Protein p4 (gp4) is the 125-residue early regulatory protein that operates the early-to-late transcriptional switch of Bacillus phage phi29. The phage encodes no polymerase and no sigma factor of its own: all of its genes are read by the host Bacillus subtilis sigma-A RNA polymerase, and p4 redirects that enzyme. Early genes lie at the two ends of the genome and the structural, morphogenesis and lysis genes lie in the middle, transcribed from a single late promoter, A3. p4 is a homodimer that binds three sites in the 219 bp region separating the early promoters A2b and A2c from A3, recognising each site as an inverted repeat of two 6 bp arms through an N-terminal beta-turn N-hook that enters the major groove and makes base-specific contacts, aided by a helix-turn-helix motif and by a pronounced bend that p4 both reads and deepens. At A3, whose own -35 element is too poor to be used, bound p4 stabilises RNA polymerase as a closed complex and switches the promoter on; at A2c it binds immediately upstream of the polymerase and shuts the promoter off by blocking promoter clearance rather than polymerase binding; at A2b it displaces the polymerase. All three effects run through a single protein-protein contact, between the basic C-terminal tail of p4 and the C-terminal domain of the polymerase alpha subunit, with Arg120 the critical residue, so one interaction surface activates at one promoter and represses at another depending on where p4 sits relative to the enzyme. p4 does not act alone: it binds cooperatively with the abundant nucleoid protein p6 across the whole A2c-to-A3 interval, defining the phase and the boundaries of the p6 filament through its C-terminus and building the nucleoprotein hairpin that enforces the switch. Because A3 is the only late promoter, p4 is indirectly required for everything made late in infection, including virion assembly and host lysis, but its own activity is transcriptional regulation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IDA PMID:2107318 Bend induced by the phage phi 29 transcriptional activator i... | MODIFY | Summary: Direct binding of p4 to the late A3 promoter, mapped by gel retardation, DNase I footprinting, methylation interference and circular permutation to a region between -50 and -100 relative to the A3 start site that is intrinsically curved and is bent further on binding. The site is an inverted repeat bound by two p4 monomers, and the later crystal structure shows the recognition is base-specific. Reason: The claim is directly demonstrated, but the root term is less specific than the evidence supports and the refinement is informative here. p4 does not bind DNA generically: it recognises inverted-repeat target sites that lie in the regulatory region of three promoters, with sites overlapping the -35 boxes of A2c and A2b and the activation site upstream of the A3 start, and the N-hook motif makes base-specific major-groove contacts in the 41 bp A3-site co-crystal. GO:0000987 cis-regulatory region sequence-specific DNA binding is therefore the supported term and is a descendant of GO:0003677 by way of GO:0043565. It is not refined further to the transcription-regulator activity terms, which are not DNA-binding descendants and are proposed separately as NEW rows below. Proposed replacements: cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:2107318 Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. PMID:2107318 The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. PMID:2107318 two monomers of protein p4 bind to the inverted recognition sequences PMID:16600871 The two N-hooks enter the major groove of the double helix, establishing base-specific contacts. PMID:16600871 The crystal structure of p4 alone and in complex with a 41 bp DNA, including the A3 promoter binding site, helps us to understand how the phage cycle is controlled. file:BPPH2/4/4-uniprot.txt Interacts with DNA; binds to the A2b, A2c and A3 promoters |
| GO:0003677 DNA binding | IEA GO_REF:0000043 | MODIFY | Summary: Keyword-derived row (KW-0238 DNA-binding) asserting the same claim as the IDA row above, which is correct and experimentally grounded. UniProt curates an H-T-H motif at residues 77-96 for this entry in addition to the structurally defined N-hook. Reason: Same claim and same refinement as the IDA row: the binding that is demonstrated is to sequence-specific target sites inside the A2b/A2c/A3 regulatory region, so the row is refined to GO:0000987 rather than left at the root. The two rows are kept consistent deliberately; neither is removed as redundant, since each is an independent line of support for one true claim. Proposed replacements: cis-regulatory region sequence-specific DNA binding Supporting Evidence: file:BPPH2/4/4-uniprot.txt /note="H-T-H motif" file:BPPH2/4/4-uniprot.txt DNA_BIND 77..96 PMID:16600871 The two N-hooks enter the major groove of the double helix, establishing base-specific contacts. |
