Protein rI (gpRI) is a 97-amino-acid periplasmic antiholin of bacteriophage T4 that serves as the primary sensor and effector of lysis inhibition (LIN) in response to superinfection. Synthesized as a precursor with a cleavable 24-residue N-terminal Sec signal peptide, mature soluble RI is released into the Escherichia coli periplasm where it binds the periplasmic C-terminal domain of holin T (gpT) to form a heterotetrameric T2-RI2 complex. By stabilizing holin T in an inactive state—further reinforced by binding periplasmic DNA released from superinfecting virions—RI blocks holin oligomerization and inner-membrane pore formation, delaying host cell lysis and extending intracellular virion maturation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000043 | KEEP AS NON CORE | Summary: In complex with the periplasmic domain of holin T, RI participates in binding periplasmic double-stranded DNA from superinfecting phages as a lysis inhibition signal. Reason: DNA binding is an accessory sensory property of the assembled holin-antiholin (T-RI) complex during superinfection rather than the primary intrinsic function of RI, which is inhibition of holin T pore-forming activity. Supporting Evidence: file:BPT4/rI/rI-uniprot.txt The genomic DNA from the superinfecting phage bound to the complex holin-antiholin probably serves as a signal for the lysis inhibition and blocks the holin multimerization. |
| GO:0031640 killing of cells of another organism | IEA GO_REF:0000043 | MODIFY | Summary: RI is an antiholin that inhibits holin T and delays host cell lysis upon superinfection rather than killing the host cell. Reason: Killing of cells of another organism (GO:0031640) has the opposite biological direction to RI's actual function; RI suppresses holin-mediated cytolysis in response to secondary phage infection, which is specifically represented by suppression by virus of host cell lysis in response to superinfection (GO:0039639). Proposed replacements: suppression by virus of host cell lysis in response to superinfection Supporting Evidence: file:BPT4/rI/rI-uniprot.txt Involved in lysis inhibition. Senses superinfections and inhibits the holin, thereby delaying the host cell lysis timing. file:BPT4/rI/rI-deep-research-falcon.md **Primary function:** RI is a **T-holin inhibitor (antiholin)** that binds the holin and prevents holin **oligomerization/triggering**, delaying membrane permeabilization and therefore delaying endolysin access to peptidoglycan |
| GO:0044229 host cell periplasmic space | IEA GO_REF:0000044 | ACCEPT | Summary: Following cleavage of its N-terminal signal peptide by signal peptidase I, mature RI localizes to the Escherichia coli periplasmic space. Reason: Experimental and sequence evidence establish that mature RI is released into the host cell periplasmic space where it binds the periplasmic domain of holin T. Supporting Evidence: PMID:34456892 Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. |
| GO:0140678 molecular function inhibitor activity | IEA GO_REF:0000120 | ACCEPT | Summary: RI directly binds the periplasmic C-terminal domain of holin T and inhibits its oligomerization and pore-forming activity. Reason: Molecular function inhibitor activity (GO:0140678) accurately captures the core antiholin activity of RI in inhibiting holin T. Supporting Evidence: PMID:17693511 At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. file:BPT4/rI/rI-deep-research-falcon.md **Primary function:** RI is a **T-holin inhibitor (antiholin)** that binds the holin and prevents holin **oligomerization/triggering**, delaying membrane permeabilization and therefore delaying endolysin access to peptidoglycan |
| GO:0044229 host cell periplasmic space | EXP PMID:34456892 The Phage T4 Antiholin RI Has a Cleavable Signal Peptide, No... | ACCEPT | Summary: RI is processed by signal peptidase I to release the mature soluble antiholin into the host cell periplasmic space. Reason: Direct experimental evidence in PMID:34456892 demonstrates that RI carries a cleavable signal peptide and is released into the host periplasm as a mature protein. Supporting Evidence: PMID:34456892 Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. |
| GO:0020002 host cell plasma membrane | IDA PMID:17693511 The T4 RI antiholin has an N-terminal signal anchor release ... | KEEP AS NON CORE | Summary: Uncleaved RI precursor associates transiently with the host inner membrane prior to signal peptide cleavage, and mature RI is recruited to the inner membrane through complex formation with the integral membrane holin T. Reason: Although PMID:17693511 detected inner-membrane-associated RI and proposed a SAR domain model, subsequent work (PMID:34456892) showed that RI has a cleavable signal peptide and that its mature functional form is a soluble periplasmic protein that associates with the inner membrane via binding to holin T. Supporting Evidence: PMID:17693511 At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. PMID:34456892 Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. |
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Download this section (compressed HTML)Q: What regulates the rapid turnover of mature periplasmic RI in the absence of superinfection if DegP is not required for its proteolytic degradation?
Q: How does transient periplasmic passage of superinfecting phage genomic DNA or internal capsid proteins allosterically stabilize the periplasmic T2-RI2 heterotetramer in vivo?
Experiment: Perform pulse-chase radiolabeling of mature periplasmic RI across a panel of Escherichia coli periplasmic protease knockouts (degP, ptrA, tsp/prc,degQ, degS) during T4 infection.
Hypothesis: A periplasmic protease distinct from DegP—or spontaneous conformational unfolding of free mature RI—rapidly clears uncomplexed RI unless stabilized by superinfection signals.
Experiment: Measure binding kinetics and complex half-life of purified mature RI (residues 25-97) and the periplasmic domain of holin T by surface plasmon resonance in the presence and absence of linear dsDNA fragments.
Hypothesis: Binding of dsDNA across the composite T-RI positively charged groove reduces the dissociation rate constant (k_off) of the T-RI complex by more than an order of magnitude.
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