Terminal protein p3 (TP, gp3) is the 266-residue early protein of Bacillus phage phi29 that makes protein-primed DNA replication possible. It is covalently attached to both 5' ends of the linear phage genome through a phosphoester bond between the hydroxyl of Ser-232 and the terminal 5'-dAMP. A free molecule of TP forms a heterodimer with the phage DNA polymerase; the heterodimer recognises the origins at the two genome ends, and the polymerase deoxyadenylylates Ser-232 to give the TP-dAMP initiation product, which is then elongated as the 5' end of the nascent strand. The TP is not a passive acceptor: its C-terminal priming domain occupies the polymerase cleft that duplex DNA later uses, excludes initiation at internal genome sites, and determines which internal template thymine directs the initiating nucleotide, after which the TP-dAMP product slides back to recover the terminal nucleotide. The protein has three parts: a disordered N-terminal domain (1-73) that binds DNA without sequence specificity, an intermediate domain (74-172) that confers specificity for the polymerase, and the priming domain (173-266) carrying Ser-232. In infected Bacillus subtilis the N-terminal domain tethers both the parental genome and the priming TP/polymerase heterodimer to the bacterial nucleoid and recruits the DNA polymerase there, which is required for efficient replication in vivo; the complex later redistributes along MreB-dependent helical paths. As the covalently attached genome-end protein, p3 also compacts and, with the packaging ATPase gp16, supercoils the DNA ends before the genome is taken up by the prohead. Terminal proteins of phi29 and of several unrelated phages additionally carry nuclear localisation signals that are functional when the proteins are expressed in mammalian cells, a property of unknown significance for the phage life cycle that has been proposed to favour horizontal gene transfer to eukaryotes.
Definition: The function of a protein that supplies a hydroxyl group on one of its own amino acid side chains as the primer for DNA strand synthesis: a DNA polymerase covalently links the initiating deoxynucleoside monophosphate to that hydroxyl in a template-directed reaction, and the resulting protein-dNMP adduct is elongated as the 5' end of the nascent DNA strand, leaving the protein covalently attached to that end.
Justification: Protein priming is a distinct solution to the primer requirement of DNA polymerases, used by the terminal proteins of phi29 and related phages, of PRD1 and Bam35, of adenoviruses, and of linear plasmids of bacteria, fungi and plants. GO represents it only as a process (GO:0006269 DNA replication, synthesis of primer, whose definition assumes a short nucleotide chain made by a primase) and has no molecular function term for the priming protein itself, so terminal proteins can presently be annotated only to DNA binding or to process terms. A molecular function term would let the priming role be stated as an activity and distinguished from the polymerase's nucleotidyltransferase activity.
Parent term: molecular_function
Supporting Evidence:
Definition: The region of a host prokaryotic cell to which its DNA is confined. The host is defined as the larger of the organisms involved in a symbiotic interaction.
Justification: Phage and other prokaryote-infecting symbiont proteins that localise to the host nucleoid currently have no term to be annotated to. GO:0009295 nucleoid and GO:0043590 bacterial nucleoid describe the organism's own nucleoid, not the host's, and GO:0033643 host cell part has no nucleoid child, so the only available host-chromatin-like term is GO:0042025 host cell nucleus - which is taxonomically wrong for a bacterial host and is how phi29 terminal protein came to carry a nucleus annotation. At least two phage terminal proteins (phi29 in Bacillus subtilis and PRD1 in Escherichia coli) localise to the host nucleoid independently of other phage proteins, and the phage DNA polymerase is recruited to the same compartment.
