Polosa PL , Deceglie S , Falkenberg M , Roberti M , Di Ponzio B , Gadaleta MN , Cantatore P .

	Figure 1. Cloning strategy of the sea urchin mtRNAP cDNA. The 4266 bp ORF is represented by a thick line, the untranslated sequences by thin lines. Below the complete cDNA are positioned the cDNA fragments isolated by PCR on the cDNA library with degenerate, inosine-containing primers and vector- or internal-specific primers (striped bars); the fragment obtained by hybridization-screening of the cDNA library (heavy-shaded bar); the fragments obtained by RACE, hybridization-screening of the genomic library and PCR on genomic clones (open bars, with the shaded portion indicating the untranslated sequences). The position of the initiating ATG and stop TAA codons is shown. I, inosine-containing primers; Sp, specific primers; NUP (Nested Universal Primer, Clontech), 5′-RACE commercial primer.
	Figure 2. Multiple alignment of mtRNA polymerases. Amino-acid sequences of mtRNAP from P. lividus (EF068167), Pl; S. purpuratus (29), Sp; H. sapiens (U75370), Hs; X. laevis (AF200705), Xl, and D. melanogaster (AE003587), Dm, were aligned with the ClustalW program. The multiple alignment was performed at NPS@ Web server of the PBIL using GONNET weight matrix and formatted with the Web tool ESPript 2.2. Red boxes include amino acids identical in all sequences; yellow boxes include amino acids similar or identical (red letters) in at least three of the five sequences; black letters, non-conserved residues. The 10 conserved blocks and the two putative PPR motifs are indicated by continuous lines; the DX2GR motif, that is placed inside block III, is indicated by a striped box The amino acids of the P. lividus enzyme implicated in the catalysis, as inferred from T7 mutant studies, are indicated by asterisks.
	Figure 3. Purification of the sea urchin mtRNAP from baculovirus-infected insect cells and functional assay. (A) Purification of mtRNAP by metal chelate affinity chromatography. The soluble portion of the insect cell lysate expressing the sea urchin mtRNAP was purified by Ni2+-NTA chromatography; cleared lysate, C.lys, flow-through, FT, wash, W, 3–5, fractions eluted at 250 mM imidazole, were separated on a 10% SDS–PAGE and revealed by immunoblotting as described in ‘Materials and Methods’ section. (B) Purification profile of mtRNAP as obtained by Heparin–Sepharose chromatography. Peak fractions from Ni2+-NTA column were pooled and subjected to Heparin–Sepharose chromatography. Input to the column (I) and fractions eluting between 0.75 and 0.9 M NaCl were analyzed by 7.5% SDS–PAGE and Coomassie Brilliant Blue stained. The molecular weight marker Precision Plus Protein Standards (Bio-Rad) is shown (M). The arrow inside the picture indicates the mtRNAP-containing band, as assessed by MALDI-TOF analysis. (C) Immunoblotting assay of input to the column (I) and Heparin–Sepharose eluted fractions. (D) Transcriptional activity of purified mtRNAP. The indicated Heparin–Sepharose fractions were assayed in the presence of [α-32]PUTP, as described in ‘Materials and Methods’ section. On the top it is shown the diagram of the 71-bp tailed template, named 71bpDNA, with the open bar referring to the duplex DNA portion and the thin line to the 3′-tail. Run-off transcripts are indicated by arrowed line. Radiolabeled transcripts were separated on a 12% polyacrylamide/7M urea mini-gel followed by phosphorimaging analysis. 15 + R, fraction 15 treated with RNase A. RNA markers corresponding to the 10 nt ladder are indicated on the left.
	Figure 4. Transcription termination assays with recombinant mtRNAP and mtDBP. (A) Gel mobility shift analysis of recombinant mtDBP purified from baculovirus-infected insect cells. The assay was performed as described in ‘Materials and Methods’ section, with the indicated amounts of purified mtDBP and 50 fmol of end-labeled probe TermNCR(F) (see below). (B) Effect of mtDBP on the elongation activity of mtRNAP on templates TermNCR(F/R). (Top) Scheme of the 98-bp 3′-tailed DNA constructs, named TermNCR(F) and TermNCR(R), used in the transcription assays. The filled boxes indicate the 38-bp region in the Non-Coding Region (NCR) of P. lividus mtDNA (the sequence and nucleotide position are shown); the mtDBP-binding site, as from DNase I footprinting analysis (35), is underlined. The arrow enclosed in the boxes marks the orientation of mtDBP target site with respect to transcription direction. The open boxes represent the flanking sequences (41 and 19 bp, respectively), which are unrelated to sea urchin mtDNA. Run-off and terminated transcripts are indicated by arrowed lines. The thickness of the lines referring to the terminated molecules (Term), obtained with TermNCR(F) template, represents the relative abundance of the transcripts. (Bottom) Transcription termination assay. Transcription reactions were performed as described in ‘Materials and Methods’ section in the presence of 2 pmol of the indicated template, about 0.15 pmol of recombinant mtRNAP and the indicated amounts of mtDBP. DBPTerm refers to the terminated products generated by mtDBP, indicated by the line with two consecutive arrows in the top scheme. RNA markers corresponding to the 32P-5′-end labeled RNA ladder, Decade Markers, Ambion, are shown on the left. (C) Transcription assay performed on template NoTerm. The construct is a 98-bp 3′-tailed DNA lacking mtDBP-binding site. Reactions were performed as in (B). (D) Transcription assay on templates TermNCRbis(F/R). (Top) Schematic representation of the 98-bp 3′-tailed constructs, named TermNCRbis(F) and TermNCRbis(R). The regions flanking the mtDBP-binding site, indicated with striped boxes, are different in sequence from those of TermNCR(F/R). The sequence and nucleotide position of the region containing the mtDBP-binding site are as in (B). The thickness of the lines that represent the terminated molecules (Term), obtained with the forward template, indicates the relative abundance of the transcripts. (Bottom) Transcription termination assay. Reactions were performed as reported in (B). DBPTerm, RNA molecules terminated by mtDBP. (E) Pulse-chase of transcription elongation by mtRNAP in the presence of mtDBP. Assays were carried out as described in ‘Materials and Methods’. After a pulse-label of 15 min with [α−32]PUTP, the reaction was chased with a 2300-fold excess of unlabeled UTP. Samples (20 μl) were taken at the time points indicated and analyzed on a 12% polyacrylamide/7M urea, followed by phosphorimaging analysis.
	Figure 5. Schematic diagram showing the mitochondrial transcription termination in sea urchin NCR. The region of P. lividus mtDNA containing the NCR, with the bound mtDBP, and some adjacent genes is shown. Numbers mark the position of the mtDBP-binding site. Arrows inside the open boxes indicate the transcription direction of the genes. Arrow on the top indicates the direction of H-strand DNA synthesis; the possible DNA portion of the newly synthesized strand is indicated as a bold line, the remaining RNA portion (primer) as a thin line (44). OH, DNA replication origin. Arrows below the scheme represent sequence-dependent and protein-dependent termination events, with points of transcription arrest indicated by dashed vertical lines. H-strand readthrough transcription can cause dislodging of mtDBP and resumption of H-DNA synthesis (40).

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