[4] In Organello mitochondrial protein and RNA synthesis systems from Saccharomyces cereuisiae

Poyton, Robert O.; Bellus, Gary A.; McKee, Edward E.; Sevarino, Kevin A.; Goehring, Bradley

doi:10.1016/s0076-6879(96)64006-3

Cited by 12 publications

(10 citation statements)

References 17 publications

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“…DNA was fixed to the nylon membrane using UV light generated from a UV Stratalinker 2400 (Stratagene). Hybridization experiments were performed essentially as described by Poyton et al (41). Membranes were prehybridized in 0.15 ml of hybridization solution (65% formamide, 0.2% SDS, 0.4 M NaCl, 20 mM piperazine-N, NЈ-bis (2-ethanesulfonic acid) [PIPES] [pH 6.4]/cm 2 , 0.1 mg of torula yeast RNA/ml, and 0.1 mg of denatured salmon sperm DNA/ml) for 30 min at 50°C.…”

Section: Methodsmentioning

confidence: 99%

UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

Militello

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2000

Molecular and Cellular Biology

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Although primary transcripts are polycistronic in the mitochondria of Trypanosoma brucei, steady-state levels of mature, monocistronic RNAs change throughout the parasitic life cycle. This indicates that steadystate RNA abundance is controlled by posttranscriptional mechanisms involving differential RNA stability. In this study, in organello pulse-chase labeling experiments were used to analyze the stability of different T. brucei mitochondrial RNA populations. In this system, total RNA and rRNA are stable for many hours. In contrast, mRNAs can be degraded by two biochemically distinct turnover pathways. The first pathway results in the rapid degradation of mRNA (half-life [t 1/2 ] of 11 to 18 min) and is dependent upon the presence of an mRNA poly(A) tail. Remarkably, this pathway also requires the addition of UTP and therefore is termed UTP dependent. The second pathway results in slow turnover of mitochondrial mRNA (t 1/2 of ϳ3 h) and is not dependent upon the presence of an mRNA poly(A) tail or the addition of exogenous UTP. In summary, these results demonstrate the presence of a novel, UTP-dependent degradation pathway for T. brucei mitochondrial mRNAs and reveal an unprecedented role for both UTP and mRNA polyadenylation in T. brucei mitochondrial gene expression.The regulation of RNA stability plays a pivotal role in controlling gene expression in many organisms. Although RNA stability in bacteria (12), chloroplasts (23), and the eukaryotic cytoplasm (43, 47) has been extensively studied, little is known about turnover mechanisms of mitochondrial mRNAs. A common theme in all systems examined to date is the regulation of mRNA stability through polyadenylation (32). In the eukaryotic cytoplasm, polyadenylation increases the stability of mRNAs in a mechanism dependent upon the poly(A)-binding protein I (25, 48). Conversely, in bacteria and chloroplasts, polyadenylation destabilizes mRNAs and their endonucleolytic cleavage products (13,23,49). Although mitochondrial mRNAs are polyadenylated in many systems (6), little is known about the role of mitochondrial polyadenylation and its possible effect on RNA turnover. The only two studies, to our knowledge, that directly address this subject are the recent reports by Gagliardi and Leaver (18) and Lupold et al. (31), which provide evidence that polyadenylation destabilizes mitochondrial mRNAs in plants.Both mitochondrial and nuclear gene expression in the protozoan parasite Trypanosoma brucei are primarily regulated at the posttranscriptional level (42, 55). Reverse transcriptase PCR and Northern blot experiments have demonstrated that primary transcripts in the mitochondria are polycistronic and contain at least two different open reading frames (17,28,35,44,53). The generation of mature mitochondrial RNAs requires several posttranscriptional processing events, and the order of these events is variable. Polycistronic RNAs require cleavage to release individual, monocistronic RNAs with correct 5Ј and 3Ј ends. mRNAs are also modified by the addition of either a sh...

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Section: Methodsmentioning

confidence: 99%

UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

Militello

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2000

Molecular and Cellular Biology

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“…4), indicating that Sls1p is not simply a contaminant in the fraction. In addition, its nucleoid association was dependent on the presence of an energy-regenerating system that is needed for optimal in organello translation under these conditions (24). Furthermore, in the rpo41⌬3 strain, a mutant in which the ATD is deleted and does not interact with Nam1p (10), Sls1p no longer associated with the nucleoid fraction (Fig.…”

Section: Sls1p and Nam1p Collaborate In A Pathway Required Formentioning

confidence: 98%

“…Mitochondria competent for in organello translation were prepared and stored as described (24). Mitochondria (2 mg of mitochondrial protein/ sample) were thawed, and in organello translation reactions were performed as described (24), except 20 mM HEPES was substituted for Tris in the protein synthesis medium.…”

