TRANSCRIPTION OF YEAST MITOCHONDRIAL DNA11This work was supported in part by USPHS Grants HL0442 and HL09172 and Grant NP-281 from the American Cancer Society.

Levens, David; Edwards, John C.; Locker, Joseph; Lustig, Arthur J.; Merten, S; Morimoto, Richard I.; Synenki, R. M.; Rabinowitz, Murray

doi:10.1016/b978-0-12-198780-0.50024-6

Cited by 18 publications

(25 citation statements)

References 22 publications

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“…The mitochondrial (mt) 2 genome of the yeast (Saccharomyces cerevisiae) is transcribed by nuclear DNA-encoded RNA polymerase (RNAP) subunits that produce ribosomal RNAs, transfer RNA, and mRNAs of the proteins of the oxidative phosphorylation machinery (1). The yeast transcription machinery consists of the core RNAP subunit, a 153-kDa protein called Rpo41, and the transcription factor, a 40-kDa protein called Mtf1 (2)(3)(4)(5).…”

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The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

Paratkar¹,

Deshpande²,

Tang³

et al. 2011

Journal of Biological Chemistry

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Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ϳ80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ϳ50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1-270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1-380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.The mitochondrial (mt) 2 genome of the yeast (Saccharomyces cerevisiae) is transcribed by nuclear DNA-encoded RNA polymerase (RNAP) subunits that produce ribosomal RNAs, transfer RNA, and mRNAs of the proteins of the oxidative phosphorylation machinery (1). The yeast transcription machinery consists of the core RNAP subunit, a 153-kDa protein called Rpo41, and the transcription factor, a 40-kDa protein called Mtf1 (2-5). Rpo41 is evolutionarily related to the single-subunit phage T7/T3 RNAP (6). Although the ϳ80-kDa C-terminal domain of Rpo41 is highly homologous to the phage T7/T3 RNAPs and conserved across the mitochondrial RNAPs of yeasts, plants, animals, and humans, the ϳ50-kDa N-terminal domain (NTD) is only conserved in few yeasts and absent in single subunit T7/T3 RNAPs (supplemental Table 1) (6, 7).The yeast Rpo41 NTD has been implicated previously to play a role in RNA processing and translation (8). However, whether the NTD of the Rpo41 plays a role in transcription related functions has not been determined. Deletion of 1-200 amino acids (aa) causes temperature sensitive petite phenotype in S. cerevisiae and results in mitochondrial genome instability, whereas deletions beyond 200 aa result in the RPO41 null phenotype (9). Subsequently, it was shown that the region 118 -208 NTD of the Rpo41 harbors a binding site for the Nam1p protein, which is involved in ...

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“…The yeast transcription machinery consists of the core RNAP subunit, a 153-kDa protein called Rpo41, and the transcription factor, a 40-kDa protein called Mtf1 (2)(3)(4)(5). Rpo41 is evolutionarily related to the single-subunit phage T7/T3 RNAP (6).…”

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confidence: 99%

The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

Paratkar¹,

Deshpande²,

Tang³

et al. 2011

Journal of Biological Chemistry

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“…In Saccharomyces cerevisiae, release from glucose repression and the consequent increase in respiratory activity [I -31 involve important changes in mitochondrial morphology [4] and strong increases in the level of mitochondrial RNA polymerase [5] and in the rates of synthesis of mitochondrial respiratory enzymes [6]. In an attempt to elucidate the mitochondrial events accompanying glucose repression, we had previously compared the mitochondrial transcripts in growing repressed and derepressed cells; differences specific for the transcripts of various mitochondrial genes were noted [7].…”

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Mitochondrial transcription and processing of transcripts during release from glucose repression in ‘resting cells’ of Saccharomyces cerevisiae

