Rpo41 is the nuclear-encoded, single-subunit, bacteriophage T7-like DNA-directed RNA polymerase of Schizosaccharomyces pombe mitochondria. It is imported into mitochondria via an N-terminal transit peptide and, together with the transcription factor Mtf1 as a holoenzyme, recognizes the core promoter element of the mitochondrial promoters (Pma, Pmi, Pin) and initiates transcription at the same start sites used in vivo. Unlike its budding-yeast ortholog, purified S. pombe Rpo41 can weakly bind promoters and initiate transcription on its own, with Mtf1 strongly stimulating both. Rpo41 is required for transcription of the mitochondrial genome and, because S. pombe is petite-negative, for viability.
Existing Annotations Review
GO Term
Evidence
Action
Reason
GO:0001018 mitochondrial promoter sequence-specific DNA binding
EXP PMID:21357609 Identification and characterization of the mitochondrial RNA...
ACCEPT
Summary: Purified Rpo41 binds S. pombe mitochondrial promoters in EMSA; binding is weak alone, strong with Mtf1, and depends on the core promoter sequence.
Reason: Direct biochemical evidence on the S. pombe protein. Rpo41 alone gives a weak retarded species on Pma and Pmi, Mtf1 converts this to a strong super-shift, and three-base mutations in the core promoter element abolish or reduce binding, so the binding is sequence-specific. Promoter recognition is a property of the Rpo41-Mtf1 holoenzyme, but Rpo41 itself contacts promoter DNA, so 'enables' is appropriate.
Mtf1 alone did not cause a detectable shift of the Pma, Pmi and Pin promoter DNA but Rpo41 could generate a weak retarded species in the gel on its own with Pma, Pmi and Pin promoter DNA
Mtf1 and Rpo41 with the mutant promoters exhibited a significantly reduced band shift compared to the wild-type promoters (Figure 4). This suggests that Mtf1 and Rpo41 bind to the promoters specifically and that the core sequences of the promoters are required for recognition by Mtf1 and Rpo41.
GO:0001018 mitochondrial promoter sequence-specific DNA binding
IBA GO_REF:0000033
ACCEPT
Summary: Phylogenetic inference of mitochondrial promoter binding, placed at a node whose only experimental donor is rpo41 itself.
Reason: The PAINT node (PTN000010147) is distinct from, and shallower than, the node carrying polymerase activity and mitochondrial transcription (PTN000010146), so curators deliberately restricted promoter-binding transfer. rpo41's own experimental annotation is the grounding donor, which is expected and not circular. Consistent with the direct EMSA evidence.
Mtf1 and Rpo41 with the mutant promoters exhibited a significantly reduced band shift compared to the wild-type promoters (Figure 4). This suggests that Mtf1 and Rpo41 bind to the promoters specifically and that the core sequences of the promoters are required for recognition by Mtf1 and Rpo41.
Summary: InterPro2GO DNA binding from the phage-type RNA polymerase signature.
Reason: Correct but general. The S. pombe protein has experimentally shown sequence-specific binding to mitochondrial promoters (GO:0001018, already annotated), which is a descendant of DNA binding and the informative term.
Mtf1 and Rpo41 with the mutant promoters exhibited a significantly reduced band shift compared to the wild-type promoters (Figure 4). This suggests that Mtf1 and Rpo41 bind to the promoters specifically and that the core sequences of the promoters are required for recognition by Mtf1 and Rpo41.
EXP PMID:21357609 Identification and characterization of the mitochondrial RNA...
ACCEPT
Summary: DNA-directed RNA polymerase activity is the core catalytic activity of Rpo41.
Reason: Purified recombinant Rpo41 transcribes the Pma, Pmi and Pin mitochondrial promoters in vitro, alone at low efficiency and more efficiently with Mtf1, with start sites matching those mapped in vivo. This is direct evidence that Rpo41 is the catalytic polymerase.
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
We show biochemically, that purified Mtf1 and Rpo41 together can bind to the S. pombe mitochondrial promoters and can support transcription from the S. pombe mitochondrial promoters in vitro.
Summary: DNA-directed RNA polymerase activity is the core catalytic activity of Rpo41.
Reason: PAINT node PTN000010146 (PANTHER PTHR10102:SF0, the mitochondrial/phage single-subunit RNA polymerase subfamily that includes S. cerevisiae RPO41 and human POLRMT) carries the polymerase activity. rpo41 lies within the clade, retains the PS00900/PS00489 phage RNA polymerase signatures and the annotated active-site residues, and has direct experimental support.
