Multifunctional replication protein gpAlpha (777 aa) is the autonomous replication initiator, DNA helicase, and DNA primase of satellite bacteriophage P4. It comprises an N-terminal zinc-binding prokaryotic DNA primase domain that synthesizes RNA primers on single-stranded DNA, a central NTP-driven DNA helicase domain that unwinds the replication fork, and a C-terminal winged-helix origin-binding domain (P4-OBD) that binds Type I iterons at the P4 replication origin (ori) and cis replication region (crr) under negative regulation by Cnr.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000043 | MODIFY | Summary: Broad keyword mapping from KW-0547 (Nucleotide-binding) to nucleotide binding. Reason: Too general; gpAlpha specifically binds ATP (GO:0005524) to fuel its helicase activity and ribonucleoside triphosphates for primer synthesis. Proposed replacements: ATP binding Supporting Evidence: PMID:8253092 Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. |
| GO:0000428 DNA-directed RNA polymerase complex | IEA GO_REF:0000043 | REMOVE | Summary: Automated keyword mapping from KW-0240 (DNA-directed RNA polymerase) to DNA-directed RNA polymerase complex. Reason: False keyword mapping; gpAlpha is a multifunctional replication primase-helicase-origin-binding protein and is not part of a transcriptional DNA-directed RNA polymerase complex. Supporting Evidence: PMID:8253092 alpha Protein of satellite phage P4 of Escherichia coli is multifunctional in P4 replication with three activities. |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | ACCEPT | Summary: Combined InterPro and keyword annotation to DNA binding. Reason: Directly supported by biochemical and NMR structural studies showing that the C-terminal winged-helix origin-binding domain (P4-OBD) specifically binds Type I iterons in the P4 replication origin (ori) and cis replication region (crr). Supporting Evidence: PMID:8253092 First, the protein (subunit M(r) = 84,900) complexes specifically the P4 origin and the cis replication region required for replication. PMID:11929537 The origin-binding domain of the gpalpha protein of phage P4 (P4-OBD) mediates origin recognition and regulation of gpalpha activity by the protein Cnr. |
| GO:0003678 DNA helicase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC mapping from EC:3.6.4.12 to DNA helicase activity. Reason: Ziegelin et al. (1993) biochemically demonstrated that purified gpAlpha possesses NTP-hydrolysis-dependent DNA helicase activity required for P4 origin unwinding. Supporting Evidence: PMID:8253092 Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. |
| GO:0003899 DNA-directed RNA polymerase activity | IDA PMID:1618804 A common sequence motif, -E-G-Y-A-T-A-, identified within th... | ACCEPT | Summary: Experimental annotation from Strack et al. (1992) to DNA-directed RNA polymerase activity representing P4 DNA primase activity. Reason: Strack et al. (1992) and Ziegelin et al. (1993) demonstrated that gpAlpha synthesizes short oligoribonucleotide primers on single-stranded DNA templates; in GO, DNA primase catalytic activity is represented by GO:0003899 (DNA-directed RNA polymerase activity) coupled to GO:0006269 (DNA replication, synthesis of primer). Supporting Evidence: PMID:1618804 The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. PMID:8253092 Finally, the primase activity already demonstrated for alpha protein is due to synthesis of RNA primers. |
| GO:0004386 helicase activity | IEA GO_REF:0000120 | MODIFY | Summary: Broad InterPro and keyword mapping to helicase activity. Reason: Less specific than its child term GO:0003678 (DNA helicase activity), which is already present on gpAlpha. Proposed replacements: DNA helicase activity Supporting Evidence: PMID:8253092 Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. |
| GO:0005524 ATP binding | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword mapping from KW-0067 (ATP-binding) to ATP binding. Reason: gpAlpha contains a canonical Walker A nucleotide-binding motif (residues 496-503) that binds and hydrolyzes ATP to power DNA unwinding. Supporting Evidence: PMID:8253092 Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. |