Parent term: host cell part
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000043 | MODIFY | Summary: Keyword-derived row (KW-0238 DNA-binding). The terminal protein's disordered N-terminal domain binds double-stranded DNA in a sequence-independent manner, and this activity is the molecular basis of its nucleoid-tethering role; the DNA-binding residues have been mapped by mutagenesis. The assays are all on duplex DNA, so the row is refinable to GO:0003690. Reason: The claim is directly demonstrated rather than merely predicted, but the root term is less specific than the evidence supports. Residues 1-73 of the phi29 TP bind DNA without sequence specificity and that binding is what places the protein on the Bacillus subtilis nucleoid (PMID:20823229); mutating the basic residues of this domain correlates loss of DNA binding with loss of nucleoid localisation, loss of DNA polymerase recruitment and reduced phage DNA replication (PMID:24205926, PMID:25722367). Every binding assay behind the claim used duplex substrates: gel mobility shift assays with a 297-bp phage right-end fragment and, to establish sequence independence, a 216-bp fragment of the host chromosome, both double stranded. GO:0003690 double-stranded DNA binding is therefore the supported term, and it carries no sequence-specificity requirement. The sequence-specific descendants are what the evidence excludes: GO:0003688 DNA replication origin binding does not apply, because origin recognition at the phage DNA ends proceeds through protein-protein contact with the parental TP rather than through sequence-specific DNA binding by this domain. Single-stranded binding is not claimed and was not tested. Proposed replacements: double-stranded DNA binding Supporting Evidence: PMID:20823229 possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association PMID:20823229 only wild-type TP and the truncated mutant proteins bearing the N-terminal region were able to interact with the dsDNA fragment PMID:25722367 has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo |
| GO:0006260 DNA replication | IEA GO_REF:0000120 | TOO GENERAL | Summary: Root-level DNA replication term from the combined automated pipeline. True of the protein, but entailed by the more specific replication terms accepted elsewhere in this review. Reason: The gene product's replication role is captured at a useful level by GO:0039693 viral DNA genome replication, GO:0006270 DNA replication initiation and GO:0006269 DNA replication, synthesis of primer, all accepted here. GO:0006260 is an ancestor of all three and adds nothing beyond them; the action judges the term's informativeness, not the soundness of the claim, which is not in doubt. Supporting Evidence: PMID:22210885 the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232 |
| GO:0006269 DNA replication, synthesis of primer | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO row from IPR008770 (DNA terminal protein Gp3). The priming step of phi29 replication is exactly the step this term names: the polymerase covalently links the initiating dAMP to Ser-232 of the TP, and the TP-dAMP adduct is the primer that is then extended. Reason: The signature is this protein's own family signature, so the mapping is not an over-propagation. The term definition is written with nucleotide primers in mind (a short chain made by a primase), and GO has no term for protein priming - GO:0006269 is the only primer-synthesis term in the ontology - but the fit is substantive rather than approximate: a nucleotide (dAMP) is added on the DNA template to form the primer, the TP supplies the 3'-OH-bearing moiety that the polymerase extends, and the TP's priming domain selects which internal template nucleotide directs the reaction (PMID:19011105), so the TP does part of the work of making the primer. The missing molecular function term is filed under proposed_new_terms. Core. Supporting Evidence: PMID:16511564 The DNA polymerase of bacteriophage phi29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. PMID:19011105 we show that this domain is the main structural determinant that dictates the internal 3' nucleotide used as template during initiation |
| GO:0006270 DNA replication initiation | IDA PMID:6813861 Initiation of phage phi 29 DNA replication in vitro: formati... | ACCEPT | Summary: CACAO IDA row. Penalva and Salas 1982 is the paper that demonstrated the phi29 initiation reaction: incubating extracts of phi29-infected Bacillus subtilis with labelled dATP produces a labelled protein with p3's electrophoretic mobility, and chemical and enzymatic dissection shows that the label is a covalently bound 5'-dAMP on p3. The resulting p3-dAMP complex is elongated on addition of dNTPs. Reason: What the paper shows for this gene product is the initiation step itself, assayed directly on p3: nuclease-, phosphatase- and RNase-resistant but proteinase K-sensitive label; piperidine release of 5'-dAMP; abolition by anti-p3 serum; and a requirement for the phi29 DNA-protein p3 template. The CACAO term matches that assay at the right granularity - GO:0006270 DNA replication initiation is the process the p3-dAMP complex constitutes, and the dideoxy-terminated elongation products of 9 and 12 bases confirm that initiation occurred at the two genome ends. This is not a phenotype read as a process: the molecular event is observed, not inferred from an outcome. Core, and the same paper carries the UniProt IDA row for GO:0039693, which is also accepted. The cached record is abstract-only, so figure-level detail could not be inspected; the GONUTS wiki page that might record the student's evidence is offline. Supporting Evidence: PMID:6813861 a covalent complex between protein p3 and 5'-dAMP is formed in vitro PMID:6813861 was strongly inhibited by anti-p3 serum and required the preence of phi 29 DNA-protein p3 complex PMID:6813861 can be elongated by addition of 40 microM dNTPs |
