scholarly journals Glucose repression of yeast mitochondrial transcription: kinetics of derepression and role of nuclear genes.

1994 ◽  
Vol 14 (2) ◽  
pp. 1160-1170 ◽  
Author(s):  
T L Ulery ◽  
S H Jang ◽  
J A Jaehning
Keyword(s):  
Rna Polymerase ◽  
Glucose Repression ◽  
Nuclear Gene ◽  
Transcript Abundance ◽  
Nuclear Genes ◽  
Mitochondrial Rna ◽  
Pulse Labelling ◽  
Mitochondrial Dna Copy Number ◽  
Mitochondrial Transcript

Yeast mitochondrial transcript and gene product abundance has been observed to increase upon release from glucose repression, but the mechanism of regulation of this process has not been determined. We report a kinetic analysis of this phenomenon, which demonstrates that the abundance of all classes of mitochondrial RNA changes slowly relative to changes observed for glucose-repressed nuclear genes. Several cell doublings are required to achieve the 2- to 20-fold-higher steady-state levels observed after a shift to a nonrepressing carbon source. Although we observed that in some yeast strains the mitochondrial DNA copy number also increases upon derepression, this does not seem to play the major role in increased RNA abundance. Instead we found that three- to sevenfold increases in RNA synthesis rates, measured by in vivo pulse-labelling experiments, do correlate with increased transcript abundance. We found that mutations in the SNF1 and REG1 genes, which are known to affect the expression of many nuclear genes subject to glucose repression, affect derepression of mitochondrial transcript abundance. These genes do not appear to regulate mitochondrial transcript levels via regulation of the nuclear genes RPO41 and MTF1, which encode the subunits of the mitochondrial RNA polymerase. We conclude that a nuclear gene-controlled factor(s) in addition to the two RNA polymerase subunits must be involved in glucose repression of mitochondrial transcript abundance.

Glucose repression of yeast mitochondrial transcription: kinetics of derepression and role of nuclear genes

1994 ◽  
Vol 14 (2) ◽  
pp. 1160-1170
Author(s):  
T L Ulery ◽  
S H Jang ◽  
J A Jaehning
Keyword(s):  
Rna Polymerase ◽  
Glucose Repression ◽  
Nuclear Gene ◽  
Transcript Abundance ◽  
Nuclear Genes ◽  
Mitochondrial Rna ◽  
Pulse Labelling ◽  
Mitochondrial Dna Copy Number ◽  
Mitochondrial Transcript

Yeast mitochondrial transcript and gene product abundance has been observed to increase upon release from glucose repression, but the mechanism of regulation of this process has not been determined. We report a kinetic analysis of this phenomenon, which demonstrates that the abundance of all classes of mitochondrial RNA changes slowly relative to changes observed for glucose-repressed nuclear genes. Several cell doublings are required to achieve the 2- to 20-fold-higher steady-state levels observed after a shift to a nonrepressing carbon source. Although we observed that in some yeast strains the mitochondrial DNA copy number also increases upon derepression, this does not seem to play the major role in increased RNA abundance. Instead we found that three- to sevenfold increases in RNA synthesis rates, measured by in vivo pulse-labelling experiments, do correlate with increased transcript abundance. We found that mutations in the SNF1 and REG1 genes, which are known to affect the expression of many nuclear genes subject to glucose repression, affect derepression of mitochondrial transcript abundance. These genes do not appear to regulate mitochondrial transcript levels via regulation of the nuclear genes RPO41 and MTF1, which encode the subunits of the mitochondrial RNA polymerase. We conclude that a nuclear gene-controlled factor(s) in addition to the two RNA polymerase subunits must be involved in glucose repression of mitochondrial transcript abundance.

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

1986 ◽  
Vol 6 (7) ◽  
pp. 2543-2550
Author(s):  
D F Bogenhagen ◽  
B K Yoza
Keyword(s):  
Mitochondrial Dna ◽  
Xenopus Laevis ◽  
Rna Polymerase ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Xenopus Laevis Oocytes ◽  
Mitochondrial Rna ◽  
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.

scholarly journals Anti-viral nucleotide incorporation by recombinant human mitochondrial RNA polymerase is predictive for increased in vivo mitochondrial toxicity risk

2016 ◽  
pp. AAC.01253-16 ◽  
Author(s):  
Martijn Fenaux ◽  
Xiaodong Lin ◽  
Fumiaki Yokokawa ◽  
Zachary Sweeney ◽  
Oliver Saunders ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mitochondrial Dysfunction ◽  
Elevated Serum ◽  
Nucleoside Analogs ◽  
Hepatic Function ◽  
Gene Signature ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B and - since the FDA approval of sofosbuvir in 2013 - also for hepatitis C (HCV). However, many promising nucleotides have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of1, a monophosphate prodrug of a 2’ -ethynyluridine developed for the treatment of HCV.1inhibits multiple HCV genotypes in vitro (EC50= 0.05-0.1 μM) with a selectivity index of >300 (CC50= 30 μM in MT-4 cells). The active triphosphate metabolite of1,2, does not inhibit human α, β or γ DNA polymerases, but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of1resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of1as an anti-HCV compound was terminated.

scholarly journals The human mitochondrial 12S rRNA m4C methyltransferase METTL15 is required for mitochondrial function

2020 ◽  
Vol 295 (25) ◽  
pp. 8505-8513 ◽  
Author(s):  
Hao Chen ◽  
Zhennan Shi ◽  
Jiaojiao Guo ◽  
Kao-jung Chang ◽  
Qianqian Chen ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Small Subunit ◽  
12S Rrna ◽  
Mitochondrial Rna ◽  
Small Subunit Rrna ◽  
Mitochondrial Ribosomes

Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro. We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes.

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

2011 ◽  
Vol 286 (18) ◽  
pp. 16109-16120 ◽  
Author(s):  
Swaroopa Paratkar ◽  
Aishwarya P. Deshpande ◽  
Guo-Qing Tang ◽  
Smita S. Patel
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Full Length ◽  
Mitochondrial Rna ◽  
Terminal Domain ◽  
Mitochondrial Rna Polymerase

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 ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-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.

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

1986 ◽  
Vol 6 (7) ◽  
pp. 2543-2550 ◽  
Author(s):  
D F Bogenhagen ◽  
B K Yoza
Keyword(s):  
Mitochondrial Dna ◽  
Xenopus Laevis ◽  
Rna Polymerase ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Xenopus Laevis Oocytes ◽  
Mitochondrial Rna ◽  
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.

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.

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