Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7

Masters, Brian S.; Stohl, Lori L.; Clayton, David A.

doi:10.1016/0092-8674(87)90013-4

Cited by 398 publications

(240 citation statements)

References 43 publications

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“…Several key enzymes involved in mtDNA maintenance are closely related to their prokaryotic counterparts; these include mitochondrial RNA polymerase (3,21), SSB protein (4,9,20,31), the high-mobility-group-box DNA binding protein mitochondrial transcription factor A (25), and the catalytic subunit of DNA pol ␥ (17,26,27,34). Nevertheless, the observation that the small subunit of DNA pol ␥ is related to prokaryotic aminoacyl-tRNA synthetases is surprising, since the latter represent a different class of enzyme involved in nucleic acid metabolism.…”

Section: Resultsmentioning

confidence: 99%

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

Carrodeguas

Kobayashi

Lim

et al. 1999

Molecular and Cellular Biology

102

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Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase ␥ (pol ␥) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol ␥ have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol ␥ purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol ␥ stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions. Mitochondrial DNA (mtDNA) is replicated by a DNA polymerase, DNA polymerase ␥ (pol ␥), that is distinct from nuclear DNA polymerases ␣, ␤, ␦, ε, and . Since DNA pol ␥ represents only a small fraction of total cellular DNA polymerase, purification and characterization of the subunit composition of this enzyme have been difficult. Molecular cloning has contributed greatly to understanding the structure of DNA pol ␥ in different organisms. The catalytic subunits of DNA pol ␥ have been cloned for several organisms (6,16,17,27,34) and have been found to resemble family A of DNA polymerases, related to Escherichia coli DNA pol I. In Saccharomyces cerevisiae DNA pol ␥ is composed of a single polypeptide, while in Drosophila melanogaster DNA pol ␥ is comprised of two different polypeptides, a catalytic subunit of 125 kDa and an accessory subunit of 41 kDa (24, 33). The Drosophila subunits copurify and have been shown to interact, but the recombinant proteins have not yet been shown to be functional. The function of the small subunit, which we refer to as pol ␥B, is unknown. It has been proposed to influence the processivity of the catalytic subunit. Putative mammalian homologs of the Drosophila accessory subunit have been identified in sequence databases. One published purification scheme for human DNA pol ␥ suggested the existence of a small subunit (11), but the potential relationship between this polypeptide and Drosophila pol ␥B has not been established. Recently, the catalytic subunit of human DNA pol ␥ was expressed in an active form (10,19). Surprisingly, the recombinant catalytic subunit alone displayed most of the characteristics of the enzyme purified from human cells, which did not appear to contain a stoichiometric amount of a small subunit. Thus, it is not clear what role, if any, is played by putative human DNA pol ␥B.We hav...

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Section: Resultsmentioning

confidence: 99%

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

Carrodeguas

Kobayashi

Lim

et al. 1999

Molecular and Cellular Biology

102

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“…In this study, we demonstrated that spontaneous suppressor mutations partially rescuing the functioning of the mitochondrial genetic system in degradosome-deficient suv3⌬ strains occur in the MTF1 (YMR228W) and RPO41 (YFL036W) genes encoding the two subunits of the mitochondrial RNA polymerase (Kelly et al, 1986;Masters et al, 1987;Jang and Jaehning, 1991;Ulery and Jaehning, 1994). They are temperature-sensitive, recessive, partial loss-offunction (hypomorphic) mutations that reduce the transcrip- tion rate at normal temperature and nearly abolish the mitochondrial RNA polymerase function at elevated temperature.…”

Section: Discussionmentioning

confidence: 99%

“…The SDB9 strain, compared with the parental strain BWG1, contained the G2932T substitution in the RPO41 gene, resulting in the V978F mutation in the amino acid sequence. Neither of these mutations has been previously described, they are both located in regions of the Rpo41p sequence displaying homology to the T3 and T7 phage RNA polymerases (Masters et al, 1987). .…”

Section: The Suppressor Mutations Are Novel Single Amino Acid Substitmentioning

confidence: 99%

“…The posttranscriptional mechanisms affecting mitochondrial gene expression, including RNA turnover, are of particular importance because transcriptional control is relatively simple and rudimentary. The single RNA polymerase (RNAP) of Saccharomyces cerevisiae mitochondria is composed of only two nuclear-encoded protein subunits-the core enzyme encoded by the RPO41 gene and a transcription initiation factor encoded by the MTF1 gene (Masters et al, 1987;Jang and Jaehning, 1991). The RNAP holoenzyme recognizes a simple nonanucleotide promoter sequence (Osinga et al, 1982;Mangus et al, 1994) and initiates synthesis of at least 13 primary multicistronic transcripts that undergo extensive processing to form mature RNAs (Christianson and Rabinowitz, 1983;Tzagoloff and Myers, 1986;Foury et al, 1998;Gagliardi et al, 2004;Schafer, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