| GO:0003899 DNA-directed RNA polymerase activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO row from IPR008771 "Phi-29-like late genes activator", the family that is p4 itself. Checked live against the InterPro API: the entry maps to GO:0003899 and GO:0016987 as well as GO:0006355, and its description still reads that the protein "may function as a sigma-like component of the host RNA polymerase". p4 has no catalytic activity of any kind. Reason: p4 is not a polymerase and has no nucleotidyltransferase chemistry: it is a 125-residue DNA-binding regulator whose entire mechanism is to bind a target site and touch the C-terminal domain of the alpha subunit of the host enzyme, which does the transcribing. The transcription it controls is carried out by Bacillus subtilis sigma-A RNA polymerase, and UniProt's own curated FUNCTION says p4 stabilises the binding of that host polymerase rather than being one. Its crystal structure is a unique alpha/beta DNA-binding fold with no polymerase domain. The row is a family-level mapping of a superseded sigma-like-subunit reading and is removed rather than refined, because nothing on this branch is true of the protein; the defect is recorded against InterPro. Supporting Evidence: file:BPPH2/4/4-uniprot.txt Mediates, together with protein p6, the early to late transcriptional switch by stabilizing the binding of host RNA polymerase (RNAP) to the late A3 promoter. PMID:21614197 Phage Γ29 gene expression is directed by the Bacillus subtilis ΟA-RNAP PMID:8799127 Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. PMID:16600871 The crystal structure of p4 alone and in complex with a 41 bp DNA, including the A3 promoter binding site, helps us to understand how the phage cycle is controlled. |
| GO:0006355 regulation of DNA-templated transcription | IDA PMID:8617213 Activation and repression of transcription at two different ... | MODIFY | Summary: The defining experiment for p4: in vitro transcription and footprinting with purified p4, purified host RNA polymerase and the phi29 promoter region show that p4 activates the late A3 promoter and represses the early A2b and A2c promoters, and that site-directed substitution of Arg120 abolishes both. Reason: The claim is correct and is the gene's core function, but the term is less specific than the evidence supports and both specific descendants are demonstrated in the same experiments, so both are proposed rather than one being chosen. Activation of A3 is positive regulation (GO:0045893); repression of A2b and A2c is negative regulation (GO:0045892). This is a genuinely bidirectional regulator, not a case of picking a sign: the same p4 surface, the Arg120-containing domain, activates at one promoter and represses at another according to where p4 binds relative to the polymerase, and a gene-4 nonsense mutant shows both arms failing in vivo at once. Proposed replacements: positive regulation of DNA-templated transcription negative regulation of DNA-templated transcription Supporting Evidence: PMID:8617213 Phage phi29 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. PMID:8617213 Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Arg120 can activate or repress transcription, depending on the promoter. PMID:11689446 In bacteria harbouring a nonsense mutant in gene 4, transcription from promoters A2b and A2c increases steadily during infection while the transcripts arising from promoter A3 are almost undetectable file:BPPH2/4/4-uniprot.txt Activates transcription from the late A3 promoter and represses the main early promoters A2b and A2c |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000120 | MODIFY | Summary: Automatic row for the same claim, from the p4 family signature (IPR008771) and from the sigma factor keyword. The InterPro half of its basis is sound for this term even though the keyword half is not: IPR008771 is p4's own family and transcription regulation is what the family does. Reason: Same claim and same refinement as the IDA row above, kept consistent with it: both specific directions are demonstrated for p4, so both are proposed. The row is not removed on account of the defective KW-0731 component of its basis, because the claim itself is correct and independently established; the keyword defect is recorded on the GO:0016987 row, which is the row that only the keyword supports. Proposed replacements: positive regulation of DNA-templated transcription negative regulation of DNA-templated transcription Supporting Evidence: PMID:16600871 Transcriptional switch by p4 is achieved by activating the late promoter A3 and repressing the early promoters A2b and A2c. PMID:21614197 Protein p4 binds to specific target sites and is required for the activation of the late promoter A3 and for the repression of early promoters A2b and A2c |