| GO:0006270 DNA replication initiation | IMP PMID:6402761 Factors involved in the initiation of phage phi 29 DNA repli... | ACCEPT | Summary: CACAO IMP row. Blanco et al. 1983 dissected the in vitro requirements for forming the p3-dAMP initiation complex using extracts of Bacillus subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17. p3 and the phi29 DNA-protein p3 template are required alongside the gene 2 product; the products of genes 5, 6 and 17 are not. Reason: The paper's title and headline claim are about gene 2 (the DNA polymerase), which makes this row look at first like a paper about another gene - but the abstract states the requirement for this gene product explicitly, and the mutant-extract design is what makes IMP the correct evidence code. The CACAO term matches the assay: the readout is formation of the initiation complex, which is the process GO:0006270 names, so there is no phenotype-as-process or granularity problem. The row is a useful independent, mutant-based complement to the IDA row from PMID:6813861. The cached record is abstract-only (PMC holds only scanned images for this 1983 article) and GONUTS is offline, so the student's figure-level evidence could not be checked; the claim is nonetheless corroborated by the direct assay in PMID:6813861 and by the mutagenesis of the priming serine (PMID:3135531). Core. Supporting Evidence: PMID:6402761 The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 DNA-protein p3 as template. PMID:6402761 extracts from B. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used PMID:3135531 The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | REMOVE | Summary: Keyword row (KW-0378 Hydrolase) that traces to UniProt's second FUNCTION line, hydrolysis of host peptidoglycans during virus entry, cited to Moak and Molineux 2004. That attribution of the phi29 virion muralytic activity to gp3 has been superseded: the enzyme is the tail protein gp13. Reason: Moak and Molineux assayed muralytic activity associated with whole virions and placed the phi29 activity on gp3. The phi29 tail knob protein gp13 was subsequently solved at 1.8 A and shown to carry a lysozyme-like glycosidase domain and a metallo-endopeptidase domain, positioned at the distal tip of the tail where peptidoglycan is encountered (PMID:18606992); UniProt itself annotates gp13 (UniProtKB:P15132) as a lysozyme-like glycosidase plus a probable metalloendopeptidase. The p3 structure (PDB 2EX3) is a three-domain priming protein with no hydrolase fold and no candidate catalytic residues, and no hydrolytic activity has ever been assayed on purified p3. The row is an electronic keyword mapping, not an experimental annotation, and the keyword rests on an attribution that the later structural work reassigns, so removal rather than over-annotation is appropriate. Independently, bare GO:0016787 would be uninformative even if the activity were real. Supporting Evidence: PMID:18606992 whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively PMID:18606992 this enzyme is located at the distal end of the phi29 tail knob PMID:14763988 lies in the terminal protein gp3 |
| GO:0019073 viral DNA genome packaging | IDA PMID:18674782 DNA packaging motor assembly intermediate of bacteriophage p... | KEEP AS NON CORE | Summary: UniProt IDA row from the phi29 packaging-motor assembly paper. The parental TP is the covalently bound genome-end protein that the packaging machinery engages, and gp3 actively condenses the DNA ends: DNA-gp3 sediments faster than its proteinase K-treated counterpart in a gp3-dependent way, and gp3 together with the packaging ATPase gp16 supercoils the ends before the prohead takes up the genome. Reason: Participation is satisfied rather than assumed: gp3 does not merely ride along on the substrate but compacts it, and the lariat and supercoiled forms that precede efficient prohead interaction require gp3 (PMID:9086269). The cited paper is principally about gp16 oligomerisation on the prohead/pRNA, and its gp3 content is that the assembled prohead/pRNA/gp16 motor supercoils DNA-gp3; the direct gp3-dependence is in PMID:9086269, added here as a supporting reference. Kept as non-core because the protein's defining and evolutionarily primary role is priming replication, with the genome-end role in packaging a consequence of the covalent linkage established during replication. Supporting Evidence: PMID:9086269 the faster sedimentation was both gp3 and Mg2+-dependent PMID:9086269 DNA-gp3 has a maturation pathway in which the packaging proteins gp3 and gp16 supercoil the DNA ends, probably as a prerequisite for efficient interaction with the prohead. PMID:18674782 The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone |
| GO:0039693 viral DNA genome replication | IDA PMID:6813861 Initiation of phage phi 29 DNA replication in vitro: formati... | ACCEPT | Summary: UniProt IDA row on the same paper that supplies the CACAO GO:0006270 row. Formation of the covalent p3-dAMP complex in phi29-infected extracts, and its elongation into the expected terminal oligonucleotides, places p3 directly in replication of the phage genome. Reason: This is the core process term for the gene: p3 is the primer from which both strands of the phi29 genome are replicated, and the reaction was observed directly on the protein. The elongation products of 9 and 12 bases match the sequences at the left and right genome ends, so the assay reports replication of the viral genome specifically and not generic DNA synthesis. Supporting Evidence: PMID:6813861 a covalent complex between protein p3 and 5'-dAMP is formed in vitro PMID:16511564 this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome |
| GO:0039693 viral DNA genome replication | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword row (KW-1194 Viral DNA replication) for the same claim that the IDA and IMP rows on this term carry. Correct and at the right level. Reason: The keyword restates what the experimental rows on this term establish, so the electronic mapping is sound; rows sharing a term reinforce each other and this one is kept with the same action as its experimental counterparts. Core. Supporting Evidence: PMID:22210885 the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232 |
| GO:0039693 viral DNA genome replication | IMP PMID:4630506 Temperature-sensitive mutants affected in DNA synthesis in p... | ACCEPT | Summary: CACAO IMP row from the 1972 Talavera, Salas and Vinuela survey of phi29 temperature-sensitive mutants that fail to make phage DNA. Gene 3 is one of the phi29 cistrons whose mutants are DNA-negative, which is the phenotype this row records. Reason: The cached record for this paper carries no abstract at all - only title, authors, journal and DOI - and PMC has no text for it, so what the student read could not be inspected; the GONUTS wiki page for PMID:4630506, which may hold the evidence, returns only the site's temporary-offline notice. The term and evidence code are nonetheless right for the gene and for a ts-mutant DNA-synthesis screen, which is a legitimate IMP for involvement in genome replication rather than a downstream phenotype read as a process. Per the curator-deference rule this is accepted rather than left UNDECIDED, because the claim is independently established for gene 3 by the in vitro initiation assays (PMID:6813861, PMID:6402761) and by the replication defect of N-terminal-domain and priming-site mutants (PMID:20823229, PMID:3135531). Core. Re-check the cited paper's own content if GONUTS returns. Supporting Evidence: PMID:4630506 Temperature-sensitive mutants affected in DNA synthesis in phage phi29 of Bacillus subtilis. PMID:6402761 extracts from B. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used |
| GO:0039693 viral DNA genome replication | IMP PMID:820555 Bacillus subtilis phage phi29. Characterization of gene prod... | ACCEPT | Summary: CACAO IMP row from the 1976 Carrascosa et al. characterisation of phi29 gene products and functions, which assigned proteins to cistrons and identified the cistrons whose mutants fail to synthesise phage DNA. Gene 3 is among them, and this is the paper that paired the p3 band with its gene. Reason: The abstract states that mutants in six genes do not synthesise phage DNA and that ten of the seventeen cistrons were matched to their protein products, but does not name gene 3 explicitly, and the cached record is abstract-only with GONUTS offline, so the student's reading could not be verified. The claim is correct for this gene and the evidence code fits a mutant-based assignment, so the row is accepted under curator deference: gene 3 is one of the DNA-synthesis cistrons, as the companion in vitro work using gene 3 mutant extracts confirms (PMID:6402761). The term is at the right granularity - viral genome replication, not a downstream outcome such as burst size or lysis, which this paper also measures for other genes. Supporting Evidence: PMID:820555 Mutants in six genes do not synthesize phage DNA. PMID:820555 The protein products of 10 out of the 17 cistrons detected in the genome of phage phi29 have been identified. |
