In vitro transcription of human mitochondrial DNA: accurate termination requires a region of DNA sequence that can function bidirectionally.

Abstract

Mammalian mitochondrial genomes have a presumptive transcription termination site at the 16S rRNAtRNALU gene boundary. We have developed an in vitro system from human KB cells that terminates transcription at this gene boundary. By S1 nuclease protection, the 3' ends of terminated transcripts were mapped 3 and 4 base pairs upstream of the 16S rRNA-tRNAL" gene boundary, in agreement with in vivo data.By high-resolution sizing of transcripts, the 3' end was mapped 7 ± 1 base pairs downstream from the gene boundary. Termination occurs with equal efficacy from transcriptional initiation at the heavy-or light-strand promoter. AU template nucleotide sequence information necessary for termination appears to be located near the termination site itself. An unexpected observation is that the termination region functions bidirectionally.Transcription of mammalian mtDNA initiates from promoters in the displacement (D)-loop region, producing polycistronic RNA precursors that are processed into mature transcripts by cleavages between the gene sequences (1). Two lines of evidence indicate that transcription of the heavy (H) strand can either terminate at the 3' end ofthe 16S rRNA gene or continue transcribing H-strand genes. First, the ribosomal gene region appears to be transcribed 50-100 times more frequently than the other H-strand genes (2). Second, the heterogeneity of the 3' ends of 16S rRNA of several species (3-5) suggests that 16S rRNA termini may be the result of transcription termination, rather than a precise processing likely responsible for the 3' ends of most mature mitochondrial transcripts. We describe here an in vitro system derived from human mitochondria and show promoter-independent, bidirectional termination at the 16S rRNA-tRNAL"u gene boundary. MATERIALS AND METHODSClone Construction. A Hae II fragment inserted into pACYC177 (6) supplied mtDNA from nucleotides (nt) 3160 to 4534 (7). Clone L3'A-91, containing the mtDNA sequence from nt 495 to 741, and L5'lA-28 with the mtDNA sequence from nt 1 to 440, provided the promoter sequences (8). From this source material, three template clones were constructed with standard techniques (9). Clone A consists of nt 495-739, a 16-base-pair (bp) linker, and nt 3165-4121, all inserted between the BamHI and EcoRI sites of pBR322. Clone B is similar, except that nt 440-324 and a 10-bp linker precede the nt 3165-4121 fragment. Clone C consists of nt 440-324joined without linkers to nt 3316-3165, all inserted between the BamHI and EcoRI sites of pBR322.Transcription Termination Reactions. For the transcription termination reactions, mtRNA polymerase was prepared as described (10). The transcription mixture was typically in a 50-1.l vol containing 10 mM Tris HCl (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, bovine serum albumin at 100 pg/ml, 180 ,M CTP and UTP, 360 AuM ATP, 0.01 AM [a-32P]GTP (3000 Ci/mmol; 1 Ci = 37 GBq), 0.5 ,ug of DNA (10 gg/ml), and 7-14 A1 of the polymerase preparation; reactions were incubated at 15°C for 45 min and terminated by adding 10,ul o...

Genetic aspects of mitochondrial genome evolution

“…It ends at proximal, so-called mt-TERM signal beyond the rrnL rRNA gene within the adjacent tRNA-Leu gene (Christianson and Clayton, 1986). Sequence box II, located in the D-loop, acts as a sequence-dependent termination element for transcription originating from the light strand promoter (Pham et al, 2006).…”

3D model of RNA polymerase and bidirectional transcription

“…The plasmids were transformed into Escherichia coli JM107, and purified by alkaline lysis procedure followed by Biogel-A150 chromatography, and finally extracted with the standard phenol-chloroform-isoamyl alcohol (or isopropanol) protocol followed by chloroform and ether separately [22]. The restriction fragments eluted from the gel of the plasmids were linearized with restriction enzymes, and were used for template in the in-vitro reconstituted transcription complex system [16,17]. …”

“…Mitochondrial D-loop region; In-vitro transcription; mt RNA polymerase; gel-mobility shift assay; 3D model Both human and mouse mitochondrial (mt) in-vitro reconstituted transcription system and mt lysate were shown to transcribe bidirectionally from D-loop region containing H-strandpromoter/L-strand-promoter (HSP/LSP) of mitochondrial genome .These transcripts ranged between >100bp and >3000bp in both human and mouse [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The D-loop distal genes were also transcribed in this system [16].…”

“…. In the in-vitro reconstituted transcription system (isolated by the heparin-sepharose chromatography), the HSP/LSP region (where both major and minor promoters lie within close proximity) of the mitochondrial Dloop segment in both human and mouse were reported previously to initiate transcription products which ranged between >100bp to 700bp runoff transcripts [13][14][15][16][17]19,20,29,30], although the mitochondrial lysate transcribed >2050bp ->3000bp [16] in an in vitro assay system. The mitochondrial lysate produced comparatively larger transcripts (>4-5kb) from HSP/LSP fused with larger human mitochondrial DNA segment [16].…”

“…650bp RNA was transcribed from the cloned mt noncoding sequence 95bp-1004bp [32]. Both human HSP (495bp-741bp) and LSP (440bp-324bp) were fused with human mt segment (3165bp-4121bp containing 16SrRNA-tRNA leu gene) in association with or without a 10-16bp linker consisting of those two genes boundary sequence (D-TERM), and were shown to initiate transcription products (that ranged between <60bp and >622bp) with human KB cell mt in-vitro transcription system [17]. The transcription initiation products from p405, p622 and p10 plasmids were not studied further.…”