Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

Abstract

A methylation protection assay was used in a novel manner to demonstrate a specific bovine proteinmitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a -48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end. (14). Consistent with this suggestion, closely related terminationassociated sequences (TASs) have been identified at similar positions relative to mapped D-loop DNA 3' ends in Xenopus laevis (15), cow, pig (34), and several primates (21).The current picture, therefore, is that conserved sequence domains (TAS elements) located short distances upstream of 3' ends of D-loop DNA exist in all vertebrate species and are candidate cis elements for the regulation of H-strand DNA synthesis. However, no experimental evidence regarding the potential function of TAS elements exists. Additionally, it remains unclear how such elements might function as terminators of H-strand DNA synthesis both because the number of consensus TAS domains can exceed the number of 3' D-loop termini and because TASs are found at variable distances (20 to 80 bp) upstream of these termination sites.In this study, we have adapted the methylation protection technique to assay purified bovine mitochondria for specific protein-mtDNA interactions near the D-loop 3' end in an in vivo-like environment. By this method, we identified a single protected sequence element located 58 bases upstream from the D-loop 3' end. Using partially purified protein extracts from bovine mitochondria, a 48-kDa protein(s) which binds specifically to this element was identified. In vitro DNase I footprint experiments using a series of deleted target sequences demonstrated that although seven TAS-like sequences exist in the region, only one is selectively bound. These results suggest that D-loop termination and, subsequently, mtDNA copy number may be regulated through the binding of a sequence-specific protein factor.

Expression of the Smooth Muscle Myosin Heavy Chain Gene Is Regulated by a Negative-acting GC-rich Element Located between Two Positive-acting Serum Response Factor-binding Elements

“…DNase I digestions and electrophoresis of the samples were performed as described previously (30). Location of the footprinted regions was determined by including on the gel a dideoxy sequence marker ladder, and a Maxam-Gilbert G-specific ladder of the labeled fragment was also generated to verify the integrity of the probe (data not shown).…”

3D model of RNA polymerase and bidirectional transcription

“…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].…”

“…Both RNA polymerase and RNase MRP are involved for mt DNA replication from a RNA-D-loop DNA hybrid [1][2][3][4][5][6][7][8][9][10], whereas these two enzymes in addition to RNase P are involved in transcription as well as RNA (mRNA, rRNA and tRNA) processing of human and mouse mt.RNA [1][2][3][4][5][6][7][8][9][10][11][12]23]. RNase MRP cleaves mRNA substrate at an adjacent conserved decamer sequence 5′-CGACCCCUCC-3′ (relative to CSB2 in D-loop), whereas RNase P is involved in 5′ and 3′ end-processing of mt tRNA [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. This conserved decamer sequence 5′-CGACCCCUCC-3′ is also present in MRP RNA [3,4,7,[11][12][13].…”

“…The mouse homologs (or orthologs) mTfb1m and mTfb2m were identified [13]. The transcription termination was reported to occur by 34kd mt-TERM/mtTERF DNA binding protein [14,15], although 24kd Tf1 transcription factor, 48kd TAS-A protein (which binds to termination associated sequence of mt D-loop region) as well as other putative binding proteins (TAS-B, -C, -D, -E, -F, -G; A/B and F/G which bind to partially overlapping sites) in the mt D-loop segment were reported [15,16]. Two-to-three putative mitochondrial transcription termination 1 Corresponding author: Pradip Bhattacharya, Room Nos.…”

“…. 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].…”