Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the packaging of 2 base-pairs of phi 29 DNA, or 9 X 10(3) ATP molecules per virion. The hydrolysis of ATP by gp16 was both prohead and DNA-gp3 dependent. gp16 contained both the "A-type" and the "B-type" ATP-binding consensus sequences (Walker et al., 1982) and the predicted secondary structure for ATP binding. The A-type sequence of gp16 was "basic-hydrophobic region-G-X2-G-X-G-K-S-X7-hydrophobic", and similar sequences were found in the phage DNA packaging proteins gpA of lambda, gp19 of T7 and gp17 of T4. Having both the ATP-binding and potential magnesium-binding domains, all of these proteins probably function as ATPases and may have common prohead-binding capabilities. The phi 29 protein gp3, covalently bound to the DNA, may be analogous in function to proteins gpNul of lambda and gpl of phi 21 that bind the DNA.
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PMID:Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29. 296 Aug 20

The products of the bacteriophage T4 terminase genes 16 and 17 are known to mediate cutting and packaging of concatemeric vegetative DNA. We show here that the larger of these genes, 17, yields multiple protein species. The complex expression of the T4 terminase genes includes overlapping transcripts, probably initiated from multiple promoters, RNA processing at certain preferred sites and translation initiation from multiple ribosome binding sites (RBS). Translation initiation from these RBS may be modulated by inverted repeat (IR) sequences whose folding can be predicted to differ in different RNA species. In T4 infected bacteria, genes 16 and 17 are probably co-transcribed from several near-consensus late promoters upstream from gene 16, and processed at multiple sites. Additional 5' ends of late transcripts are located downstream from a near-consensus late promoter inside gene 17 and further downstream, unrelated to any known promoter consensus sequence. The gene 17 transcripts that are initiated or cleaved internally contain RBS for shorter open reading frames (ORFs) in the same frame as full-length gene product (gp) 17 of 70 kDa. The truncated proteins, a 59-kDa gp17' and a 45-kDa gp17", are synthesized from cloned gene 17 segments in which the first gene 17 RBS is deleted. Expression of gene 17 is different in BL21(DE3) or W3110[pACT7] host bacteria. The gp17' and gp17" proteins are predicted to contain one or more of the ATPase motifs that are common among large subunits of other phage terminases. They lack a predicted single stranded (ss) DNA binding motif that is unique the large terminase proteins in T4 gp17, and that has been implicated in recognizing ssDNA regions in replicating and recombining T4DNA destined to be packaged. We hypothesize that a truncated gene 17' is an evolutionary precursor of the full-size T4 gene 17. Its function may have been maintained to allow processive packaging from double stranded (ds) DNA ends.
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PMID:Expression of the bacteriophage T4 DNA terminase genes 16 and 17 yields multiple proteins. 892 65

Bacteriophage terminases are oligomeric multifunctional proteins that bind to vegetative DNA, cut it and, together with portal proteins, translocate the DNA into preformed heads. Most terminases are encoded by two partially overlapping genes. In phage T4 they are genes 16 and 17. We have shown before that the larger of these, gene 17, can yield, in addition to a full-length 70 kDa product, several shorter peptides. At least two of these, gene product (gp) 17' and gp17", are initiated in the same reading frame as the 70 kDa gp17 from internal ribosome binding sites. Most of the shorter gp17 s contain predicted ATPase motifs, but only the largest (70 kDa) peptide has a predicted single-stranded DNA binding domain. Here we describe the DNA binding and cutting properties of the purified 70 kDa protein, expressed from two different clones containing gene 17 but no other T4 gene. Epitope-specific antibodies, which recognize several different gene 17 products in extracts of induced clones or of T4-infected cells, precipitate the purified 70 kDa gp17. When Mg2+ is chelated by EDTA this 70 kDa protein binds to single-stranded DNA, preferentially to junctions of single- and double-stranded DNA segments. It does not bind to blunt-ended double-stranded DNA. When Mg2+ is present the purified 70 kDa gp17 digests single-stranded segments preferentially up to junctions with double-stranded DNA. A 70 kDa gp17 from a P379L temperature sensitive (ts) mutant, which has lost the nuclease and ATPase activities, retains the single-stranded DNA binding activity. Taken together with earlier findings these results support a model for packaging of T4 DNA from single-stranded regions in recombinational or replicative intermediates, which occur at nearly random positions of the genome. This mechanism may be an alternative to site-specific initiation of packaging proposed by other investigators.
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PMID:The largest (70 kDa) product of the bacteriophage T4 DNA terminase gene 17 binds to single-stranded DNA segments and digests them towards junctions with double-stranded DNA. 953 79

