Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

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Template sequences required for transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters

Molecular and Cellular Biology ◽

10.1128/mcb.8.7.2917-2924.1988 ◽

1988 ◽

Vol 8 (7) ◽

pp. 2917-2924

Author(s):

D F Bogenhagen ◽

M F Romanelli

Keyword(s):

Mitochondrial Dna ◽

Xenopus Laevis ◽

Consensus Sequence ◽

Point Mutations ◽

In Vitro Mutagenesis ◽

Start Site ◽

Sequence Matching ◽

Bidirectional Promoters

Previous work from our laboratory has shown that transcription of Xenopus laevis mitochondrial DNA initiates both in vivo and in vitro from bidirectional promoters located between the gene for tRNA(Phe) and the 5' termini of displacement loop DNA strands. A consensus sequence matching the octanucleotide ACGTTATA surrounds each transcription start site. In the present study, we used in vitro mutagenesis to define sequences required for specific transcription in vitro. First, cloned mitochondrial DNA templates generated by deletion mutagenesis were transcribed in vitro to define the limits of functional promoters. The bidirectional promoter located approximately 33 nucleotides upstream from the gene for tRNA(Phe), termed promoter 1, was studied in greatest detail. The results confirmed the hypothesis that the consensus octanucleotide sequence surrounding each start site is an essential promoter element. A duplex 18-base-pair oligonucleotide encoding the symmetrical promoter 1 region was synthesized and cloned in a plasmid vector. This synthetic oligonucleotide was sufficient to support bidirectional transcription. Point mutations within this oligonucleotide were used to identify critical residues within the consensus sequence.

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Template sequences required for transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.8.7.2917 ◽

1988 ◽

Vol 8 (7) ◽

pp. 2917-2924 ◽

Cited By ~ 24

Author(s):

D F Bogenhagen ◽

M F Romanelli

Keyword(s):

Mitochondrial Dna ◽

Xenopus Laevis ◽

Consensus Sequence ◽

Point Mutations ◽

In Vitro Mutagenesis ◽

Start Site ◽

Sequence Matching ◽

Bidirectional Promoters

Previous work from our laboratory has shown that transcription of Xenopus laevis mitochondrial DNA initiates both in vivo and in vitro from bidirectional promoters located between the gene for tRNA(Phe) and the 5' termini of displacement loop DNA strands. A consensus sequence matching the octanucleotide ACGTTATA surrounds each transcription start site. In the present study, we used in vitro mutagenesis to define sequences required for specific transcription in vitro. First, cloned mitochondrial DNA templates generated by deletion mutagenesis were transcribed in vitro to define the limits of functional promoters. The bidirectional promoter located approximately 33 nucleotides upstream from the gene for tRNA(Phe), termed promoter 1, was studied in greatest detail. The results confirmed the hypothesis that the consensus octanucleotide sequence surrounding each start site is an essential promoter element. A duplex 18-base-pair oligonucleotide encoding the symmetrical promoter 1 region was synthesized and cloned in a plasmid vector. This synthetic oligonucleotide was sufficient to support bidirectional transcription. Point mutations within this oligonucleotide were used to identify critical residues within the consensus sequence.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00356-08 ◽

2008 ◽

Vol 28 (18) ◽

pp. 5795-5802 ◽

Cited By ~ 10

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.

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Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3603 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3603-3613 ◽

Cited By ~ 25

Author(s):

J C Kennell ◽

A M Lambowitz

Keyword(s):

Mitochondrial Dna ◽

Neurospora Crassa ◽

Transcription Initiation ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Transcription System ◽

Group I ◽

Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

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Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3603-3613.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3603-3613

Author(s):

J C Kennell ◽

A M Lambowitz

Keyword(s):

Mitochondrial Dna ◽

Neurospora Crassa ◽

Transcription Initiation ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Transcription System ◽

Group I ◽

Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

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A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.580 ◽

1995 ◽

Vol 15 (1) ◽

pp. 580-589 ◽

Cited By ~ 88

Author(s):

B Xu ◽

D A Clayton

Keyword(s):

Mitochondrial Dna ◽

Dna Replication ◽

Rna Polymerase ◽

In Vitro Transcription ◽

Mitochondrial Rna ◽

Hybrid Formation ◽

Dna Replication Origin ◽

Mitochondrial Replication ◽

Mitochondrial Dna Polymerase

Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5' end of the rG region. The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required. The efficiency of hybrid formation depends on the length of RNA synthesized 5' to CSBII and the type of RNA polymerase employed. Once made, the RNA strand of an RNA-DNA hybrid can serve as an effective primer for mitochondrial DNA polymerase. These results reveal a new mechanism for persistent RNA-DNA hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA hybrid.

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Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1467 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1467-1478 ◽

Cited By ~ 27

Author(s):

S A Shaaban ◽

B M Krupp ◽

B D Hall

Keyword(s):

Amino Acid ◽

Rna Polymerase ◽

Transcription Initiation ◽

Transcription Termination ◽

Rna Polymerase Iii ◽

In Vitro Transcription ◽

The Other ◽

Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.7032 ◽

1995 ◽

Vol 15 (12) ◽

pp. 7032-7042 ◽

Cited By ~ 59

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.

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