA binding of TFAM. (A) Location, on the P. lividus mtDNA map, of the AT-rich sequences (circled AT) and fragments (gray boxes) used as probes in footprinting experiments. Probe ends on mtDNA sequence are the following: NCR/tRNAPro (NCR/Pro) nt 1090–1307; tRNAVal/tRNAMet (Val/Met) nt 1731–1947; ND2/16S nt 4201–4410; A6/COIII nt 9244–9468; tRNASer(UCN)/ND3 (Ser/ND3) nt 10062–10370; ND6/Cytb nt 14453–14680. OH and OL, origins of DNA replication. Black arrows indicate the transcription direction of the two DNA strands. (B) DNase I footprinting analysis of TFAM-mtDNA complexes. The L strand of fragment Ser/ND3, nt 10062–10370, was fluorescently labeled; DNA (45 fmol) was incubated without TFAM (lanes 1 and 5) and with increasing amounts of TFAM (2.5, 4.9 and 12.6 pmol in lanes 2–4, respectively) and treated with DNase I as described in the Materials and methods section. Samples were loaded onto a 6% sequencing gel, which was analyzed by phosphor imaging. Shaded regions to the left indicate sites of DNase I protection by TFAM. Genes and nucleotide positions on mtDNA are indicated to the right. (C) Same as described above for panel B, except that labeled DNA (L strand) was fragment A6/COIII, nt 9244–9468. (D) Same as in (B) except that probe was fragment NCR/Pro, nt 1090–1307, labeled at the H strand. (E) Alignment of the DNase I protected sequences. Conserved nucleotides are highlighted in black; nucleotides identical in two sequences are highlighted in gray. The inverted motif is indicated above the sequences.

S. Deceglie et al. / Mitochondrion 14 (2014) 34–41

39

40

S. Deceglie et al. / Mitochondrion 14 (2014) 34–41

tRNASer(UCN) and ND3, nt 10229–10241; ND6 and Cytb, nt 14534– 14546); the remaining two AT-rich sequences are located inside genes, that is, at the 3′ end of ND2 (nt 4271–4283) and at the 3′ end of ATPase 6 (nt 9369–9378) (Cantatore et al., 1989). Probes of similar length (about 200 bp), which contained those short AT-rich sequences (see legend of Fig. 4 for name and position on mtDNA), were fluorescently end-labeled and used in DNase I footprinting experiments. As reported in Fig. 4B, a nearly complete DNase I protection was observed with probe Ser/ND3 at position nt 10159–10189. The protected region, which was detectable already at lower concentrations of TFAM, is located inside the tRNASer(UCN) gene and is 40 nt from the AT-rich sequence placed between tRNASer(UCN) and ND3 genes. For probe A6/COIII we observed a domain of DNase I protection, which spans nt 9305–9334 and is placed about 40 nt apart from the AT-rich sequence (Fig. 4C). Interestingly, the footprint (nt 9305–9334) matches that produced, on S. purpuratus sea urchin mtDNA, by a heparin–sepharose fraction from a S. purpuratus mitochondrial lysate (Qureshi and Jacobs, 1993). The DNA binding activity, named PBP1, was associated to a single polypeptide of apparent molecular weight 25 kDa. The identity of the footprinted regions on both sea urchin mtDNAs strongly suggests that PBP1 could be S. purpuratus TFAM. A domain of DNase I protection, observable at a high protein/DNA ratio, was detectable on fragment NCR/ Pro. The protected region (nt 1252–1293) is located inside the tRNAPro gene and is about 30 nt from the AT-rich region (Fig. 4D). No clear footprints were evident on the Val/Met, ND2/16S and ND6/Cytb fragments at any TFAM–DNA ratio (not shown). We aligned the sequences of the protected regions and found a considerable similarity, which concerns mainly the first six and the last ten bases (Fig. 4E). No homology could be detected with the sequences in the vicinity of those three AT-rich regions of P. lividus mtDNA that did not produce any footprint. In the protected sequences we found the trinucleotide inverted motif AGT/A–TGA separated by 9–12 bp. Similarly, in humans comparison of HSP, LSP and other two 28-bp TFAM binding sites found in the D-loop region, named X and Y (Fisher et al., 1987), revealed some homology. These regions contain the trinucleotide inverted repeat AAC - 10 bp - CAA, which in LSP is involved in the intercalation of TFAM (Rubio-Cosials et al., 2011). Given this parallelism, we suggest that the inverted repeat in the region contacted by sea urchin TFAM could be involved in the protein intercalation. 3.4. Structural and functional features of sea urchin TFAM The data reported in this paper provide interesting information on the structural and functional features of sea urchin TFAM. Comparison of the primary and tridimensional structures of the human and sea urchin proteins shows that, with the exception of the C-terminal tail that in sea urchin is very short, they share similar structural properties, including a similar distribution of the surface potential. DNase I footprinting data indicate that sea urchin TFAM, differently from yeast Abf2 and similarly to the human homologue, could be included among those HMGB proteins that specifically bind DNA; interestingly for both species there is an inverted repeat within the contacted sequences. The above observations allow predicting that sea urchin TFAM could share with the human protein the mtDNA-binding mode. Recently, Malarkey et al. (2012) have shown that a mutant of human TFAM lacking the C-terminal tail bent both promoter and non-specific sequences to a reduced degree compared to the full-length protein. Furthermore, previous experiments showed that deprivation of the C-tail in human TFAM, as well as point mutation of Arg232, reduced the transcription activation capacity (Dairaghi et al., 1995), highlighting the importance of the tail in stimulation of transcription. It should be noted that the C-terminal tail of sea urchin TFAM, besides being short, lacks that critical Arg residue. Therefore, by virtue of the peculiar features of its tail, sea urchin TFAM might impart a less enhanced bending of the DNA and it might even play a non-stringent role in mitochondrial transcription. On the other hand, although the sequence-specific DNA