| GO:0016987 sigma factor activity | IEA GO_REF:0000120 | REMOVE | Summary: Automatic row resting on the UniProt keyword KW-0731 "Sigma factor" and on the same InterPro family, whose description calls p4 a possible "sigma-like component of the host RNA polymerase". GO:0016987 is defined as the promoter-specificity subunit of the polymerase, which binds promoter elements only as part of the holoenzyme and is released once elongation begins. None of that describes p4. Reason: p4 is not a subunit of RNA polymerase. It binds DNA on its own, at its own target sites, independently of the enzyme, and it acts on the holoenzyme from outside by contacting the C-terminal domain of the alpha subunit, the same surface that conventional activators and repressors use; the contact is dispensable for DNA binding and is mapped to a single residue, Arg120, whose substitution leaves DNA binding intact while abolishing both activation and repression. Nothing about p4 matches the sigma-factor concept: it confers no promoter specificity on core polymerase, is not part of the holoenzyme, and is not released at elongation but stays bound to its site. The claim is false rather than imprecise, so the row is removed; the functional claim it was reaching for is captured instead by the activator and repressor activity rows proposed below. Two resources carry the defect, so two reports are filed. Supporting Evidence: PMID:8799127 Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues. PMID:8617213 Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Arg120 plays an essential role. PMID:8617213 Mutation of protein p4 residue Arg120, which relieves the contact between the two proteins, leads to a loss of repression. file:BPPH2/4/4-uniprot.txt R->Q: 80% loss of transcription activation from the A3 promoter and 80% loss of transcription repression from the A2c promoter. Complete loss of interaction with host RNAP. |
| GO:0044659 viral release from host cell by cytolysis | IMP PMID:820555 Bacillus subtilis phage phi29. Characterization of gene prod... | MARK AS OVER ANNOTATED | Summary: CACAO row (2025) from the 1976 Carrascosa et al. nonsense-mutant characterisation of phi29 gene products. That paper reports one lysis cistron, whose mutants show delayed lysis and a normal or larger burst after artificial lysis, and the 2025 campaign placed GO:0044659 on two entries from it: gene 4 and gene 15. Gene 4 is the early transcription activator, and p4 is indirectly required for lysis because the holin and endolysin genes are late genes read from the single promoter it switches on. Reason: The term is not false of p4, which is why it is marked over-annotated rather than removed: no p4 means no A3 transcription, hence no gp14 holin and no gp15 endolysin, so p4 is genetically upstream of lysis with a positive effect. But it is an over-annotation of a transcription regulator to a downstream outcome. A3 is the only late promoter, so by the same reasoning p4 would be upstream of capsid assembly, scaffolding, tail morphogenesis and DNA packaging as well; annotating lysis alone records no property specific to p4, and what p4 actually does is already said by the transcription-regulation rows. The cited evidence is also another gene's. The 1976 abstract reports a single lysis cistron, and its mutant produced a burst size similar to or higher than wild type after artificial lysis, which a gene-4 mutant cannot do: a gene-4 nonsense mutant makes almost no A3 transcript, hence no structural proteins and no progeny to release, so the reported phenotype excludes gene 4 by construction. The cistron is a late lysis gene, and three independent lines put it on gene 14, the holin (UniProtKB:P11188): gene 14 is the gene with a sus allele in that region, sus14(1241); its product is the one shown to be required for efficient lysis of B. subtilis; and delayed lysis with an undiminished burst is the canonical holin-null phenotype, gene 15 being the lysozyme the holin releases. Gene 14 carries no CACAO row at all. Neither of the two records that would settle it outright could be read: the 1976 full text is not in PMC and its two open-access locations were unreachable from here, and the GONUTS page that held the student's reasoning is permanently gone, gowiki.tamu.edu now returning HTTP 410 with "GONUTS, the GO wiki, has been retired" and the Wayback Machine holding no snapshot of it. The source defect is therefore PROBABLE rather than CONFIRMED. Supporting Evidence: PMID:820555 A cistron, probably involved in the final lysis of the infected bacteria, has been found. PMID:820555 Mutants in this gene give place, under restrictive conditions, to delayed lysis and produce, after artificial lysis, a burst size similar or higher than that obtained after wild-type phage infection. PMID:11689446 In bacteria harbouring a nonsense mutant in gene 4, transcription from promoters A2b and A2c increases steadily during infection while the transcripts arising from promoter A3 are almost undetectable PMID:16600871 The synthesis of DNA replication proteins and p4 takes place in the early phase, while structural, morphogenesis, and lysis proteins are synthesized in the late phase. PMID:21614197 Late genes are expressed from a single promoter, A3. PMID:8432697 Phage phi 29 gene 14, the product of which is required for efficient lysis of Bacillus subtilis, was cloned in Escherichia coli. PMID:3027653 The g14 open reading frame (ORF) was confirmed by sequencing a sus14(1241) mutant. PMID:7831803 The 131-codon gene 14 of Bacillus subtilis phage phi 29 encodes the holin function, whereas the downstream gene 15 codes for a lysozyme. |