In bacteriophage T4, the terminase complex constituted by the large subunit gp17 (69 kDa) and the small subunit gp16 (18 kDa) is a critical component of the ATP-driven DNA-packaging pump that translocates DNA into an empty capsid shell. Evidence suggests that the large subunit gp17 is the critical component and consists of a number of the functional sites required for DNA-packaging. It exhibits a terminase activity that introduces non-specific cuts into DNA, a portal vertex binding site that allows linkage of cleaved DNA to an empty prohead, an in vitro DNA-packaging activity, and an ATPase activity. In addition, a consensus metal-binding motif and two consensus ATP-binding sites have been identified by sequence analysis. In order to understand the mechanism of action of the multifunctional gp17, we developed an expression-based selection strategy to select for mutants that are defective in terminase function. Characterization of one of the mutants revealed a unique phenotype in which a single H436R mutation resulted in a dramatic loss of both the terminase and the DNA-packaging functions. Indeed, in vivo substitution of H436 with any of the 12 amino acids for which a suppressor is available was lethal to T4 development. According to one hypothesis, H436 is part of a metal-binding motif that is essential for gp17 function. This hypothesis was tested by introducing mutations at each of the three histidine pairs, the H382-X2-H385 pair, the H411-X2-H414 pair and the H430-X5-H436 pair, which constitute the histidine-rich region near the C terminus of gp17. A mutation at either the H411 pair or the H430 pair resulted in a loss of gp17 function, whereas a mutation at the H382 pair had no effect. In addition to the putative metal-binding motif, substitutions at residue K166 within the putative N terminus-proximal ATP-binding site also resulted in a loss of gp17 function. We propose that a metal-binding motif involving the histidine residues within the sequence H411-X2-H414-X15-H430-X5-H436 is essential for gp17 function. Metal-terminase interactions may be required for structural alignment and stabilization of functional sites in phage T4 terminase and other double-stranded DNA phage terminases.
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PMID:Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4. 971 36

Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell. Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated. Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations. The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni(2+)-agarose chromatography and were shown to be highly active for packaging DNA in vitro. The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity. Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold. The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis. A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16. The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.
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PMID:Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4. 1096 92

Double-stranded DNA packaging in bacteriophages is apparently driven by the most powerful molecular motor ever measured. Although it is widely accepted that a translocating ATPase powers the DNA packaging machine, the identity of the ATPase that generates this driving force is unknown. Evidence suggests that the large terminase protein gp17, which possesses two consensus ATP binding motifs and an ATPase activity, is a strong candidate for the translocating ATPase in bacteriophage T4. This hypothesis was tested by a PCR-directed combinatorial mutagenesis approach in which mutant libraries consisting of all possible codon combinations were constructed at the signature residues of the ATP binding motifs. The impact on gp17 function of each randomly selected mutant was evaluated by phenotypic analysis following recombinational transfer into the viral genome. The precise mutation giving rise to a particular phenotype was determined by DNA sequencing. The data showed that the N-terminal ATP binding site I (SRQLGKT(161-167)), but not the ATP binding site II (TAAVEGKS(299-306)), is critical for gp17 function. Even conservative substitutions such as G165A, K166R, and T167A were not tolerated at the GKT signature residues, which are predicted to interact with the ATP substrate. Biochemical analyses of the mutants showed a complete loss of in vitro DNA packaging activity but not the terminase (DNA-cutting) activity. The purified K166G mutant showed a loss of gp17-ATPase activity. The data, for the first time, implicated a specific ATPase center in the viral dsDNA packaging.
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PMID:The N-terminal ATPase site in the large terminase protein gp17 is critically required for DNA packaging in bacteriophage T4. 1184 54