| GO:0006260 DNA replication | IEA GO_REF:0000120 | ACCEPT | Summary: Combined InterPro and keyword annotation to DNA replication. Reason: Directly supported by genetic and biochemical studies showing that gpAlpha is the essential replication initiator, helicase, and primase for phage P4 DNA replication. Supporting Evidence: PMID:3309336 This segment of DNA contains two open reading frames of 106 and 777 amino acid residues; the latter of which is the coding sequence for the Mr 84,841 alpha protein, which is necessary for P4 DNA replication and is thought to act as a P4-specific DNA primase. PMID:8253092 alpha Protein of satellite phage P4 of Escherichia coli is multifunctional in P4 replication with three activities. |
| GO:0006269 DNA replication, synthesis of primer | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword mapping from KW-0639 (Primase) to DNA replication, synthesis of primer. Reason: Directly supported by biochemical assays showing that the N-terminal primase domain of gpAlpha synthesizes RNA primers on ssDNA templates to initiate P4 DNA replication. Supporting Evidence: PMID:1618804 The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. PMID:8253092 Finally, the primase activity already demonstrated for alpha protein is due to synthesis of RNA primers. |
| GO:0008270 zinc ion binding | IEA GO_REF:0000120 | ACCEPT | Summary: Combined InterPro and keyword mapping to zinc ion binding. Reason: The N-terminal prokaryotic DNA primase domain of gpAlpha contains a conserved Cys4 zinc-binding motif (IPR013237) involved in ssDNA template recognition. Supporting Evidence: PMID:1618804 The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. |
| GO:0016740 transferase activity | IEA GO_REF:0000043 | MODIFY | Summary: Broad keyword mapping from KW-0808 (Transferase) to transferase activity. Reason: Too general; should be refined to the specific catalytic activity GO:0003899 (DNA-directed RNA polymerase activity), which is already present on this record. Proposed replacements: DNA-directed RNA polymerase activity Supporting Evidence: PMID:1618804 The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. |
| GO:0016779 nucleotidyltransferase activity | IEA GO_REF:0000043 | MODIFY | Summary: Broad keyword mapping from KW-0548 (Nucleotidyltransferase) to nucleotidyltransferase activity. Reason: Less specific than its child term GO:0003899 (DNA-directed RNA polymerase activity), which is already present on this record. Proposed replacements: DNA-directed RNA polymerase activity Supporting Evidence: PMID:1618804 The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | MODIFY | Summary: Broad keyword mapping from KW-0378 (Hydrolase) to hydrolase activity. Reason: Too general; gpAlpha specifically catalyzes NTP/ATP hydrolysis (GO:0016887) coupled to DNA helicase activity (GO:0003678), both of which are already present on this record. Proposed replacements: DNA helicase activity ATP hydrolysis activity Supporting Evidence: PMID:8253092 Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea mapping to ATP hydrolysis activity. Reason: Directly supported by biochemical assays showing that gpAlpha hydrolyzes ATP/GTP to fuel DNA unwinding. Supporting Evidence: PMID:8253092 Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | MODIFY | Summary: Broad keyword mapping from KW-0479 (Metal-binding) to metal ion binding. Reason: Less specific than its child term GO:0008270 (zinc ion binding), which is already present on gpAlpha. Proposed replacements: zinc ion binding Supporting Evidence: PMID:1618804 The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. |
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Download this section (compressed HTML)Q: How does the hexameric central helicase ring of gpAlpha coordinate architecturally with its C-terminal dimeric winged-helix origin-binding domains during origin melting between ori and crr?
Suggested experts: Erich Lanka, Gianni Dehò, Daniela Ghisotti
Experiment: Determine the cryo-EM structure of full-length P4 gpAlpha bound to a forked DNA substrate containing P4 Type I iterons in the presence and absence of Cnr.
Hypothesis: Cnr heterodimerization with the C-terminal P4-OBD sterically blocks assembly of the hexameric helicase-primase ring on ori iterons.
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