Terminase small subunit (gpNu1) of Escherichia phage lambda that forms the viral DNA recognition and regulatory component of the heterotrimeric terminase holoenzyme (one gpA large subunit associated with two gpNu1 small subunits). Using an N-terminal winged helix-turn-helix domain, gpNu1 binds and bends specific R elements within the cosB sub-site of concatemeric viral DNA cooperatively with host integration host factor (IHF), positioning the catalytic gpA subunit at cosN to initiate site-specific maturation cleavage and ATP-driven viral DNA packaging into the procapsid. It also possesses a low-affinity, DNA-stimulated ATPase activity that modulates cos binding and cleavage dynamics.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000043 | MODIFY | Summary: gpNu1 specifically binds and hydrolyzes ATP at a low-affinity nucleotide-binding site, so the broad keyword mapping to nucleotide binding should be refined to ATP binding. Reason: Biochemical and mutational analyses demonstrate that gpNu1 contains a low-affinity, DNA-stimulated ATP-binding and hydrolysis center [PMID:2965248, PMID:8611586]. Because the specific nucleotide bound is ATP (already captured by GO:0005524), this general keyword-derived term is refined to GO:0005524 (ATP binding). Proposed replacements: ATP binding Supporting Evidence: PMID:8611586 In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA. |
| GO:0003677 DNA binding | IEA GO_REF:0000043 | MODIFY | Summary: gpNu1 binds specifically to the R1, R2, and R3 recognition elements of the phage lambda cosB site and bends the DNA duplex, so the general DNA binding term should be refined to sequence-specific DNA binding, bending. Reason: Circular permutation and footprinting assays show that gpNu1 recognizes specific elements within cosB and introduces a strong bend in cos-containing DNA [PMID:16618107]. Accordingly, the general keyword-mapped term GO:0003677 (DNA binding) is refined to its specific descendant GO:0044374 (sequence-specific DNA binding, bending). Proposed replacements: sequence-specific DNA binding, bending Supporting Evidence: PMID:16618107 Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. |
| GO:0005524 ATP binding | IEA GO_REF:0000043 | ACCEPT | Summary: gpNu1 possesses a low-affinity ATP-binding site that stimulates cos cleavage and regulates terminase-DNA interactions. Reason: Purified gpNu1 and the terminase holoenzyme exhibit a low-affinity, DNA-stimulated ATP-binding and ATPase center in the small subunit [PMID:2965248, PMID:8611586]. Supporting Evidence: PMID:8611586 In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA. |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | MODIFY | Summary: The hydrolase activity of gpNu1 is specifically ATP hydrolysis activity, so this broad keyword mapping should be refined to GO:0016887. Reason: gpNu1 catalyzes DNA-stimulated ATP hydrolysis with a low turnover number [PMID:2965248, PMID:8611586]. The broad GO:0016787 (hydrolase activity) annotation is therefore refined to its specific descendant GO:0016887 (ATP hydrolysis activity). Proposed replacements: ATP hydrolysis activity Supporting Evidence: PMID:2965248 The small subunit of the bacteriophage lambda terminase enzyme, the product of the phage's Nu1 gene, is shown to contain amino acid segments homologous to those present in a large number of ATPases. In keeping with these predictions, the purified protein has been found to hydrolyze ATP with a relatively low turnover number. |
| GO:0016887 ATP hydrolysis activity | IDA PMID:2965248 Prediction of an ATP reactive center in the small subunit, g... | ACCEPT | Summary: Purified gpNu1 directly catalyzes ATP hydrolysis with a low turnover number. Reason: Direct biochemical assays of purified gpNu1 confirm intrinsic ATPase activity [PMID:2965248]. Supporting Evidence: PMID:2965248 The small subunit of the bacteriophage lambda terminase enzyme, the product of the phage's Nu1 gene, is shown to contain amino acid segments homologous to those present in a large number of ATPases. In keeping with these predictions, the purified protein has been found to hydrolyze ATP with a relatively low turnover number. |
| GO:0016887 ATP hydrolysis activity | IDA PMID:8611586 Kinetic and mutational dissection of the two ATPase activiti... | ACCEPT | Summary: Kinetic and mutational analysis of terminase confirms a low-affinity, DNA-stimulated ATPase catalytic center in gpNu1. Reason: Kinetic and site-directed mutagenesis studies dissect the two ATPase sites of lambda terminase and establish that gpNu1 houses the low-affinity, DNA-stimulated ATPase activity [PMID:8611586]. Supporting Evidence: PMID:8611586 In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA. |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea reaction mapping accurately reflects the experimentally demonstrated ATP hydrolysis activity of gpNu1. Reason: Consistent with direct biochemical measurements showing that gpNu1 hydrolyzes ATP [PMID:2965248, PMID:8611586]. Supporting Evidence: PMID:2965248 In keeping with these predictions, the purified protein has been found to hydrolyze ATP with a relatively low turnover number. |
| GO:0019073 viral DNA genome packaging | IDA PMID:15755448 Self-association properties of the bacteriophage lambda term... | ACCEPT | Summary: As part of the terminase holoenzyme, gpNu1 is essential for site-specific cos recognition, cleavage, and viral DNA packaging into procapsids. Reason: Purified heterotrimeric terminase (gpA1-gpNu1_2) and its higher-order assembly directly catalyze cos cleavage and viral DNA packaging in vitro [PMID:15755448]. Supporting Evidence: PMID:15755448 Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. |
| GO:0030430 host cell cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: gpNu1 operates in the Escherichia coli cytoplasm during the lytic DNA packaging stage of infection. Reason: Lambda terminase assembles on concatemeric viral DNA together with host cytoplasmic integration host factor (IHF) in the bacterial cytoplasm to package viral genomes into procapsids [PMID:15755448, PMID:16618107]. Supporting Evidence: PMID:15755448 Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. |
| GO:0044374 sequence-specific DNA binding, bending | IDA PMID:16618107 Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins... | ACCEPT | Summary: gpNu1 binds specifically to the R elements within cosB and introduces a strong bend into cos-containing viral DNA. Reason: Circular permutation and gel-shift assays demonstrate that gpNu1 binds specifically to cos recognition elements and bends cos DNA cooperatively with E. coli IHF [PMID:16618107]. Supporting Evidence: PMID:16618107 The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. PMID:16618107 Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. |
| GO:0097710 viral terminase, small subunit | IDA PMID:15755448 Self-association properties of the bacteriophage lambda term... | ACCEPT | Summary: gpNu1 is the small subunit of the bacteriophage lambda terminase complex, associating in a 2:1 stoichiometry with the large subunit gpA. Reason: Sedimentation equilibrium and velocity analyses show that recombinant lambda terminase forms a heterotrimeric protomer composed of one gpA subunit and two gpNu1 small subunits [PMID:15755448]. Supporting Evidence: PMID:15755448 We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). |
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Download this section (compressed HTML)Q: How does ATP binding and hydrolysis at the low-affinity site in gpNu1 allosterically trigger the transition of the terminase holoenzyme from a site-specific cos-cleavage complex to a mobile DNA translocation motor?
Experiment: Determine high-resolution cryo-EM structures of the full-length lambda terminase heterotrimer (gpA1-gpNu1_2) bound to cos DNA and IHF in apo, ATP-analog-bound, and post-hydrolysis states to resolve inter-subunit allosteric coupling.
Hypothesis: Nucleotide occupancy in gpNu1 remodels the gpNu1-cosB interface to release the motor after cosN cleavage and allow gpA to initiate processive translocation.
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