Gene Ontology annotation based on Enzyme Commission mapping
Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
Automatic Gene Ontology annotation based on Rhea mapping
Combined Automated Annotation using Multiple IEA Methods
Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained.
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P1 lysogens maintain the prophage as a low-copy plasmid and lose it at about 1e-5 per cell per generation; a plasmid-encoded mechanism kills the cells that lose it.
"Here we show that a significant part of this remarkable stability can be attributed to a plasmid-encoded mechanism that causes death of cells that have lost P1."
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doc encodes the 126-residue killer and phd the 73-residue protector; the two form an operon with translational coupling.
"The two genes constitute an operon and the synthesis of Doc appears to be translationally coupled to that of Phd."
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Killing by Doc is independent of the SOS response and of recA.
"Expression of doc is not SOS-inducing and killing by Doc is recA-independent."
Corepression of the P1 addiction operon by Phd and Doc.
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Doc does not bind DNA. The regulation-competent, non-toxic DocH66Y protein failed to bind DNA in vitro while strongly enhancing Phd's operator binding.
"DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator."
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Doc deepens Phd-mediated repression of the operon about fourfold in vivo.
"transcription was repressed 40-fold, while in the presence of Phd, transcription was repressed only 10-fold, indicating that under these conditions, Doc enhanced repression about fourfold."
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At high Doc levels the regulation inverts and the operon is partially derepressed.
"Strikingly, higher levels of Doc, obtained by adding higher concentrations of IPTG, partially derepressed the operon"
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Doc enters the Phd-operator complex, retarding it further, and mediates cooperative interaction between the two adjacent Phd-binding sites.
"Mobility of the protein-DNA complex is further retarded in the presence of Doc, indicating that Doc may directly participate in the protein-DNA complex"
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Host ClpXP degrades the antidote Phd, which is what liberates Doc in a plasmid-free segregant.
"Degradation of antidote by the host-encoded ClpXP protease liberates the toxin, which then poisons the plasmid-free segregant"
Postsegregational killing mediated by the P1 phage "addiction module" phd-doc requires the Escherichia coli programmed cell death system mazEF.
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Post-segregational killing by the P1 phd-doc module requires the host mazEF system.
"Here we show that the postsegregational killing mediated by the P1 phd-doc module depends on the presence of the E. coli mazEF system."
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Host protein synthesis is inhibited under phd-doc post-segregational killing conditions.
"we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E. coli is inhibited"
Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit.
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Inducing Doc in E. coli arrests growth and inhibits translation specifically, without perturbing transcription or replication.
"Doc expression resulted in rapid cell growth arrest and marked inhibition of translation without significant perturbation of transcription or replication."
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Unlike the mRNA interferase toxins, Doc does not cleave mRNA; it stabilises polysomes and increases mRNA half-life.
"However, Doc did not cleave mRNA as do other addiction-module toxins whose activities result in translation inhibition."
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Doc was proposed to act by binding the 30S ribosomal subunit at the hygromycin B site in helix 44, blocking elongation non-catalytically.
"Doc toxicity is the result of inhibition of translation elongation, possibly at the translocation step, through its interaction with the 30S ribosomal subunit."
Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation.
Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity.
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Monomeric Doc engages two Phd dimers at two unrelated binding sites.
"Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites."
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Doc's binding to Phd's intrinsically disordered C-terminal domain orders that domain and thereby structures Phd's N-terminal DNA-binding domain - the allosteric basis of corepression, and the reason the DNA-binding function belongs to Phd and not to Doc.
"The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain"
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Rising Doc tightens repression up to the point where toxin accumulation triggers Phd production, which is the conditional cooperativity switch.
"In this way, higher levels of Doc tighten repression up to a point where the accumulation of toxin triggers the production of Phd to counteract its action."
The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu.
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Doc is a protein kinase, not an AMPylating Fic protein, and inhibits translation by phosphorylating EF-Tu Thr382 so that EF-Tu can no longer bind aminoacylated tRNA.
"Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs."
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In a reconstituted translation system Doc had little effect on ribosomes or on preformed ternary complexes; it acts on free EF-Tu and blocks ternary complex formation.
"These results indicated that Doc does not interfere with tRNA binding to ribosomes, as was suggested earlier13 but rather inhibits formation of the ternary tRNA:EF-Tu:GTP complex."
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Doc uses either ATP or GTP as phosphate donor and turns over about 20 EF-Tu molecules per second.
"We found that Doc phosphorylated EF-Tu (using either ATP or GTP) at a relatively high rate of ~20 EF-Tu molecules per second by one molecule of Doc"
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Phd blocks de novo phosphorylation but cannot reverse it; Doc itself reverses the modification when ADP or GDP is present, suggesting a route out of dormancy.
"in the presence of ADP or GDP (the levels of which are likely elevated during dormancy), the phosphorylation of EF-Tu could be reverted"
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Translation inhibition by Doc requires no component of the translation machinery other than EF-Tu, which supersedes the earlier ribosome-occlusion model.
"Inhibition of translation by Doc is the direct result of phosphorylation of the translation elongation factor EF-Tu without the involvement of the ribosome itself or of any other player of the translation machinery."
Doc toxin is a kinase that inactivates elongation factor Tu.
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Doc transfers the gamma- and not the alpha-phosphate of ATP, so it is a kinase rather than an adenylyltransferase; the activity is lost in the Fic-motif mutant H66Y and is blocked by Phd.
"potent phosphorylation of a single product migrating just above 40 kDa was observed, indicating that Doc is in fact a kinase"
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EF-Tu is the only Doc substrate detectable in E. coli whole-cell lysate.
"EF-Tu is the sole target of Doc toxin."
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Mass spectrometry at 100% EF-Tu coverage, searched for phosphoserine, phosphothreonine and phosphotyrosine, gave a single site, Thr-382, and the T382A mutant is not a substrate - so the only demonstrated acceptor residue is a threonine.
"Sample analysis resulted in 100% protein coverage and yielded a single EF-Tu phosphorylation site in this Doc-treated sample at Thr-382"
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Kirromycin, which traps EF-Tu on the ribosome and binds near Thr-382, completely blocks phosphorylation by Doc.
"We observed complete inhibition of phosphorylation by kirromycin"
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The degenerate Fic motif of the Doc family is what distinguishes these kinases from the AMPylating Fic proteins, so Doc-family toxins are predicted to be kinases generally.
"By extension, all Doc toxins with this Fic domain are predicted to function as kinases as well."