| GO:2000142 regulation of DNA-templated transcription initiation | IEA GO_REF:0000108 | MODIFY | Summary: Inter-ontology inference: GO_REF:0000108 derived this row logically from the GO:0016987 sigma factor activity row, which this review removes. The conclusion is nonetheless true, and for better reasons than its premise: p4 acts specifically at the initiation step, stabilising RNA polymerase at A3 as a closed complex and displacing it from A2b. Reason: A true claim reached from a false basis. The row's stated support is the sigma factor row, which is removed, so the inference no longer stands; but p4 really does regulate transcription initiation, and the demonstrated directions are more specific than the parent, so the row is refined rather than discarded. Positive: activation of A3 works by stabilising the polymerase at the promoter as a closed complex, which is initiation complex formation (GO:2000144). Negative: at A2b the p4-containing complex displaces the polymerase from the promoter, abolishing the RNAP footprint there, which is negative regulation at the same step (GO:2000143). The repression of A2c is deliberately not counted here: that promoter is blocked at promoter clearance, with polymerase binding and an initiated complex both allowed, so it is not an initiation-step effect and is carried by the GO:0045892 replacement instead. GOA and GO Central should re-derive this row from the direct evidence once the sigma factor row is withdrawn. Proposed replacements: positive regulation of DNA-templated transcription initiation negative regulation of DNA-templated transcription initiation Supporting Evidence: PMID:8617213 Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Arg120 plays an essential role. PMID:11689446 Concomitantly, the RNAP-derived hypersensitive band at promoter A2b disappeared (band at PMID:11689446 RNAP binds to promoters A2c and A2b, but not to promoter A3 that lacks a PMID:8799127 Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. |
| GO:0001216 DNA-binding transcription activator activity | IDA PMID:8617213 Activation and repression of transcription at two different ... | NEW | Summary: Proposed row. GOA carries no molecular-function term for what p4 is: its only MF rows are DNA binding plus the two wrong terms removed above. p4 is a sequence-specific DNA-binding protein that turns on the late A3 promoter, whose own -35 element is too poor for the host polymerase to use, by binding upstream and stabilising the enzyme there as a closed complex. Reason: The activity is directly demonstrated with purified components and is p4's defining property, and the gene performs the work itself: it binds the cis-regulatory site and makes the protein-protein contact that recruits and stabilises the polymerase, rather than being required for the process at a distance. Site-directed substitution of a single p4 residue, Arg120, abolishes activation while leaving DNA binding intact, which pins the activator function on p4's own surface. The comparator check supports the term: GO:0001216 is used for exactly this class of bacterial and phage activators that contact the polymerase alpha C-terminal domain from an upstream site. It is not a descendant or ancestor of any other term proposed here: GO:0000987, the replacement on the DNA-binding rows, sits under GO:0003677, while GO:0001216 sits under GO:0140110. Supporting Evidence: PMID:8617213 Phage phi29 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. PMID:8617213 Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Arg120 plays an essential role. PMID:11689446 RNAP binds to promoters A2c and A2b, but not to promoter A3 that lacks a file:BPPH2/4/4-uniprot.txt Mediates, together with protein p6, the early to late transcriptional switch by stabilizing the binding of host RNA polymerase (RNAP) to the late A3 promoter. |
| GO:0001217 DNA-binding transcription repressor activity | IDA PMID:8617213 Activation and repression of transcription at two different ... | NEW | Summary: Proposed row for the other half of the same activity. Bound at its target sites in the A2c and A2b promoters, p4 shuts both down: at A2c it allows the polymerase to bind and initiate but blocks promoter clearance, and at A2b it displaces the polymerase outright. Reason: Repression is demonstrated in the same purified in vitro transcription and footprinting experiments as activation, and p4 does the work: it occupies the cis-regulatory site and holds the polymerase at the promoter through the Arg120 contact with the alpha C-terminal domain, which is the repressive mechanism rather than a downstream consequence. Both directions are needed because they are separate molecular-function claims that one surface of one protein performs at different promoters; keeping only the activator term would lose the repression of the early operons, which is half the switch and the half the in vivo gene-4 mutant phenotype shows most clearly. Not an ancestor or descendant of GO:0001216, its sibling under GO:0003700. Supporting Evidence: PMID:8617213 We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding PMID:8799127 Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. PMID:11689446 Concomitantly, the RNAP-derived hypersensitive band at promoter A2b disappeared (band at file:BPPH2/4/4-uniprot.txt Activates transcription from the late A3 promoter and represses the main early promoters A2b and A2c |
