Mitochondrial RNA Polymerase Activity Research Articles

Decreasing mitochondrial RNA polymerase activity reverses biased inheritance of hypersuppressive mtDNA

Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that some regions of rho+ mtDNA persisted while others were lost after a specific hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced biased inheritance of a subset of hypersuppressive genomes. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance over a specific hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.

A study on the human mitochondrial RNA polymerase activity points to existence of a transcription factor B-like protein

In the present work, the RNA polymerase activity of the human mitochondrial RNA polymerase mature protein (h-mtRPOLm) is shown, and its molecular activity calculated (2.1±0.9 min−1). An activity analysis of h-mtRPOLm and deleted versions of it has demonstrated that the entire recombinant protein is required for this activity. In addition, h-mtRPOLm alone or in presence of the known mitochondrial transcription factors (human mitochondrial transcription factor A and/or human mitochondrial transcription termination factor) is not able to initiate transcription from the specific human mitochondrial promoters pointing to the existence of a human mitochondrial transcription factor B-like protein.

Identification of a Mitochondrial RNA Polymerase in the CrustaceanArtemia franciscana

Mitochondrial RNA polymerase activity has been isolated from the crustaceanArtemia franciscanaat two stages of development, dormant embryo and developing larva. The preparations were obtained from purified mitochondria and the polymerase activity was purified by heparin–Sepharose chromatography. The presumed polymerase has a molecular mass of about 120 kDa and a 7.4S sedimentation coefficient. The biochemical characterization of the enzymatic reaction identified our RNA polymerase preparations as mitochondrial. The transcription initiation sites ofArtemiamtDNA were characterized recently in our laboratory (J. A. Carrodeguas and C. G. Vallejo,Eur. J. Biochem.250, 514–523, 1997).ArtemiamtDNA fragments comprising the transcription initiation sites were transcribed by the partially purified polymerase preparation from the two developmental stages, but the transcription turned out to be unspecific. DNAse I footprinting analysis of a main transcription initiation site-containing DNA fragment revealed a protected region around the initiation site +1 position, when using a crude polymerase preparation. However, the protected region was not observed with the purified preparation. The results altogether suggest that a specificity factor is lost during purification. Based on the footprinting data, we suggest that the sequence from positions −6 to +13 of the main transcription initiation site in theArtemiamitochondrial DNA is the binding site of the homologous RNA polymerase holoenzyme.

The Synthesis of mRNA in Isolated Mitochondria can be Maintained for Several Hours and is Inhibited by High Levels of ATP

The dependence for the maintenance of the synthesis and maturation of mitochondrial RNA on the supply of nucleo-cytoplasmic factors has been investigated by a novel in organello RNA synthesis system. We found that mitochondrial DNA transcription can be maintained for several hours in isolated mitochondria. Analysis of the individual mitochondrial RNA species revealed that: the processing of the rRNA precursors and the stability of the mature rRNAs, but not the transcription itself, is severely impaired after short periods of incubation, indicating that these processes are strongly dependent on the mitochondrial interaction with the nucleo-cytoplasmic compartment; the events that lead to the synthesis, processing and turnover of the mitochondrial mRNAs do not require the continuous supply of nucleo-cytoplasmic factors, that are accumulated in excess by mitochondria. Furthermore, we present evidence indicating an inhibition of high ATP levels on the mitochondrial RNA polymerase activity, both in organello and in vitro. Consequently, it is proposed that mitochondrial mRNA synthesis can be regulated in response to changes in intramitochondrial ATP levels. This regulation of mitochondrial mRNA synthesis together with their very rapid turnover described here and elsewhere [Gelfand, R. & Attardi, G. (1981) Mol. Cell Biol. 1, 497-511], could represent a mechanism that would allow each individual mitochondrion to adjust its optimal levels of mRNA, and hence its translation capacity, in response to local energetic demands.

In vitro transcription of human mitochondrial DNA. Identification of specific light strand transcripts from the displacement loop region.

The displacement loop region of human mitochondrial DNA contains the origin of heavy strand DNA replication and is the most likely site of promotion of transcription of both heavy and light strands. In order to identify relevant control regions for initiation of transcription, a partially purified human mitochondrial RNA polymerase activity was isolated and utilized in a runoff transcription assay using a cloned portion of the displacement loop region as the DNA template. Analysis of the transcription products from differentially cleaved DNA templates reveals that specific light strand transcripts are synthesized and no heavy strand transcripts are detectable. The 5' ends of the light strand transcripts map within a unique trinucleotide site on the heavy strand template at a position which overlaps the pentanucleotide map position of the 5' ends of in vivo 7 S RNA light strand transcripts. By using templates that have been truncated at the 5' or 3' end, an upper limit on the size of template sequence required for synthesis of the specific light sequence required for synthesis of the specific light strand transcripts can be defined as the 433-nucleotide genomic region between the 5' 10 nucleotides of the 12 S rRNA gene and a BalI restriction site in the displacement loop region that is 352 nucleotides from the gene boundary for tRNAPhe. Two of the previously identified conserved sequences of the mammalian displacement loop region are not required for synthesis of the light strand transcripts. The location of the in vitro light strand transcripts is consistent with a functional role in either in vivo transcription or priming of heavy strand DNA replication.

