scholarly journals Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation.

1986 ◽  
Vol 6 (9) ◽  
pp. 3262-3267 ◽  
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
High Rate ◽  
In Vitro Assays ◽  
Regulatory Sequences ◽  
Sufficient Information ◽  
Mitochondrial Rna ◽  
Loop Region ◽  
Heavy Strand

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species.

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation

1986 ◽  
Vol 6 (9) ◽  
pp. 3262-3267
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
High Rate ◽  
In Vitro Assays ◽  
Regulatory Sequences ◽  
Sufficient Information ◽  
Mitochondrial Rna ◽  
Loop Region ◽  
Heavy Strand

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species.

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.

scholarly journals Distinct roles for two purified factors in transcription of Xenopus mitochondrial DNA.

1995 ◽  
Vol 15 (12) ◽  
pp. 7032-7042 ◽  
Author(s):  
I Antoshechkin ◽  
D F Bogenhagen
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Sequence Similarity ◽  
Protein A ◽  
Mitochondrial Rna ◽  
Basal Transcription ◽  
Hmg Box ◽  
Mitochondrial Rna Polymerase ◽  
Dnase I Footprinting

Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA. xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB. An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters. We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB.

scholarly journals Non-DNA-Templated Addition of Nucleotides to the 3′ End of RNAs by the Mitochondrial RNA Polymerase of Physarum polycephalum

2008 ◽  
Vol 28 (18) ◽  
pp. 5795-5802 ◽  
Author(s):  
Mara L. Miller ◽  
Dennis L. Miller
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Rna Editing ◽  
Physarum Polycephalum ◽  
Mitochondrial Gene ◽  
In Vitro Transcription ◽  
Open Reading Frames ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

ABSTRACT Mitochondrial gene expression is necessary for proper mitochondrial biogenesis. Genes on the mitochondrial DNA are transcribed by a dedicated mitochondrial RNA polymerase (mtRNAP) that is encoded in the nucleus and imported into mitochondria. In the myxomycete Physarum polycephalum, nucleotides that are not specified by the mitochondrial DNA templates are inserted into some RNAs, a process called RNA editing. This is an essential step in the expression of these RNAs, as the insertion of the nontemplated nucleotides creates open reading frames for the production of proteins from mRNAs or produces required secondary structure in rRNAs and tRNAs. The nontemplated nucleotide is added to the 3′ end of the RNA as the RNA is being synthesized during mitochondrial transcription. Because RNA editing is cotranscriptional, the mtRNAP is implicated in RNA editing as well as transcription. We have cloned the cDNA for the mtRNAP of Physarum and have expressed the mtRNAP in Escherichia coli. We have used in vitro transcription assays based on the Physarum mtRNAP to identify a novel activity associated with the mtRNAP in which non-DNA-templated nucleotides are added to the 3′ end of RNAs. Any of the four ribonucleoside triphosphates (rNTPs) can act as precursors for this process, and this novel activity is observed when only one rNTP is supplied, a condition under which transcription does not occur. The implications of this activity for the mechanism of RNA editing are discussed.

Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro

1989 ◽  
Vol 9 (8) ◽  
pp. 3193-3202
Author(s):  
G T Marczynski ◽  
P W Schultz ◽  
J A Jaehning
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
In Vitro Transcription ◽  
Catalytic Rna ◽  
Mitochondrial Rna ◽  
Promoter Sequences ◽  
Sequence Recognition ◽  
Mitochondrial Rna Polymerase

We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.

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