| GO:0070063 RNA polymerase binding | IPI PMID:8799127 Protein p4 represses phage phi 29 A2c promoter by interactin... | NEW | Summary: Proposed row for the protein-protein contact that is the mechanism of both p4 activities. p4 binds the C-terminal domain of the alpha subunit of Bacillus subtilis RNA polymerase; the contact was mapped with a reconstituted polymerase lacking the last 15 residues of alpha-CTD and with purified alpha subunit in isolation, and on the p4 side it maps to Arg120. Reason: Added as a specific, informative binding term rather than bare protein binding: it names the partner, and the partner identity is the whole point, since contacting the host polymerase from a fixed upstream site is how a 125-residue phage protein reprogrammes a bacterial transcription machine. The evidence is direct and reciprocal: p4 forms a complex with purified wild-type alpha subunit but not with the C-terminal deletion, and p4 R120Q loses the interaction with host RNAP altogether while retaining DNA binding. The term is not a parent or child of the activator and repressor terms; it records the interaction those activities run through. The contact with the nucleoid protein p6, which is equally real, is deliberately not proposed as a row, because the informative consequence of that interaction is already carried by the regulation rows. Supporting Evidence: PMID:8799127 Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues. PMID:8799127 Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. file:BPPH2/4/4-uniprot.txt Interacts with host RNA polymerase (via C-terminus) file:BPPH2/4/4-uniprot.txt R->Q: 80% loss of transcription activation from the A3 promoter and 80% loss of transcription repression from the A2c promoter. Complete loss of interaction with host RNAP. |
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Download this section (compressed HTML)Q: Which residues of p6 form the interface with the C-terminal tail of p4? The p4 side is mapped residue by residue, but no p6 variant has ever been tested for the contact.
Suggested experts: Fernando Rojo, Ana Camacho
Q: What makes the switch happen when it does? p4 and p6 are both early proteins, yet the late promoter turns on only around 20 minutes after infection. Is the trigger simply p6 accumulating to the concentration at which p4-nucleated polymerisation becomes stable, or is something else rate-limiting in vivo?
Suggested experts: Ana Camacho, Fernando Rojo
Q: Why does one p4 surface activate at A3 and repress at A2c? The Arg120 contact with alpha-CTD is shared, and the stated explanation is the relative geometry of p4 and the polymerase at each promoter. Would a structure of p4 bound to DNA together with the polymerase, at each of the two promoters, show two different alpha-CTD contacts or one contact used from two orientations?
Suggested experts: Miguel Coll, Mario Mencia
Experiment: Charge-reversal and alanine scanning across the solvent-exposed surface of p6, scored in three assays with purified components: cooperative binding with wild-type p4 over the A2c-to-A3 fragment by DNase I footprinting, formation of the p4-p6-DNA complex by band shift, and p6-assisted repression of A2c by in vitro transcription on TP-DNA. Intrinsic p6 DNA binding and dimerisation are measured in parallel so that interface mutants are distinguished from destabilised or DNA-binding-defective protein, and candidate interface mutants are then tested for charge-swap rescue against the complementary p4 substitutions.
Hypothesis: The p4-p6 contact is mediated by acidic or polar surface residues of p6 pairing with the basic C-terminal tail of p4 (R116, R118, R120, R121, K122).
Type: site-directed mutagenesis with in vitro reconstitution
Experiment: Supply gp14 and gp15 from a host-borne inducible construct in cells infected with a gene-4 nonsense mutant, and ask whether lysis timing is restored to wild type while virion production stays blocked. Separating the lysis phenotype from the rest of the late programme in this way would show directly that p4 contributes nothing to lysis beyond transcription of the lysis genes, and would settle whether any process term beyond transcriptional regulation belongs on p4.
Hypothesis: p4's requirement for host lysis is entirely indirect, through activation of the single late promoter A3, and is not a separate activity of the protein.
Type: trans-complementation of a nonsense mutant
Experiment: Cryo-EM of reconstituted complexes of Bacillus subtilis sigma-A RNA polymerase with p4 on DNA fragments carrying either the A3 or the A2c promoter, compared against a charge-swap matrix pairing p4 C-terminal substitutions with alpha-CTD substitutions and scored for activation at A3 and repression at A2c, to test whether the two outcomes use one contact from two geometries or two distinct contacts.
Hypothesis: p4 bound at A3 and p4 bound at A2c engage different surfaces of the RNA polymerase alpha C-terminal domain.
Type: structural biology with genetic complementation
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