| GO:0042025 host cell nucleus | EXP PMID:23091024 Functional eukaryotic nuclear localization signals are wides... | MARK AS OVER ANNOTATED | Summary: UniProt experimental row for a nuclear location. The underlying experiment is heterologous: phi29 TP, like the terminal proteins of Nf, PRD1, Bam35 and Cp-1, localises to the nucleus when expressed in mammalian cells, through a bona fide NLS in residues 1-37. Mammalian cells are not phi29's host. Reason: GO:0042025 is the nucleus of the host, and the host of phi29 is Bacillus subtilis (recorded as such in the UniProt entry's host-organism line), which has no nucleus. The observation is real and interesting - it is the basis of using TP-DNA for gene delivery into eukaryotic nuclei - but it is a property of the protein in a non-host eukaryotic cell, not a location it occupies in the organism it infects. The protein's actual in-host location is the bacterial nucleoid, for which GO has no host-scoped term (GO:0009295 nucleoid and GO:0043590 bacterial nucleoid are not host-qualified, and GO:0033643 host cell part has no nucleoid child); that gap is filed under proposed_new_terms. The row is marked over-annotated rather than removed because the localisation itself is well demonstrated and the NLS is mapped by mutagenesis of Lys-25 and Lys-27, and rather than modified because no existing GO term states the claim correctly. Supporting Evidence: PMID:23091024 were found to localize to the nucleus when expressed in mammalian cells PMID:23091024 gene delivery into the eukaryotic nucleus is enhanced by the presence of PMID:20823229 associate with the host bacterial nucleoid independently of other viral-encoded proteins |
| GO:0042025 host cell nucleus | IEA GO_REF:0000044 | MARK AS OVER ANNOTATED | Summary: Subcellular-location keyword row (SL-0414 Host nucleus) propagating the same heterologous nuclear localisation as the EXP row above. Reason: Same judgment as the EXP row on this term, applied consistently: the SL-0414 assignment derives from PMID:23091024, where the TP was expressed in mammalian cells, and phi29's host is a bacterium with no nucleus. The electronic mapping from the subcellular-location vocabulary to GO is faithful; the problem is upstream, in treating a heterologous-expression result as a host location, so the row is marked over-annotated rather than removed. Supporting Evidence: PMID:23091024 were found to localize to the nucleus when expressed in mammalian cells |
| GO:0043493 viral terminase complex | IDA PMID:18674782 DNA packaging motor assembly intermediate of bacteriophage p... | KEEP AS NON CORE | Summary: UniProt IDA row placing gp3 in the viral terminase complex; the UniProt FUNCTION line calls it the small terminase protein during packaging. The experimental basis is the gp16-containing packaging machinery that engages DNA-gp3, and the co-sedimenting DNA-gp3-gp16 complexes that are packaged preferentially into proheads. Reason: The claim is defensible: phi29 has no conventional small terminase subunit, pRNA and gp3 between them supply the functions a small terminase performs, and gp16 co-sediments with DNA-gp3 in complexes that are the preferred packaging substrate (PMID:9086269). It is kept rather than accepted as core because gp3's role here is secondary to priming, and kept rather than removed or modified because the complex membership is consistent with the cited work. The granularity caveat is worth recording: the cited paper's own reconstruction is of prohead/pRNA/gp16, with DNA-gp3 as the substrate it acts on, so the part_of claim rests on the co-sedimentation data in PMID:9086269 and on the small-terminase analogy rather than on a structure that contains gp3. Supporting Evidence: PMID:9086269 cosedimented with the fast-sedimenting DNA-gp3 fragments, and the putative L-DNA-gp3-gp16 complexes were packaged preferentially into proheads in the defined in vitro system file:BPPH2/3/3-uniprot.txt Part of a DNA-gp3-gp16 complex (PubMed:9086269). |
| GO:0046718 symbiont entry into host cell | IEA GO_REF:0000043 | REMOVE | Summary: Keyword row (KW-1160 Virus entry into host cell) carried by P03681 only because of the superseded peptidoglycan-hydrolase attribution. p3 has no known role in getting the phage or its genome across the host envelope. Reason: The keyword set that generates this row (KW-1160, KW-1235, KW-1236) accompanies UniProt's FUNCTION statement about hydrolysing host peptidoglycans during virus entry, cited to PMID:14763988. The cell-wall-penetrating enzyme of phi29 is the tail knob protein gp13, which carries lysozyme-like and metallo-endopeptidase domains at the distal tail tip (PMID:18606992) and is annotated with those activities in UniProt (P15132). p3 is a virion component by virtue of being covalently attached to the genome ends, but being injected along with the DNA is not performing a step of entry, and no entry function has been assayed for it. The row is an electronic keyword mapping resting on a reassigned activity, so it is removed; the upstream fix is recorded as a source defect on the GO:0016787 row. Supporting Evidence: PMID:18606992 gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall PMID:14763988 In most cases, the lack of genetic and structural characterization of the phage precludes making a definitive identification of the enzymatic protein species. |