Section: Translation In Organello and Isolation Of Mitochondrial Nuclmentioning

confidence: 99%

“…Mitochondria (2 mg of mitochondrial protein/ sample) were thawed, and in organello translation reactions were performed as described (24), except 20 mM HEPES was substituted for Tris in the protein synthesis medium. ATP, GTP, ␣-ketoglutarate, phosphoenolpyruvate, and pyruvate kinase (all obtained from Sigma) comprise the energy-regenerating system in the protein synthesis medium.…”

Section: Translation In Organello and Isolation Of Mitochondrial Nuclmentioning

confidence: 99%

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Multiple Interactions Involving the Amino-terminal Domain of Yeast mtRNA Polymerase Determine the Efficiency of Mitochondrial Protein Synthesis

Rodeheffer

Shadel

2003

Journal of Biological Chemistry

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The amino-terminal domain (ATD) of Saccharomyces cerevisiae mitochondrial RNA polymerase has been shown to provide a functional link between transcription and post-transcriptional events during mitochondrial gene expression. This connection is mediated in large part by its interactions with the matrix protein Nam1p and, based on genetic phenotypes, the mitochondrial membrane protein Sls1p. These observations led us to propose previously that mtRNA polymerase, Nam1p, and Sls1p work together to coordinate transcription and translation of mtDNA-encoded gene products. Here we demonstrate by specific labeling of mitochondrial gene products in vivo that Nam1p and Sls1p indeed work together in a pathway that is required globally for efficient mitochondrial translation. Likewise, mutations in the ATD result in similar global reductions in mitochondrial translation efficiency and sensitivity to the mitochondrial translation inhibitor erythromycin. These data, coupled with the observation that the ATD is required to co-purify Sls1p in association with mtDNA nucleoids, suggest that efficient expression of mtDNA-encoded genes in yeast involves a complex series of interactions that localize active transcription complexes to the inner membrane in order to coordinate translation with transcription.The ϳ80-kb Saccharomyces cerevisiae mitochondrial genome encodes seven oxidative phosphorylation subunits destined for the inner mitochondrial membrane, one ribosomal protein, two rRNAs, and a full complement of tRNAs (1). Transcription of yeast mtDNA is initiated at multiple promoters by a dedicated mtRNA polymerase that is encoded by the RPO41 gene (2) and homologous to the bacteriophage T7 family of RNA polymerases (3). Most mitochondrial transcripts are polycistronic and require extensive processing to release the mature RNA species for proper gene expression (4). In addition, introns are often present in certain messages that also must be excised. In the case of the COX1 and COB genes, this is a complex process that requires intron-encoded maturases that help catalyze splicing (5, 6). Once fully processed, mRNAs are translated within the matrix by ribosomes associated with the mitochondrial inner membrane (7,8). Finally, the newly synthesized mtDNA-encoded proteins are assembled with the corresponding nucleus-encoded subunits to form the oxidative phosphorylation enzyme complexes in the inner membrane. The execution and coordination of these events are necessary for normal cellular respiration.We previously examined the mechanism of mitochondrial gene expression in the budding yeast S. cerevisiae and elucidated a pathway of transcription-coupled events that involves an amino-terminal domain (ATD) 1 of mtRNA polymerase (9 -11). Over the course of these experiments, several mutations in the yeast mtRNA polymerase ATD have been characterized, including two deletion mutations, rpo41⌬2 and rpo41⌬3, and several single or double point mutations, which result in mitochondrial petite phenotypes and defects in mitochondrial RNA processing...

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“…Subunits I, II, and III are products of mitochondrial genes (COX1, COX2, and COX3, respectively) (1,6) and make up the catalytic core of the enzyme (7,8). All three of these polypeptides are translated on mitochondrial ribosomes and are inserted co-translationally into the mitochondrial inner membrane (9), a process termed mitochondrial export (10,11). Subunits IV, V, Va or Vb, VI, VII, and VIIa are encoded by nuclear COX genes (COX4, COX5a or 5b, COX6, COX7, COX8, and COX9, respectively).…”

mentioning

confidence: 99%

A Role for Pet100p in the Assembly of Yeast Cytochrome c Oxidase

Church¹,

Goehring

Forsha

et al. 2005

Journal of Biological Chemistry

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[4] In Organello mitochondrial protein and RNA synthesis systems from Saccharomyces cereuisiae

Cited by 12 publications

References 17 publications

UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

Multiple Interactions Involving the Amino-terminal Domain of Yeast mtRNA Polymerase Determine the Efficiency of Mitochondrial Protein Synthesis

A Role for Pet100p in the Assembly of Yeast Cytochrome c Oxidase

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