Effect of aldosterone on ribonucleic acid polymerase activity in rat kidney cortical and medullary mitochondria

The involvement of mitochondria in the mechanism of action of aldosterone is suggested by the ability of this hormone to alter the activity of certain mitochondrial enzymes. Since the mitochondrion is able to synthesize some of its own macromolecules, we investigated whether the mineralocorticoid effects mitochondrial RNA synthesis in isolated kidney mitochondria. Rat kidney mitochondria were purified and then separated into two populations by sucrose density gradient centrifugation; mitochondria sedimenting at 1.2 M sucrose were predominantly of medullary origin, while mitochondria sedimenting at 1.4 M sucrose were mainly from the cortex. RNA polymerase activity in medullary mitochondria was almost twice that of cortical mitochondria. The activity was characterized as α-amanitin resistant and rifampicin sensitive DNA-dependent RNA polymerase. Adrenalectomy resulted in a 40 per cent decrease in the RNA polymerase activity of cortical mitochondria 5 days later (P < 0.001), but no decrease occurred in medullary mitochondrial activity, compared to sham-operated controls. Three hr after aldosterone administration ( 2 μmg/100 g body wt, i.p.) the RNA polymerase activities of medullary and cortical mitochondria were increased 20 and 50 per cent, respectively, above the adrenalectomized values (P < 0.05, P < 0.001). Spironolactone, an antagonist of aldosterone (7 mg/100 g body wt, i.p., 30 min prior to aldosterone administration), blocked the stimulation of RNA polymerase by aldosterone (P < 0.05). Steroid hormones other than aldosterone did not produce significant enhancement of mitochondrial RNA polymerase activity. No change in RNA polymerase activity occurred after direct addition of aldosterone or spironolactone to the assay medium. The results suggest that the mechanism of mineralocorticoid action is at least in part mediated through its effects on mitochondrially synthesized proteins.

Germ-tube formation by atypical strains of Candida albicans

Atypical isolates of Candida albicans which failed to produce germ tubes in routine diagnostic procedures were examined for their ability to produce germ tubes in various media. Bovine serum was more effective than defined media for induction of germ tubes in the majority of isolates. A few strains formed appreciable germ tubes only in bovine serum with added thioglycollate or cysteine. One strain did not produce germ tubes in any medium. Germ-tube maturation appeared to be dependent upon mitochondrial RNA polymerase activity. The failure by an isolate to produce germ tubes, particularly in tests without strictly controlled conditions, does not preclude the possibility that the organism is C. albicans.

Some properties of rat liver mitochondrial RNA polymerase.

A rapid method suitable for purifying large amounts of mitochondria from rat liver using isopycnic zonal centrifugation is described. The RNA polymerase isolated from the purified mitochrondria was found associated with one peak when resolved by DEAE Sephadex chromatography. The enzyme was next fractionated on a phosphocellulose column followed by glycerol gradient centrifugation. A 600-fold purification was achieved when the enzyme was finally filtered through agarose gel. This final enzyme fraction consisted of one polypeptide chain as shown by polyacrylamide gel electrophoresis profiles. The enzyme has a greater preference for poly [d(A-T)] templates than for rat liver mitochondrial DNA. Inhibition of the enzyme activity required high concentrations of the inhibitors. The resistance of the enzyme to alpha-amanitin indicated that there was no contamination from nuclear RNA polymerase II. The conclusion is drawn that the mitochondrial RAN polymerase activity is associated with a single polypeptide.

Differential effects of -amanitin on RNA polymerase activity in nuclei and mitochondria.

Concentrations of α-amanitin which strongly inhibit nuclear RNA polymerase activity in presence of Mn2+ and ammonium sulfate did not produce any significant inhibition of RNA polymerase activity in mitochondria from rat brain, spleen, liver, and kidney under identical conditions. Much higher concentrations of α-amanitin, however, inhibited the mitochondrial RNA polymerase. The effects of α-amanitin on the mitochondrial RNA polymerase activity in presence of Mg2+ were similar to those on nuclear enzyme under the same conditions; in both cases only relatively very high concentrations produced significant inhibition.

Partial purification of mitochondrial RNA polymerase from rat liver.

Mitochondrial RNA polymerase activity from rat liver has previously been demonstrated in intact organelles. This activity has now been solubilized, partially purified, and shown to be a true polymerase, free of nuclease. The enzyme is derived from mitochondria and is not from contaminating bacteria or nuclear components. The enzyme is distinguished from its nuclear counterparts by its behavior on ammonium sulfate fractionation and lack of inhibition by alpha-amanitin. Rifamycin inhibits the crude enzyme, but only inconsistently inhibits the more purified preparation.