| GO:0098932 symbiont entry into host cell via disruption of host cell wall peptidoglycan | IEA GO_REF:0000043 | REMOVE | Summary: Keyword row (KW-1236 Degradation of host peptidoglycans during virus entry) for the specific peptidoglycan-degrading mode of entry. This is the claim that the gp13 work reassigns most directly. Reason: The term asserts that this gene product breaks down host peptidoglycan to let the phage in. No peptidoglycan hydrolase activity has been demonstrated on purified p3; the 2004 virion-level assay that attributed phi29 muralytic activity to gp3 (PMID:14763988) is superseded by the 1.8 A structure and cryoEM localisation of gp13 at the distal tail knob, which identify it as the enzyme that cleaves both the glycan backbone and the peptide cross-links (PMID:18606992). The p3 crystal structure (PDB 2EX3) has no hydrolase fold. Removed as a demonstrably misassigned electronic keyword mapping, consistently with the parent terms GO:0098994 and GO:0046718 and with GO:0016787. Supporting Evidence: PMID:18606992 gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall PMID:16511564 The 3.0 A resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. |
| GO:0098994 symbiont entry into host cell via disruption of host cell envelope | IEA GO_REF:0000043 | REMOVE | Summary: Keyword row (KW-1235 Degradation of host cell envelope components during virus entry), the envelope-level parent of the peptidoglycan term, from the same superseded attribution. Reason: Removed for the same reason as GO:0098932 and GO:0046718: the envelope-degrading activity of the phi29 virion belongs to the tail protein gp13 (PMID:18606992), not to the terminal protein, and p3 has no assayed activity on any host envelope component. Keeping the parent while removing the child would leave the same wrong claim in a vaguer form. Supporting Evidence: PMID:18606992 this enzyme is located at the distal end of the phi29 tail knob |
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Download this section (compressed HTML)Q: Should UniProt's FUNCTION statement about hydrolysing host peptidoglycans during virus entry, and the associated keywords (KW-0378, KW-1160, KW-1235, KW-1236), be moved from the phi29 terminal protein to the tail knob protein gp13, given the structural and cryoEM identification of gp13 as the peptidoglycan-degrading enzyme? Four GOA rows on P03681 derive from them.
Q: Does GO want a molecular function term for protein priming of DNA replication, and a host-scoped nucleoid cellular component term? Both gaps are currently absorbed by terms that misstate the biology: the priming role by DNA binding or by process terms, and the nucleoid location by host cell nucleus.
Q: What is the biological role, if any, of the eukaryotic-functional nuclear localisation signals found in the terminal proteins of phi29, Nf, PRD1, Bam35 and Cp-1? The proposed explanation is facilitation of horizontal gene transfer to eukaryotes, which has not been tested in a natural setting.
Suggested experts: Modesto Redrejo-Rodriguez
Q: In phi29, which protein performs the functions a small terminase would, and is the terminal protein properly a subunit of the packaging machine or only the recognition element on its substrate? pRNA, gp16 and gp3 divide these roles differently from canonical headful-packaging phages.
Suggested experts: Marc C. Morais, Shelley Grimes
Experiment: Assay purified recombinant p3 and purified gp13 side by side on Bacillus subtilis peptidoglycan sacculi and on dye-release or zymogram substrates, including the S232T priming-dead p3 as a control, and compare whole virions of wild-type and gp13-deficient phage. A negative result on p3 at loading comparable to the virion content would settle the attribution that four GOA rows depend on.
Hypothesis: Purified phi29 terminal protein has no peptidoglycan hydrolase activity, and the virion muralytic activity attributed to it in 2004 is contributed by gp13.
Type: enzyme activity assay
Experiment: Proximity-dependent labelling (BioID or APEX) from p3 and from the p3/DNA polymerase heterodimer in infected Bacillus subtilis at early and middle infection times, compared with the DNA-binding-deficient N-terminal mutants, followed by validation of candidates by co-localisation and by replication efficiency in the corresponding host deletion strains.
Hypothesis: The terminal protein is held at the host nucleoid by host factors in addition to generic DNA binding, and those factors also position the TP/polymerase complex on the MreB-dependent helical paths seen later in infection.
Type: proximity labelling and genetic validation
Experiment: Single-molecule optical-tweezers packaging assays comparing DNA-gp3 with proteinase K-treated DNA and with DNA bearing a non-cognate 5' protein, measuring initiation frequency, velocity, slipping and stalling; combined with cryoEM of a stalled prohead/pRNA/gp16/DNA-gp3 complex to ask whether gp3 occupies a defined site on the motor.
Hypothesis: Terminal protein is a functional constituent of the packaging machine rather than only the recognition mark on the substrate, so its presence changes motor kinetics and not merely substrate recruitment.
Type: single-molecule biophysics and structural biology
Experiment: Compare nuclear delivery and expression of TP-DNA carrying wild-type p3 with that carrying the K25A/K27A NLS mutant, in primary cells and in a gut or soil-derived co-culture where phage-eukaryote encounters occur, rather than in transfection of a cell line only.
Hypothesis: The eukaryotic NLS of phage terminal proteins confers a measurable advantage for transfer of phage DNA into eukaryotic nuclei under conditions resembling natural exposure.
Type: gene delivery assay
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