Synthesis of RNA in isolated mitochondria from Saccharomyces cerevisiae

Newman, Dawn; Martín, Nancy

doi:10.1016/0147-619x(82)90028-2

“…An appreciable portion of the decrease in mitochondrial RNA synthetic capacity in isolated organelles appears to be due to depression of rRNA synthesis, which accounts for only 10 to 20% of the total H-strand transcription (as estimated from densitometric measurements), instead of 85 to 90%, as is the case in vivo (4). The observed absolute rate of UMP incorporation into RNA of isolated HeLa cell mitochondria is comparable to the rates reported for isolated yeast mitochondria (6,14,22).…”

Section: Discussionsupporting

confidence: 65%

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

Gaines¹,

Attardi²

1984

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A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37°C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNAsynthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmicmitochondrial interactions in organelle gene expression.Recent work from this laboratory has shown that the heavy (H) strand of HeLa cell mitochondrial DNA (mtDNA) is the site of two overlapping transcription events (4a, 12, 19. 20). A considerable amount of evidence suggests that one of these events is responsible for the synthesis of the bulk of rRNA, whereas the other results in the synthesis of a polycistronic molecule which corresponds to almost the entire H strand and is destined to be processed to yield the mRNAs and most of the tRNAs encoded in the H strand (Fig. 1). The initiation of the two H-strand transcription events and that of light (L)-strand transcription occur in close proximity to each other in the region of mtDNA upstream of the origin of H-strand synthesis (10). This clustering of the three putative promoters may underlie the integrated control of expression of the corresponding transcription units, whereas their proximity to the origin of Hstrand synthesis may reflect the existence of a link between transcription events and mtDNA replication. To investigate further, under conditions allowing easy experimental manipulations, the molecular mechanisms, controls, and interrelationships of the mtDNA transcription and RNA processing events which occur in HeLa cell mitochondria, we developed a system f...

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“…An appreciable portion of the decrease in mitochondrial RNA synthetic capacity in isolated organelles appears to be due to depression of rRNA synthesis, which accounts for only 10 to 20% of the total H-strand transcription (as estimated from densitometric measurements), instead of 85 to 90%, as is the case in vivo (4). The observed absolute rate of UMP incorporation into RNA of isolated HeLa cell mitochondria is comparable to the rates reported for isolated yeast mitochondria (6,14,22).…”

Section: Discussionsupporting

confidence: 82%

Highly Efficient RNA-Synthesizing System That Uses Isolated Human Mitochondria: New Initiation Events and In Vivo-Like Processing Patterns

Gaines

¹

,

Attardi

²

1984

Molecular and Cellular Biology

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A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression.

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“…1B, lanes 3 and 4). Although the labeled products showed a preponderance of high molecular weight RNA relative to total mtRNA, this probably reflects an increased abundance of labeled precursors, as it does in other isolated mitochondrial systems (15)(16)(17)(18).…”

Section: Resultsmentioning

confidence: 99%

Autonomously replicating RNA in mitochondria of maize plants with S-type cytoplasm

Finnegan

¹

,

Brown

²

1986

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondria isolated from maize plants with They exist as a set of large recombining circular molecules in which some circles are more abundant than others, resulting in unequal representation of the various sequences that they comprise (4,5). Minicircular plasmid DNAs are also frequently present in plant mitochondria (6) and linear DNA episomes that recombine with specific sequences on the large circular molecules have been found as well (7,8). In these and in other organelles the only identified molecules with the potential of storing genetic information are DNAs.Another interesting feature of plant mitochondrial DNA (mtDNA) is that variation in it can result in the phenotype of male sterility. In maize there are four distinct cytoplasmic and hence mitochondrial types, S, C, T, and N; S, C, and T can confer male sterility when present in specific nuclear genetic backgrounds, whereas N cannot (9). We have used an in organello RNA synthesis system to search for differences in mitochondrial gene expression among the various maize cytoplasmic types. We have made the surprising discovery that several R$As unique to the S cytoplasm are synthesized in a DNA-independent manner. Our observations indicate that these RNAs represent an RNA-based genetic system that is endogenous to the mitochondnron. No other such system has been found in an organelle. Because these RNAs replicate independently of cellular chromosomes, we have termed them RNA plasmids. MATERIALS AND METHODSMaterials. Inbred WF9 seeds were from Marc Albertsen (Pioneer Hi-Bred International, Johnston, IA); inbred B73 seeds were from Mike Brayton Seeds (Ames, IA); Vg cytoplasm seed (open-pollinated) was from C. S. Levings III

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Synthesis of RNA in isolated mitochondria from Saccharomyces cerevisiae

Cited by 10 publications

References 36 publications

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

Highly Efficient RNA-Synthesizing System That Uses Isolated Human Mitochondria: New Initiation Events and In Vivo-Like Processing Patterns

Autonomously replicating RNA in mitochondria of maize plants with S-type cytoplasm

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