Phage DNA packaging is believed to be driven by a rotary device coupled to an ATPase 'motor'. Recent evidence suggests that the phage DNA packaging motor is one of the strongest force-generating molecular motors reported to date. However, the ATPase center that is responsible for generating this force is unknown. In order to identify the DNA translocating ATPase, the sequences of the packaging/terminase genes of coliphages T4 and RB49 and vibriophages KVP40 and KVP20 have been analyzed. Alignment of the terminase polypeptide sequences revealed a number of functional signatures in the terminase genes 16 and 17. Most importantly, the data provide compelling evidence for an ATPase catalytic center in the N-terminal half of the large terminase subunit gp17. An analogous ATPase domain consisting of conserved functional signatures is also identified in the large terminase subunit of other bacteriophages and herpesviruses. Interestingly, the putative terminase ATPase domain exhibits some of the common features found in the ATPase domain of DEAD box helicases. Residues that would be critical for ATPase catalysis and its coupling to DNA packaging are identified. Com binatorial mutagenesis shows that the predicted threonine residues in the putative ATPase coupling motif are indeed critical for function.
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PMID:Sequence analysis of bacteriophage T4 DNA packaging/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases. 1223 85

Phage T4 terminase is a two-subunit enzyme that binds to the prohead portal protein and cuts and packages a headful of concatameric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from chitin, two additional column steps completed purification. The purification yielded (i) homogeneous soluble gp17 highly active in in vitro DNA packaging ( approximately 10% efficiency, >10(8) phage/ml of extract); (ii) gp17 lacking endonuclease and contaminating protease activities; and (iii) a DNA-independent ATPase activity stimulated >100-fold by the terminase small subunit, gp16 (18 kDa), and modestly by portal gp20 and single-stranded binding protein gp32 multimers. Analyses revealed a preparation of highly active and slightly active gp17 forms, and the latter could be removed by immunoprecipitation using antiserum raised against a denatured form of the gp17 protein, leaving a terminase with the increased specific activity (approximately 400 ATPs/gp17 monomer/min) required for DNA packaging. Analysis of gp17 complexes separated from gp16 on glycerol gradients showed that a prolonged enhanced ATPase activity persisted after exposure to gp16, suggesting that constant interaction of the two proteins may not be required during packaging.
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PMID:Isolation and characterization of T4 bacteriophage gp17 terminase, a large subunit multimer with enhanced ATPase activity. 1246 75

Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid. Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging. It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid. Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses. Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis. Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT(161-167)) and Walker B (MIYID(251-255)) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function. However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function. None of the amino acid substitutions, including the highly conservative D and Q, was tolerated. Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact. The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation. Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine.
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PMID:Defining the ATPase center of bacteriophage T4 DNA packaging machine: requirement for a catalytic glutamate residue in the large terminase protein gp17. 1287 41

Double-stranded DNA packaging in bacteriophage T4 and other viruses occurs by translocation of DNA into an empty prohead by a packaging machine assembled at the portal vertex. Coordinated with this complex process is the cutting of concatemeric DNA to initiate and terminate DNA packaging and encapsidate one genome-length viral DNA. The catalytic site responsible for cutting, and the mechanisms by which cutting is precisely coordinated with DNA translocation remained as interesting open questions. Phage T4, unlike the phages with defined ends (e.g. lambda, T3, T7), packages DNA in a strictly headful manner, and exhibits no strict sequence specificity to initiate or terminate DNA packaging. Previous evidence suggests that the large terminase protein gp17, a key component of the T4 packaging machine, possesses a non-specific DNA cutting activity. A histidine-rich metal-binding motif, H382-X(2)-H385-X(16)-C402-X(8)-H411-X(2)-H414-X(15)-H430-X(5)-H436, in the C-terminal half of gp17 is thought to be involved in the terminase cleavage. Here, exhaustive site-directed mutagenesis revealed that none of the cysteine and histidine residues other than the H436 residue is critical for function. On the other hand, a cluster of conserved residues within this region, D401, E404, G405, and D409, are found to be critical for function. Biochemical analyses showed that the D401 mutants exhibited a novel phenotype, showing a loss of in vivo DNA cutting activity but not the DNA packaging activity. The functional nature of the critical residues and their disposition in the conserved loop region between two predicted beta-strands suggest that these residues are part of a metal-coordinated catalytic site that cleaves the phosphodiester bond of DNA substrate. The data suggest that the T4 terminase consists of at least two functional domains, an N-terminal DNA-translocating ATPase domain and a C-terminal DNA-cutting domain. Although the DNA recognition mechanisms may be distinct, it appears that T4 and other phage terminases employ a common catalytic paradigm for phosphodiester bond cleavage that is used by numerous nucleases.
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PMID:Defining the bacteriophage T4 DNA packaging machine: evidence for a C-terminal DNA cleavage domain in the large terminase/packaging protein gp17. 1459 98


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