“…In contrast to reported studies (19), our results show that RNA synthesis is not limited to short lengths, as both Rpo41 and Rpo41-Mtf1 make very long RNA products, including products from rolling circle RNA synthesis.…”
Section: Rpo41 and Rpo41-mtf1 Can Initiate Transcription On Ssdnacontrasting
confidence: 99%
“…Previously, it was shown that the lengths of RNAs transcribed by Rpo41 on M13 ssDNA are between 25-100 nt (19). However, we find that the dominant products synthesized by both Rpo41 and Rpo41-Mtf1 are long RNA products; among which some migrated close to the 9 kb marker (in the Rpo41-Mtf1 reaction) and others remained closer to the wells (Fig.…”
Section: Rpo41 and Rpo41-mtf1 Can Initiate Transcription On Ssdnamentioning
confidence: 45%
“…For the yeast, both ori-sequence specific and homologous recombination mediated mechanisms have been proposed for mt replication initiation (10 -16). The S. cerevisiae mt RNA polymerase Rpo41 and its transcription factor Mtf1 have been shown to prime leading strand synthesis in vitro from dsDNA promoter with ori-sequences (3,(17)(18)(19). Similarly, strand invasion and homologous recombination has been proposed to play a role in replication initiation in the Candida albicans yeast mt DNA (15,16).…”
mentioning
confidence: 99%
“…Lagging strand initiation would require priming on ssDNA covered with the single strand binding protein (SSB). It has been shown that the S. cerevisiae Rpo41 can transcribe on ssDNA (19,20). However, Rpo41 associates with Mtf1 (21), and therefore, it is important to determine whether the Rpo41-Mtf1 complex can synthesize RNA on ssDNA and more importantly prime DNA synthesis on ssDNA covered with SSB.…”
Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10 -12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3-TCC, TTC, and TTT, and the consensus sequence is 3-Pu(Py) 2-3 . Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.
“…In contrast to reported studies (19), our results show that RNA synthesis is not limited to short lengths, as both Rpo41 and Rpo41-Mtf1 make very long RNA products, including products from rolling circle RNA synthesis.…”
Section: Rpo41 and Rpo41-mtf1 Can Initiate Transcription On Ssdnacontrasting
confidence: 99%
“…Previously, it was shown that the lengths of RNAs transcribed by Rpo41 on M13 ssDNA are between 25-100 nt (19). However, we find that the dominant products synthesized by both Rpo41 and Rpo41-Mtf1 are long RNA products; among which some migrated close to the 9 kb marker (in the Rpo41-Mtf1 reaction) and others remained closer to the wells (Fig.…”
Section: Rpo41 and Rpo41-mtf1 Can Initiate Transcription On Ssdnamentioning
confidence: 45%
“…For the yeast, both ori-sequence specific and homologous recombination mediated mechanisms have been proposed for mt replication initiation (10 -16). The S. cerevisiae mt RNA polymerase Rpo41 and its transcription factor Mtf1 have been shown to prime leading strand synthesis in vitro from dsDNA promoter with ori-sequences (3,(17)(18)(19). Similarly, strand invasion and homologous recombination has been proposed to play a role in replication initiation in the Candida albicans yeast mt DNA (15,16).…”
mentioning
confidence: 99%
“…Lagging strand initiation would require priming on ssDNA covered with the single strand binding protein (SSB). It has been shown that the S. cerevisiae Rpo41 can transcribe on ssDNA (19,20). However, Rpo41 associates with Mtf1 (21), and therefore, it is important to determine whether the Rpo41-Mtf1 complex can synthesize RNA on ssDNA and more importantly prime DNA synthesis on ssDNA covered with SSB.…”
Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10 -12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3-TCC, TTC, and TTT, and the consensus sequence is 3-Pu(Py) 2-3 . Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.
“…Mitochondrial transcription by mtRNAP is coupled to respiratory activity by a simple ATP sensing kinetic mechanism [23], while RNA degradation and translational regulation provide regulatory mechanisms necessary to fine-tune the expression of particular genes [21,[24][25][26]. Additionally, mtRNAP primes mitochondrial DNA replication [27], and is thus essential for maintaining the functional ([rho + ]) mtDNA.…”
The core mitochondrial RNA polymerase is a single-subunit enzyme, that in yeast Only the deletion of the second motif results in a partial respiratory deficiency, manifested only at the elevated temperature. Our results thus indicate that the PPR motifs do not play an essential role in the function of the NTE domain of the mitochondrial RNA polymerase.
Yeast mitochondrial genes are expressed as polycistronic transcription units that contain RNAs from different classes and show great evolutionary variability. The promoters are simple, and transcriptional control is rudimentary. Posttranscriptional mechanisms involving RNA maturation, stability, and degradation are thus the main force shaping the transcriptome and determining the expression levels of individual genes. Primary transcripts are fragmented by tRNA excision by RNase P and tRNase Z, additional processing events occur at the dodecamer site at the 3′ end of protein‐coding sequences. groups I and II introns are excised in a self‐splicing reaction that is supported by protein splicing factors encoded by the nuclear genes, or by the introns themselves. The 3′‐to‐5′ exoribonucleolytic complex called mtEXO is the main RNA degradation activity involved in RNA turnover and processing, supported by an auxiliary 5′‐to‐3′ exoribonuclease Pet127p. tRNAs and, to a lesser extent, rRNAs undergo several different base modifications. This complex gene expression system relies on the coordinated action of mitochondrial and nuclear genes and undergoes rapid evolution, contributing to speciation events. Moving beyond the classical model yeast Saccharomyces cerevisiae to other budding yeasts should provide important insights into the coevolution of both genomes that constitute the eukaryotic genetic system.
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