binding ability of a protein does not indicate per se a role in transcription, the observation that the bases footprinted by sea urchin TFAM are placed at a fixed distance (about 40 bp) from the AT-rich sequences, which were supposed to act as transcription initiation sites (Cantatore et al., 1990; Elliott and Jacobs, 1989), suggests that the protein might have a functional role in transcription. Experiments with the minimal in vitro reconstituted transcription machinery should clarify the role of TFAM in promoter-specific transcription initiation of sea urchin mtDNA. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2013.10.003. Acknowledgments This work was supported by grants from Università di Bari, Progetto di Ricerca di Ateneo, and Telethon — Italy (grant GGP06233). We thank Drs Elio Biffali and Marco Borra (Molecular Biology Service, Stazione Zoologica “Anton Dohrn”, Naples, Italy) for sequencing TFAM ESTs and Prof. Rudolf J. Wiesner (Institute for Vegetative Physiology, University of Cologne, Germany) for supplying anti-TFAM antibody. We are indebted to F. Fracasso for technical assistance. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bestwick, M.L., Shadel, G.S., 2013. Accessorizing the human mitochondrial transcription machinery. Trends Biochem. Sci. 38, 283–291. Cantatore, P., Roberti, M., Rainaldi, G., Gadaleta, M.N., Saccone, C., 1989. The complete nucleotide sequence, gene organization, and genetic code of the mitochondrial genome of Paracentrotus lividus. J. Biol. Chem. 264, 10965–11097. Cantatore, P., Roberti, M., Loguercio Polosa, P., Mustich, A., Gadaleta, M.N., 1990. Mapping and characterization of Paracentrotus lividus mitochondrial transcripts: multiple and overlapping transcription units. Curr. Genet. 32, 2059–2068. Claros, M.G., Vincens, P., 1996. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241, 779–786. Dairaghi, D.J., Shadel, G.S., Clayton, D.A., 1995. 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. Diffley, J.F.X., Stillman, B., 1992. DNA binding properties of an HMG1-related protein from yeast mitochondria. J. Biol. Chem. 267, 3368–3374. 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. Elliott, D.J., Jacobs, H.T., 1989. Mutually exclusive synthetic pathways for sea urchin mitochondrial rRNA and mRNA. Mol. Cell. Biol. 9, 1069–1082. Fisher, R.P., Clayton, D.A., 1988. Purification and characterization of human mitochondrial transcription factor 1. Mol. Cell. Biol. 8, 3496–3509. Fisher, R.P., Topper, J.N., Clayton, D.A., 1987. Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements. Cell 50, 247–258. Fisher, R.P., Lisowsky, T., Parisi, M.A., Clayton, D.A., 1992. DNA wrapping and bending by a mitochondrial high mobility group-like transcriptional activator protein. J. Biol. Chem. 267, 3358–3367. Gangelhoff, T.A., Mungalachetty, P.S., Nix, J.C., Churchill, M.E.A., 2009. Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A. Nucleic Acids Res. 37, 3153–3164. Horton, P., Park, K.-J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C.J., Nakai, K., 2007. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 35, W585–W587 (Web Server issue). Katoh, K., Misawa, K., Kuma, K., Miyata, T., 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066. Kukat, C., Larsson, N.G., 2013. MtDNA makes a U-turn for the mitochondrial nucleoid. Trends Cell Biol. 23, 457–463. Litonin, D., Sologub, M., Shi, Y., Savkina, M., Anikin, M., Falkenberg, M., Gustafsson, C.M., Temiakov, D., 2010. Human mitochondrial transcription revisited: only TFAM and TFB2M are required for transcription of the mitochondrial genes in vitro. J. Biol. Chem. 285, 18129–18133. Lodeiro, M.F., Uchida, A., Bestwick, M., Moustafa, I.M., Arnold, J.J., Shadel, G.S., Cameron, C.E., 2012. Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro. Proc. Natl. Acad. Sci. U. S. A. 109, 6513–6518. Lu, B., Lee, J., Nie, X., Li, M., Morozov, Y.I., Venkatesh, S., Bogenhagen, D.F., Temiakov, D., Suzuki, C.K., 2013. Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA + Lon protease. Mol. Cell 49, 121–132.

