Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage .lambda.

Tomka, Mary Ann; Catalano, Carlos Enrique

doi:10.1021/bi00096a008

Cited by 69 publications

(199 citation statements)

References 21 publications

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“…Unlike SPP1 H6-G2P ATPase, the phage gpA ATPase is capable of hydrolyzing different nucleoside triphosphates (28,29) and is active in the presence of Ca 2ϩ as a divalent metal (29,33). Both gpA (28,29,33) and H6-G2P (this work) display an in vitro ATPase activity independent of proheads, whereas such hydrolysis is dependent of proheads in the 29, T3, and T7 packaging systems (see Ref.…”

Section: Discussionmentioning

confidence: 87%

“…Unlike the H6-G2P of SPP1, faithful cleavage of the P1 pac site requires phage-encoded terminase (PacA and PacB) and two E. coli chromatin-associated proteins (IHF and HU) (27). Furthermore, in the case of phage , the endonuclease activity of the terminase large subunit, gpA, is stimulated by the presence of the E. coli IHF protein (28,29) and ATP (29,30).…”

Section: Discussionmentioning

confidence: 99%

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Functional Analysis of the Terminase Large Subunit, G2P, of Bacillus subtilis Bacteriophage SPP1

Gual

Camacho²,

Alonso

2000

Journal of Biological Chemistry

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The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5-RCGG2CW-3 sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5-CTATTGCGG2C-3 sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P i . H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1P⌬N62 (lacking domain I) stimulate 3.5-and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.Initiation of packaging of double-stranded viral DNA (dsDNA) 1 involves the specific interaction of the prohead with viral DNA in a process mediated by a phage-encoded terminase protein (1-4). The terminase enzymes are usually hetero-oligomers composed of a small and a large subunit. The role of the terminase small subunit is to specifically recognize the packaging initiation site (cos or pac) and to form a nucleoprotein structure that helps to position the terminase large subunit to cleave at the pac or cos site (1-6). Two general principles for packaging of concatemeric DNA into a virus head have been proposed. The first implies a site-specific packaging that shows some constraint in DNA size, in which the recognition sequence (termed cos in phage ) plays an important role in initiation and termination of packaging. This packaging process is wellcharacterized in coliphages , T3, and T7 (1-6). The second principle implies headful packaging, in which the packaging initiates at a specific site (termed pac in phage SPP1) but with the capacity of the prohead playing a predominant role in the termination step. Within the poorly characterized headful packaging mechanism, phages P1, P22, T4, and SPP1 are included (1, 3, 7).The terminase enzyme of Bacillus subtilis phages SPP1 and SF6 is composed of a small (G1P) and a large (G2P) subunit (8). The terminase initiates unidirectional DNA packaging from a concatemeric DNA substrate by binding to pac DNA, introducing a 1-bp staggered cut, within the 83-bp pacC subsite, and encapsidating the DNA from the cleaved end into an empty prohead until no more DNA can be inserted (headful) (8 -11). DNA packaging terminates when the DNA inside the prohead is separated from the concatemer by a cutting process (headful cut). The headful cleavage...

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Functional Analysis of the Terminase Large Subunit, G2P, of Bacillus subtilis Bacteriophage SPP1

Gual

Camacho²,

Alonso

2000

Journal of Biological Chemistry

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“…The large subunits are ATP-binding proteins and are thought to carry crucial functions for DNA translocation. In the case of l, the small subunit also has ATP binding motifs (Becker & Gold 1988) and displays a DNA-stimulated ATPase (Tomka & Catalano 1993;Hwang et al 1996).…”

Section: Dna Packaging Machinerymentioning

confidence: 99%

Phage DNA packaging

1997

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Phage DNA packaging occurs by DNA translocation into a preformed protein shell-a prohead-with the aid of a packaging enzyme or a terminase. The packaging enzyme is composed of two subunits: the large subunit has ATP-binding, prohead binding, and DNA cleavage activities, and the small subunit is a DNA binding protein. DNA translocation is driven by ATP hydrolysis. In general, phage DNA replication mechanisms lead to the accumulation of concatemers. Concatemers are processed to mature DNA during and depending upon DNA packaging. This review will focus on the molecular mechanism of concatemer processing and the coupling of ATP hydrolysis to DNA translocation.

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“…the ATPase is a non-structural protein) (4). In particular, very little is known about the enzymatic action of these ATPases during packaging (7)(8)(9).…”

mentioning

confidence: 99%

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

Lísal

Kainov

Bamford

et al. 2004

Journal of Biological Chemistry

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Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid. Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid. The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive. In this study, the mechanism of RNA translocation in doublestranded RNA bacteriophages is characterized using rapid kinetic analyses. The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity. The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase. The slower phase exhibits a high activation energy and has been assigned to a conformational change. ATP binding becomes cooperative in the presence of RNA. Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity. On the other hand, ADP release is fast and RNA-independent. The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation. Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone. The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged. This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.Genome encapsidation in many viruses is realized through energy-dependent packaging into a preformed capsid (procapsid). This requires molecular motors (portal complexes) converting chemical energy (ATP hydrolysis) into mechanical work (translocation). A wealth of structural and functional data has been obtained for portal complexes of dsDNA 1 bacteriophages (e.g. 29, , SPP1, P22, T4, and T-odd phages (1-6), but the molecular basis of the mechanochemical coupling is not understood. This is partly because of the complexity of portal complexes, in which the packaging ATPase is a multifunctional enzyme and associates transiently with the packaging machinery (i.e. the ATPase is a non-structural protein) (4). In particular, very little is known about the enzymatic action of these ATPases during packaging (7-9).The packaging system of dsRNA bacteriophages from the Cystoviridae family (phages 6-14 (10, 11)) is considerably simpler. The ssRNA translocation is fueled by a single structural protein P4. P4 forms stable hexamers (12, 13), which possess nonspecific nucleotide triphosphatase activity and resides at the 5-fold vertices of the procapsid (14, 15). The dodecahedral procapsid results from co-assembly of the major structural protein P1 with P4 hexamers and minor proteins P2 (RNA polymerase) and P7 (assembly and functional cofactor) (16,17).Cystoviruses have a tripartite dsRNA genome. The procapsid-associa...

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Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage .lambda.

Cited by 69 publications

References 21 publications

Functional Analysis of the Terminase Large Subunit, G2P, of Bacillus subtilis Bacteriophage SPP1

Functional Analysis of the Terminase Large Subunit, G2P, of Bacillus subtilis Bacteriophage SPP1

Phage DNA packaging

Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages

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