Zennaro¹,

Grimaldi²,

Baldacci³

et al. 1985

European Journal of Biochemistry

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Mitochondria1 transcription and processing of transcripts have been investigated at different stages of release from glucose repression in resting cells of Saccharomyces cerevisiae.Transcripts were identified by hybridization with nick-translated or terminally labelled gene-specific probes. This allowed the determination of the steady-state levels of individual transcripts in the mitochondrial RNA population.Results showed different gene-specific patterns of response to respiratory induction: no increase in the level of transcripts (oxi2); a rapid increase in the steady-state levels of all transcripts (cob); a very strong increase in the processing of the high-molecular-mass precursors (oxi3 and oli2); an increase in the level of stable circular transcripts (oxi3). As a whole the results indicate specific and differentiated effects of release from glucose repression on the expression of the different mitochondrial genes and demonstrate the importance of processing events in mitochondrial regulation.In Saccharomyces cerevisiae, release from glucose repression and the consequent increase in respiratory activity [I -31 involve important changes in mitochondrial morphology [4] and strong increases in the level of mitochondrial RNA polymerase [5] and in the rates of synthesis of mitochondrial respiratory enzymes [6]. In an attempt to elucidate the mitochondrial events accompanying glucose repression, we had previously compared the mitochondrial transcripts in growing repressed and derepressed cells; differences specific for the transcripts of various mitochondrial genes were noted [7].Recovery of respiratory capacity after release from glucose repression can be achieved in resting cells, thus allowing an examination of the time course of respiratory induction under conditions which are not affected by variations in growth rate [8]. We have, therefore, analyzed the steady levels of the transcripts of various mitochondrial genes at different stages after release from glucose repression in resting cells. The results allowed the identification of some steps that are specifically affected by glucose repression. MATERIALS AND METHODS Strains and growth conditionsThe wild-type haploid strain D273-1 OB was grown aerobically at 28 "C in YP medium (1 % yeast extract, 1 YO peptone) containing 15% glucose or 2% galactose as the carbon source. Respiratory induction in resting cells was obtained under the following conditions: cells grown on 15% glucose were collected at the mid-exponential growth phase (about 8 -9 A600 n m units), washed three times and suspended at the same absorbance in the derepression buffer (67 mM phosphate pH 6.5, 2% lactate, 0.1 % glucose). As a control, a portion of the cells was suspended in the same buffer Abbreviation. DBM-paper, diazobenzyloximethyl-paper.containing 15% glucose as carbon source. Cultures were shaken vigorously at 28 "C. At various time intervals, samples were withdrawn for cell counting, respiration measurements and mitochondrial RNA (mtRNA) preparations.Respiration was measured with ...

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“…The nonanucleotide sequence is found in putative origins of replication (11) and in one case we have found that a nonanucleotide sequence from an origin region can serve as a promoter for transcriptional initiation (K. Osinga and H. Tabak, unpublished results). The mitochondrial RNA polymerase preparation used in these studies has been extensively purified (27). Still, the polynerase has not been purified to homogeneity so it is not clear whether the polymerase alone is capable of specificity or whether other modulating factors are necessary.…”

Section: Discussionmentioning

confidence: 99%

“…Mitochondrial RNA polymerase was purified as described by Levens et al., (27) MgCl2; 125pM GTP and UTP; 25pM ATP; and 0.5mg/ml rabbit serum albumin. The reactions were carried out at 30°C for 40 minutes.…”

Section: Methodsmentioning

confidence: 99%

Initiation of transcription of the yeast mitochondrial gene coding for ATPase subunit 9

et al. 1983

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We-have determined transcriptional initiation sites for the ATPase subunit 9 gene on the yeast mitochondrial genome. Using Si nuclease mapping, in vitro capping of primary transcripts with GTP and guanylyl transferase, and in vitro transcription analysis with purified mitochondrial RNA polymerase, we find the major site of transcriptional initiation to be at a point 630 nucleotides upstream of the coding region for the gene. In addition, we find much lower levels of initiation at a second site 78 nucleotides downstream of the first. Both initiation sites occur at the same position within a nonanucleotide sequence which we have previously found associated with initiation of rRNA synthesis. This work further supports the notion that this nonanucleotide sequence is an integral component of mitochondrial promoters and indicates that the same RNA polymerase is used for transcription of both mRNA and rRNA in yeast mi tochondri a.

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TRANSCRIPTION OF YEAST MITOCHONDRIAL DNA11This work was supported in part by USPHS Grants HL0442 and HL09172 and Grant NP-281 from the American Cancer Society.

Cited by 18 publications

References 22 publications

The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

Mitochondrial transcription and processing of transcripts during release from glucose repression in ‘resting cells’ of Saccharomyces cerevisiae

Initiation of transcription of the yeast mitochondrial gene coding for ATPase subunit 9

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