Supporting Evidence:
file:SCHPO/rpo41/rpo41-uniprot.txt
DR PANTHER; PTHR10102:SF0; DNA-DIRECTED RNA POLYMERASE, MITOCHONDRIAL; 1.
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
IDA PMID:21727087 A genome wide study in fission yeast reveals nine PPR protei...
ACCEPT
Summary: Mitochondrial localization from the PomBase-curated PPR protein study.
Reason: The cached record is abstract-only; the abstract groups the mitochondrial RNA polymerase with the PPR proteins whose mitochondrial localization was examined, and the curator read the full text. Rpo41 has a predicted N-terminal transit peptide and its activity is on mtDNA promoters, so mitochondrion is biologically coherent. An earlier ORFeome screen reported Rpo41 as cytoplasmic dots, which is compatible with mitochondrial puncta and does not contradict this.
Summary: Mitochondrial matrix by curator transfer from S. cerevisiae Rpo41 (P13433).
Reason: S. cerevisiae RPO41 is in the same PANTHER subfamily (PTHR10102:SF0) and the same PAINT clade (PTN000010146). Transcription of mtDNA requires the polymerase to act in the matrix where the nucleoid resides, and no divergence in targeting is evident in rpo41, which retains an N-terminal transit peptide. This is the most precise location supported.
Supporting Evidence:
file:SCHPO/rpo41/rpo41-uniprot.txt
FT TRANSIT 1..30
file:SCHPO/rpo41/rpo41-uniprot.txt
Combines in the mitochondrion with mitochondrial transcription factor
Reason: Correct but general. Rpo41 transcribes the mitochondrial genome; mitochondrial transcription (GO:0006390), already annotated with direct support, is the specific descendant.
Transcription of the mitochondrial genes was also reduced in the rpo41Ξ cells compared to wild-type cells, but transcription of the nuclear gene Ξ± tubulin remained at similar levels in rpo41Ξ and wild-type cells (Figure 3F).
Summary: Rpo41 is the RNA polymerase that transcribes the S. pombe mitochondrial genome.
Reason: Inherited from PTN000010146 together with the polymerase activity; supported in S. pombe directly, since rpo41 deletion reduces mitochondrial transcripts while a nuclear control is unchanged, overexpression raises them, and the purified enzyme initiates at mitochondrial promoters.
Transcription of the mitochondrial genes was also reduced in the rpo41Ξ cells compared to wild-type cells, but transcription of the nuclear gene Ξ± tubulin remained at similar levels in rpo41Ξ and wild-type cells (Figure 3F).
We next showed that transcription of the mitochondrial genes was induced when Rpo41 was over expressed, while the transcription of the nuclear gene Ξ± tubulin was not effected by over expression of Rpo41 (Figure 3H).
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
Transcription of the mitochondrial genes was also reduced in the rpo41Ξ cells compared to wild-type cells, but transcription of the nuclear gene Ξ± tubulin remained at similar levels in rpo41Ξ and wild-type cells (Figure 3F).
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
GO:0006391 transcription initiation at mitochondrial promoter
IMP PMID:21357609 Identification and characterization of the mitochondrial RNA...
ACCEPT
Summary: Rpo41 initiates transcription at the mitochondrial promoters.
Reason: Rpo41 deletion reduces mitochondrial transcripts, and purified Rpo41 (alone or with Mtf1) initiates transcription at the same nucleotides as those used in vivo at Pma, Pmi and Pin; Rpo41 performs the initiation step as the catalytic subunit.
Transcription of the mitochondrial genes was also reduced in the rpo41Ξ cells compared to wild-type cells, but transcription of the nuclear gene Ξ± tubulin remained at similar levels in rpo41Ξ and wild-type cells (Figure 3F).
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
Reason: True but general: 5'-3' RNA polymerase activity is a parent of DNA-directed RNA polymerase activity (GO:0003899), which Rpo41 carries with direct experimental support. Refine to the specific template-dependent activity.
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
We also note that both Mtf1 and Rpo41 together produce a more intense supershifted band, suggesting that the Mtf1/Rpo41 holoenzyme binds to all three promoter sequences more efficiently.
file:SCHPO/rpo41/rpo41-uniprot.txt
Combines in the mitochondrion with mitochondrial transcription factor
IDA PMID:21357609 Identification and characterization of the mitochondrial RNA...
ACCEPT
Summary: Rpo41 is the catalytic subunit of the mitochondrial RNA polymerase holoenzyme with Mtf1.
