scholarly journals A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

1995 ◽  
Vol 15 (9) ◽  
pp. 4803-4809 ◽  
Author(s):  
H E Lorimer ◽  
B J Brewer ◽  
W L Fangman
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Gene ◽  
Replication Initiation ◽  
High Density ◽  
Mitochondrial Rna ◽  
Wild Type ◽  
Mitochondrial Rna Polymerase ◽  
Origin Region ◽  
Mtdna Replication ◽  
Yeast Mitochondrial Dna

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

scholarly journals RPO41-independent maintenance of [rho-] mitochondrial DNA in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (1) ◽  
pp. 10-15 ◽  
Author(s):  
W L Fangman ◽  
J W Henly ◽  
B J Brewer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Nuclear Gene ◽  
Replication Initiation ◽  
Yeast Cells ◽  
Mitochondrial Rna ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitochondrial Rna Polymerase ◽  
Mtdna Deletion

A subset of promoters in the mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae has been proposed to participate in replication initiation, giving rise to a primer through site-specific cleavage of an RNA transcript. To test whether transcription is essential for mtDNA maintenance, we examined two simple mtDNA deletion ([rho-]) genomes in yeast cells. One genome (HS3324) contains a consensus promoter (ATATAAGTA) for the mitochondrial RNA polymerase encoded by the nuclear gene RPO41, and the other genome (4a) does not. As anticipated, in RPO41 cells transcripts from the HS3324 genome were more abundant than were transcripts from the 4a genome. When the RPO41 gene was disrupted, both [rho-] genomes were efficiently maintained. The level of transcripts from HS3324 mtDNA was decreased greater than 400-fold in cells carrying the RPO41 disrupted gene; however, the low-level transcripts from 4a mtDNA were undiminished. These results indicate that replication of [rho-] genomes can be initiated in the absence of wild-type levels of the RPO41-encoded RNA polymerase.

RPO41-independent maintenance of [rho-] mitochondrial DNA in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (1) ◽  
pp. 10-15
Author(s):  
W L Fangman ◽  
J W Henly ◽  
B J Brewer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Nuclear Gene ◽  
Replication Initiation ◽  
Yeast Cells ◽  
Mitochondrial Rna ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitochondrial Rna Polymerase ◽  
Mtdna Deletion

A subset of promoters in the mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae has been proposed to participate in replication initiation, giving rise to a primer through site-specific cleavage of an RNA transcript. To test whether transcription is essential for mtDNA maintenance, we examined two simple mtDNA deletion ([rho-]) genomes in yeast cells. One genome (HS3324) contains a consensus promoter (ATATAAGTA) for the mitochondrial RNA polymerase encoded by the nuclear gene RPO41, and the other genome (4a) does not. As anticipated, in RPO41 cells transcripts from the HS3324 genome were more abundant than were transcripts from the 4a genome. When the RPO41 gene was disrupted, both [rho-] genomes were efficiently maintained. The level of transcripts from HS3324 mtDNA was decreased greater than 400-fold in cells carrying the RPO41 disrupted gene; however, the low-level transcripts from 4a mtDNA were undiminished. These results indicate that replication of [rho-] genomes can be initiated in the absence of wild-type levels of the RPO41-encoded RNA polymerase.

scholarly journals A nuclear mutation reversing a biased transmission of yeast mitochondrial DNA.

Genetics ◽  
1991 ◽  
Vol 128 (2) ◽  
pp. 241-249 ◽  
Author(s):  
S G Zweifel ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Nuclear Gene ◽  
Diploid Strain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mtdna Replication ◽  
Clonal Lines ◽  
Yeast Mitochondrial Dna ◽  
P Cells ◽  
Allele Transmission