Rogowska

Puchta

Czarnecka

et al. 2006

MBoC

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The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3 -to-5 exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3⌬) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3⌬ strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation. INTRODUCTIONThe degradation of RNA is an essential element in the expression of genetic information. It is required to control RNA abundance and thus gene expression and to eliminate aberrant or defective molecules that inevitably form during RNA synthesis and maturation (RNA surveillance) (Vasudevan and Peltz, 2003). The posttranscriptional mechanisms affecting mitochondrial gene expression, including RNA turnover, are of particular importance because transcriptional control is relatively simple and rudimentary. The single RNA polymerase (RNAP) of Saccharomyces cerevisiae mitochondria is composed of only two nuclear-encoded protein subunits-the core enzyme encoded by the RPO41 gene and a transcription initiation factor encoded by the MTF1 gene (Masters et al., 1987;Jang and Jaehning, 1991). The RNAP holoenzyme recognizes a simple nonanucleotide promoter sequence (Osinga et al., 1982;Mangus et al., 1994) and initiates synthesis of at least 13 primary multicistronic transcripts that undergo extensive processing to form mature RNAs (Christianson and Rabinowitz, 1983;Tzagoloff and Myers, 1986;Foury et al., 1998;Gagliardi et al., 2004;Schafer, 2005). Such organization leaves little room for regulation at the transcription initiation level and makes posttranscriptional processes, including RNA degradation, key control points for mitochondrial gene expression.The enzymes controlling RNA turnover in cells and organelles show great evolutionary divergence and are only partially conserved between mitochondria of different organisms (Gagliardi et al., 2004). The enzymatic activity responsible for turnover is, however, on the basic level, similar in all the systems discovered so far-it is that of a 3Ј-to-5Ј processive exoribonuclease, either hydrolytic or phosphorolytic. In most cases, the exoribonuclease activity is contained in a larger multiprotein complex that, in addition to exoribonucleases, also contains RNA helicases, and in certain cases, endonucleases. A model example of such a complex is the eubacterial degradosome (Carpousis, 2002).The first mitoc...

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“…The yeast mtRNA polymerase represents a nuclear encoded single subunit enzyme of 145 kDa which requires a specifity factor for promoter recognition, also coded in the nucleus (4)(5). The core polymerase displays strong homology with bacteriophage RNA polymerases (6). No eubacteria-like polymerase genes have been found in any of the mitochondrial genomes sequenced so far, including the first total sequence of a higher plant mitochondrial genome from Arabidopsis thaliana (7).…”

Section: Introductionmentioning

confidence: 99%

Cloning and characterization of a cDNA encoding a bacteriophage-type RNA polymerase from the higher plant Chenopodium album

Weihe

Hedtke²,

Börner³

1997

Nucleic Acids Research

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We have cloned a full-length cDNA from the higher plant Chenopodium album coding for a single subunit bacteriophage-type RNA polymerase. The cDNA isolated from an actively growing cell suspension culture recognized a 3.8 kb transcript on Northern blots. The open reading frame comprises 987 amino acids with a predicted molecular mass of 112 kDa. A comparison of the protein sequence with those of the two known fungal mitochondrial RNA polymerases, from Saccharomyces cerevisiae and Neurospora crassa, reveals extensive homology between the three enzymes. with complete conservation of all catalytically essential amino acids. The putative mitochondrial RNA polymerase from C.album, as well as homologous sequences from rice and barley, which have been partially cloned, lack two catalytically non-essential regions of up to 176 amino acids near the C-terminus present in the two fungal mitochondrial RNA polymerases. The extreme N-terminus of the cloned C.album RNA polymerase displays features of a potential mitochondrial transit sequence. In phylogenetic trees constructed to compare the evolutionary relationships between the different single subunit RNA polymerases the C.album sequence forms a subgroup together with the S.cerevisiae and the N.crassa mitochondrial RNA polymerases, well separating from both bacteriophage enzymes and plasmid-encoded RNA polymerases found in mitochondria of many fungi and some higher plants.

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Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7

Cited by 398 publications

References 43 publications

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

Cloning and characterization of a cDNA encoding a bacteriophage-type RNA polymerase from the higher plant Chenopodium album

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