Gene Ontology annotation through association of InterPro records with GO terms
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
Combined Automated Annotation using Multiple IEA Methods
Holoenzyme proteins required for the physiological assembly and activity of telomerase.
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p65 is a stable component of the telomerase ribonucleoprotein, not a transient associate: anti-p65 immunodepletion clears the bulk of telomerase activity and telomerase RNA from extract.
"This finding demonstrates that both proteins are stable telomerase RNP components"
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p65 associates selectively with telomerase RNA rather than with RNA polymerase III transcripts generally, distinguishing it from a genuine La protein.
"The similarity of the TERT- and p65-associated RNA profiles suggests that both proteins are preferentially associated with telomerase RNA."
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Knockdown of p65 collapses telomerase RNA accumulation while leaving 5S rRNA, SRP RNA, U2, U3 and U6 untouched.
"whereas telomerase RNA level decreased in p65 knockdown strains, the accumulation of other RNA polymerase III transcripts was unaffected"
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TAP65 is an essential gene in Tetrahymena; complete disruption cannot be driven to fixation.
"The persistence of the wild-type loci through phenotypic assortment indicates that both p65 and p45 are encoded by essential genes"
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p65 knockdown produces the telomerase-loss phenotype of critically short telomeres.
"We found that telomeres were dramatically shorter in p65 and p45 knockdown strains"
A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase.
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Recombinant p65 binds TER directly with low-nanomolar affinity at physiological ionic strength.
"we determined that p65 bound TER with an affinity of ~2–5 nM under a range of conditions including physiological ionic strength"
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The sequence specificity of p65 maps to proximal stem IV of TER, and an unrelated structured RNA of similar size does not compete.
"These results demonstrate that p65 recognizes the sequence of proximal stem IV."
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TERT binds the p65-TER complex about thirty-fold more tightly and more homogeneously than it binds TER alone.
"TERT(1–516) bound to the p65–TER complex with ~30-fold greater affinity and more homogeneity than it bound to TER alone"
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The assembly-promoting effect of p65 requires the conserved central stem IV dinucleotide bulge of TER.
"These assays reveal that p65 promotes TERT RNP assembly by a mechanism requiring the TER central stem IV dinucleotide bulge."
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p65 does not alter the catalytic chemistry of the enzyme; primer extension by TERT reconstituted with TER is unchanged by p65 coassembly.
"Coassembly of p65 did not affect the primer-extension activity of T. thermophila TERT reconstituted with TER in RRL"
A novel RNA binding domain in tetrahymena telomerase p65 initiates hierarchical assembly of telomerase holoenzyme.
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Four separable modules of p65 contribute affinity for TER, with the C-terminal domain recognising the conserved central stem IV dinucleotide bulge.
"We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV."
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The C-terminal domain alone is necessary and sufficient for the TERT-recruitment function of p65.
"This C-terminal domain is necessary and sufficient for p65's function in enhancing the recruitment of TERT to TER."
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p65 and TERT load onto TER in a fixed order rather than cooperatively, establishing a hierarchical assembly pathway.
"we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding"
Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein.
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Single-molecule observation shows p65 binding induces a structural rearrangement of telomerase RNA that in turn directs TERT binding to form the functional ternary complex.
"interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs the binding of the telomerase reverse transcriptase to form the functional ternary complex"
An RPA-related sequence-specific DNA-binding subunit of telomerase holoenzyme is required for elongation processivity and telomere maintenance.
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RNase treatment of p65-tagged holoenzyme strips every other subunit while p65 remains, showing the rest of the particle is organised around TER.
"RNase treatment of p65fzz holoenzyme depleted all holoenzyme subunits except p65"
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The p65-TER-TERT catalytic core is a biologically stable assembly that docks onto a separate p75-p45-p19 subcomplex.
"a biologically stable assembly of the RNP catalytic core (p65-TER-TERT) associates directly with a second holoenzyme protein subcomplex (p75-p45-p19)"
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Depletion of p65, but not of p45 or p75, reduces accumulation of both TERT and TER in vivo.
"In vivo depletion of the La motif-containing p65, but not p45 or p75, reduced the biological accumulation of TERT and TER."
Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.
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p65 binds stems I and IV of TER and causes a conformational change that facilitates subsequent TERT binding.
"the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER"
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p65 narrows the conformational ensemble of TER so that TERT is presented with the active fold.
"by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER"
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Assembly-defective TER mutants become catalytically active only when p65 is supplied, which is chaperone-like rescue rather than passive stabilisation.
"Several TER mutants exhibited telomerase activity only in the presence of p65"
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p65 persists in the assembled particle and holds TER and TERT in catalytically active conformations.
"p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations"
Structural basis for telomerase RNA recognition and RNP assembly by the holoenzyme La family protein p65.
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The p65 xRRM recognises TER stem IV through combined single-stranded and double-stranded contacts, bending the RNA by about 105 degrees.
"RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER"
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p65 protects the 3' end of TER from degradation.
"it protects the 3’ end of TER from degradation"
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p65 acts as a chaperone that folds TER correctly for protein binding.
"it acts as a chaperone to correctly fold TER for protein binding"
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The telomerase catalytic core is a ternary complex of TER, TERT and p65.
"The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65."
The architecture of Tetrahymena telomerase holoenzyme.
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Negative-stain EM with subunit-specific labelling resolves p65 as a positioned component of the TERT-TER-p65 catalytic core within the intact holoenzyme.
"The EM results together with reconstituted enzyme activity assays suggest a subdivision of holoenzyme functional units: the TERT-TER-p65 catalytic core, p50-Teb1, and 7-1-4."
Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions.
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The Tetrahymena telomerase holoenzyme comprises TERT and TER plus six proteins, one of which is p65.
"In addition to TERT and TER, the Tetrahymena telomerase holoenzyme contains six other known proteins: p65, p75, p45, p19, p50, and Teb1"
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In the cryo-EM structure p65 bends the TER activating domain, facilitating assembly of TERT with TER into the RNP catalytic core.
"inducing a large bend in the RNA that facilitates assembly of TERT with TER to form the RNP catalytic core"
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The La-RRM1 module of p65 engages the 3' polyU tail of TER.
"The p65 La-RRM1 binds the 3′ polyU tail"