scholarly journals In vitro transcription of human mitochondrial DNA. Identification of specific light strand transcripts from the displacement loop region.

1983 ◽  
Vol 258 (2) ◽  
pp. 1268-1275 ◽  
Author(s):  
M W Walberg ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
In Vitro Transcription ◽  
Dna Identification ◽  
Human Mitochondrial Dna ◽  
Loop Region ◽  
Light Strand

scholarly journals Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts.

1986 ◽  
Vol 6 (5) ◽  
pp. 1446-1453 ◽  
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Transcriptional Control ◽  
Spatial Relationship ◽  
S1 Nuclease ◽  
Loop Region ◽  
Loop Sequence ◽  
Transcriptional Start Sites ◽  
Heavy Strand ◽  
Light Strand

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.

Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts

1986 ◽  
Vol 6 (5) ◽  
pp. 1446-1453
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Transcriptional Control ◽  
Spatial Relationship ◽  
S1 Nuclease ◽  
Loop Region ◽  
Loop Sequence ◽  
Transcriptional Start Sites ◽  
Heavy Strand ◽  
Light Strand

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.

scholarly journals Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally.

1986 ◽  
Vol 6 (1) ◽  
pp. 294-301 ◽  
Author(s):  
D D Chang ◽  
J E Hixson ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Rich Region ◽  
Human Mitochondrial Dna ◽  
Loop Region

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.

Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally

1986 ◽  
Vol 6 (1) ◽  
pp. 294-301
Author(s):  
D D Chang ◽  
J E Hixson ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Rich Region ◽  
Human Mitochondrial Dna ◽  
Loop Region

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.

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

1993 ◽  
Vol 13 (4) ◽  
pp. 2162-2171 ◽  
Author(s):  
C S Madsen ◽  
S C Ghivizzani ◽  
W W Hauswirth
Keyword(s):  
Mitochondrial Dna ◽  
Protein Binding ◽  
Loop Formation ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Loop Region ◽  
In Organello ◽  
Close Proximity ◽  
D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial 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 approximately 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.

scholarly journals The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization

2007 ◽  
Vol 176 (2) ◽  
pp. 141-146 ◽  
Author(s):  
Jiuya He ◽  
Chih-Chieh Mao ◽  
Aurelio Reyes ◽  
Hiroshi Sembongi ◽  
Miriam Di Re ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Uncharacterized Protein ◽  
Supercoiled Dna ◽  
Recombinant Fragment ◽  
Loop Region ◽  
Mitochondrial Nucleoids ◽  
Marked Preference ◽  
D Loop ◽  
Aaa Protein

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

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