S. Deceglie et al. / Mitochondrion 14 (2014) 34–41 Malarkey, C.S., Churchill, M.E.A., 2012. The high mobility group box: the ultimate utility player of a cell. Trends Biochem. Sci. 37, 553–562. Malarkey, C.S., Bestwick, M., Kuhlwilm, J.E., Shadel, G.S., Churchill, M.E.A., 2012. Transcriptional activation by mitochondrial transcription factor A involves preferential distortion of promoter DNA. Nucleic Acids Res. 40, 614–624. McCulloch, V., Shadel, G.S., 2003. Human mitochondrial transcription factor B1 interacts with the C-terminal activation region of h-mtTFA and stimulates transcription independently of its RNA methyltransferase activity. Mol. Cell. Biol. 23, 5816–5824. Ngo, H.B., Kaiser, J.T., Chan, D.C., 2011. The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA. Nat. Struct. Mol. Biol. 18, 1290–1296. Parisi, M.A., Clayton, D.A., 1991. Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. Science 252, 965–969. Parisi, M.A., Xu, B., Clayton, D.A., 1993. A human mitochondrial transcriptional activator can functionally replace a yeast mitochondrial HMG-box protein both in vivo and in vitro. Mol. Cell. Biol. 13, 1951–1961. Park, C.B., Asin-Cayuela, J., Cámara, Y., Shi, Y., Pellegrini, M., Gaspari, M., Wibom, R., Hultenby, K., Erdjument-Bromage, H., Tempst, P., Falkenberg, M., Gustafsson, C.M., Larsson, N.G., 2007. MTERF3 is a negative regulator of mammalian mtDNA transcription. Cell 130, 273–285. Qureshi, S.A., Jacobs, H.T., 1993. Characterization of a high-affinity binding site for a DNAbinding protein from sea urchin embryo mitochondria. Nucleic Acids Res. 21, 811–816. Rubio-Cosials, A., Sidow, J.F., Jiménez-Menéndez, N., Fernández-Millán, P., Montoya, J., Jacobs, H.T., Coll, M., Bernadó, P., Solà, M., 2011. Human mitochondrial transcription

41

factor A induces a U-turn structure in the light strand promoter. Nat. Struct. Mol. Biol. 18, 1281–1289. Shutt, T.E., Lodeiro, M.F., Cotney, J., Cameron, C.E., Shadel, G.S., 2010. Core human mitochondrial transcription apparatus is a regulated two-component system in vitro. Proc. Natl. Acad. Sci. U. S. A. 107, 12133–12138. Sologub, M., Litonin, D., Anikin, M., Mustaev, A., Temiakov, D., 2009. TFB2 is a transient component of the catalytic site of the human mitochondrial RNA polymerase. Cell 139, 934–944. Terzioglu, M., Ruzzenente, B., Harmel, J., Mourier, A., Jemt, E., Davila Lopez, M., Kukat, C., Stewart, J.B., Wibom, R., Meharg, C., Habermann, B., Falkenberg, M., Gustafsson, C.M., Park, C.B., Larsson, N.G., 2013. MTERF1 binds mtDNA to prevent transcriptional interference at the light-strand promoter but is dispensable for rRNA gene transcription regulation. Cell Metab. 17, 618–626. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wenz, T., Luca, C., Torraco, A., Moraes, C.T., 2009. mTERF2 regulates oxidative phosphorylation by modulating mtDNA transcription. Cell Metab. 9, 499–511. Wredenberg, A., Lagouge, M., Bratic, A., Metodiev, M.D., Spåhr, H., Mourier, A., Freyer, C., Ruzzenente, B., Tain, L., Grönke, S., Baggio, F., Kukat, C., Kremmer, E., Wibom, R., Loguercio Polosa, P., Habermann, B., Partridge, L., Park, C.B., Larsson, N.G., 2013. MTERF3 regulates mitochondrial ribosome biogenesis in invertebrates and mammals. PLoS Genet. 9, e1003178. http://dx.doi.org/10.1371/journal.pgen.1003178.

Characterization of the sea urchin mitochondrial transcription factor A reveals unusual features.

Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial tr...
1MB Sizes 0 Downloads 0 Views