Abstract The highly biased transmission of p- mitochondrial DNA that occurs in hypersuppressive matings between p- and p+ cells of the yeast Saccharomyces cerevisiae is thought to be a consequence of the replication advantage of the p- mtDNA. A nuclear gene, MGT1, that is required for this displacement of p+ mtDNA from zygotic clones has been identified through mutation. When one haploid parent carries the mgt1 allele, transmission of p- mtDNA is substantially reduced. When both haploid parents carry the mgt1 allele, p- mtDNA is essentially eliminated from the zygotic progeny. Thus in the absence of the MGT1 gene there is a switch in the transmission bias; p+ mtDNA rather than the hypersuppressive p- mtDNA is inherited by most zygotic clones. In contrast to its semi-dominant behavior in haploid matings, mgt1 behaves as a recessive allele in diploid matings since the p+ genome in MGT1/mgt1 diploids is efficiently displaced when mated with a MGT1/mgt1 hypersuppressive p- diploid strain. We find that p+ genomes can be comaintained along with hypersuppressive p- mtDNA for extended periods in clonal lines derived from MGT1 x mgt1 matings. However, as expected from the recessive nature of the mgt1 mutation, these p+ genomes are eventually eliminated. Our work indicates that MGT1 plays a crucial role in the competition for inheritance between hypersuppressive p- mtDNAs and the p+ mitochondrial genome. The MGT1 gene product may be a component of a mtDNA replication system that acts preferentially at the rep sequences found in hypersuppressive mtDNAs.

scholarly journals The PPR domain of mitochondrial RNA polymerase is a ribonuclease required for mtDNA replication

2021 ◽  
Author(s):  
Yi Liu ◽  
Zhe Chen ◽  
Zong-Heng Wang ◽  
Katherine Delaney ◽  
Juanjie Tang ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Mitochondrial Rna ◽  
Cellular Energy ◽  
Cellular Energy Metabolism ◽  
Dna And Rna ◽  
Mitochondrial Rna Polymerase ◽  
Evolutionarily Conserved ◽  
Mtdna Replication

AbstractMitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase (POLRMT) is thought to be the primase for mtDNA replication. However, it is unclear how POLRMT, which normally transcribes long polycistronic RNAs, can produce short RNA oligos to initiate mtDNA replication. Here we show that the PPR domain of Drosophila POLRMT is a 3’ to 5’ exoribonuclease. The exoribonuclease activity is indispensable for POLRMT to synthesize short RNA oligos and to prime DNA replication in vitro. An exoribonuclease deficient POLRMT, POLRMTE423P partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in POLRMT mutant background, indicating that the exoribonuclease activity is necessary for mtDNA replication. Overexpression of POLRMTE423P in adult flies leads to severe neuromuscular defects and a marked increase of mtDNA transcripts errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription. PPR domain of human POLRMT also has exoribonuclease activity, indicating evolutionarily conserved roles of PPR domain in mitochondrial DNA and RNA metabolism.

scholarly journals Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming.

1992 ◽  
Vol 12 (6) ◽  
pp. 2561-2569 ◽  
Author(s):  
L L Stohl ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Rna Processing ◽  
Mitochondrial Rna ◽  
Rna Sequence ◽  
Rnase Mrp ◽  
Processing Activity ◽  
Yeast Mitochondrial Dna ◽  
Human And Mouse

Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.

The Mitochondrial Nucleoid Protein, Mgm101p, of Saccharomyces cerevisiae Is Involved in the Maintenance of ρ+ and ori/rep-Devoid Petite Genomes but Is Not Required for Hypersuppressive ρ− mtDNA

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1389-1400
Author(s):  
Xiao Ming Zuo ◽  
G Desmond Clark-Walker ◽  
Xin Jie Chen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Rna ◽  
Mtdna Copy Number ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Nucleoids ◽  
Mtdna Replication ◽  
Sensitive Allele ◽  
Discriminatory Effect

Abstract The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional ρ+ genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a ρ+ strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have ρ− genomes that are stably maintained. Interestingly, all surviving ρ− mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS ρ− mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of ρ+ and ori/rep-devoid ρ− mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS ρ− genome is stably maintained in a mgm101, rpo41 double mutant.

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.

scholarly journals Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming

1992 ◽  
Vol 12 (6) ◽  
pp. 2561-2569
Author(s):  
L L Stohl ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Rna Processing ◽  
Mitochondrial Rna ◽  
Rna Sequence ◽  
Rnase Mrp ◽  
Processing Activity ◽  
Yeast Mitochondrial Dna ◽  
Human And Mouse

Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.

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