Reason: Purified Rpo41 and Mtf1 together form a promoter-bound species distinct from either protein alone (super-shift), and Mtf1 stimulates Rpo41 transcription, supporting an Rpo41-Mtf1 holoenzyme.
We also note that both Mtf1 and Rpo41 together produce a more intense supershifted band, suggesting that the Mtf1/Rpo41 holoenzyme binds to all three promoter sequences more efficiently.
We show biochemically, that purified Mtf1 and Rpo41 together can bind to the S. pombe mitochondrial promoters and can support transcription from the S. pombe mitochondrial promoters in vitro.
We also note that both Mtf1 and Rpo41 together produce a more intense supershifted band, suggesting that the Mtf1/Rpo41 holoenzyme binds to all three promoter sequences more efficiently.
Core Functions
Catalytic subunit of the mitochondrial RNA polymerase: synthesizes RNA on mitochondrial DNA templates, initiating at mitochondrial promoters in a holoenzyme with Mtf1.
Figure 5B shows the DNA sequencing results and indicates that Rpo41/Mtf1 or Rpo41 alone can initiate transcription in vitro. We note that the in vitro start sites for all three promoters are exactly the same as the in vivo mapped start sites for Pma, Pmi and Pin
Transcription of the mitochondrial genes was also reduced in the rpo41Ξ cells compared to wild-type cells, but transcription of the nuclear gene Ξ± tubulin remained at similar levels in rpo41Ξ and wild-type cells (Figure 3F).
Sequence-specific recognition of mitochondrial promoter core elements, weakly on its own and strongly together with Mtf1.
Mtf1 alone did not cause a detectable shift of the Pma, Pmi and Pin promoter DNA but Rpo41 could generate a weak retarded species in the gel on its own with Pma, Pmi and Pin promoter DNA
Mtf1 and Rpo41 with the mutant promoters exhibited a significantly reduced band shift compared to the wild-type promoters (Figure 4). This suggests that Mtf1 and Rpo41 bind to the promoters specifically and that the core sequences of the promoters are required for recognition by Mtf1 and Rpo41.
Q: S. pombe Rpo41 binds and initiates at mitochondrial promoters without Mtf1 in vitro, whereas S. cerevisiae Rpo41 requires Mtf1 or pre-melted promoters. Which structural features of the S. pombe enzyme (e.g. intercalating hairpin, N-terminal domain) account for this partial factor independence, and does it operate in vivo?
Q: Does S. pombe Rpo41, like budding-yeast and human mitochondrial RNA polymerases, synthesize primers for mtDNA replication?
Suggested Experiments
Experiment: Measure promoter melting and abortive initiation by purified S. pombe Rpo41 alone versus Rpo41-Mtf1 on wild-type and core-mutant Pma templates, and test active-site mutants (residues 821, 890, 1061) for loss of activity.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Review rationale: Purified S. pombe Rpo41 binds mitochondrial promoters and supports transcription together with Mtf1 (PMID:21357609). The predicted DNA binding is biologically supported. The existing mitochondrial promoter sequence-specific DNA binding annotation is more specific within the same GO aspect, so the prediction is LSP.
Supporting Evidence:
PMID:21357609: "We show biochemically, that purified Mtf1 and Rpo41 together can bind to the S. pombe mitochondrial promoters and can support transcription from the S. pombe mitochondrial promoters in vitro."
Review rationale: Purified S. pombe Rpo41 binds mitochondrial promoters and supports transcription together with Mtf1 (PMID:21357609). The predicted DNA-templated transcription is biologically supported. The existing mitochondrial transcription annotation is more specific within the same GO aspect, so the prediction is LSP.
Supporting Evidence:
PMID:21357609: "We show biochemically, that purified Mtf1 and Rpo41 together can bind to the S. pombe mitochondrial promoters and can support transcription from the S. pombe mitochondrial promoters in vitro."
Review rationale: Purified S. pombe Rpo41 binds mitochondrial promoters and supports transcription together with Mtf1 (PMID:21357609). The predicted DNA-directed RNA polymerase complex is biologically supported. The existing mitochondrial DNA-directed RNA polymerase complex annotation is more specific within the same GO aspect, so the prediction is LSP.
Supporting Evidence:
PMID:21357609: "We show biochemically, that purified Mtf1 and Rpo41 together can bind to the S. pombe mitochondrial promoters and can support transcription from the S. pombe mitochondrial promoters in vitro."