Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4

Lin, Hsingchi; Rao, Venigalla B.; Black, Lindsay W.

doi:10.1006/jmbi.1999.2781

Cited by 60 publications

(54 citation statements)

References 34 publications

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“…Furthermore, in fragment A, only nonsense mutations and mutations inferred to interfere with gene 6 translation were observed (data not shown), indicating that amino acid substitutions in the amino-terminus of the protein are well tolerated. Despite our systematic search, we did not find temperature-sensitive mutations in the SPP1 portal protein, in contrast to what was observed for the portal protein of bacteriophage T4 (Hsiao and Black, 1977;Lin et al, 1999). The frequency of this type of mutation varies significantly among proteins, most probably depending on particular features of (Table 2) according to the colour code shown on the left.…”

Section: Achievement Of a Collection Of Gp6 Lethal Missense Mutantscontrasting

confidence: 66%

The high‐resolution functional map of bacteriophage SPP1 portal protein

Isidro

Santos

Henriques

et al. 2003

Molecular Microbiology

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SummaryAn essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6 . Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.

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Section: Achievement Of a Collection Of Gp6 Lethal Missense Mutantscontrasting

confidence: 66%

The high‐resolution functional map of bacteriophage SPP1 portal protein

Isidro

Santos

Henriques

et al. 2003

Molecular Microbiology

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“…16). A different phenotype is associated with several terminase ts mutations near its C terminus: accumulation of partially DNA filled proheads (16). Such mutants are thus DNA translocation defective, similar to the ID effects seen on translocation.…”

Section: Discussionmentioning

confidence: 92%

“…Various temperature-sensitive (ts) mutations affecting DNA translocation were previously located at the C-terminal end of gp17 (reviewed in ref. 16 or newly isolated by us, in Materials and Methods). Two ts mutants showed changes at residue Y562.…”

Section: Ac Q and N Sites Coordinate With Each Other And May Be Nearmentioning

confidence: 99%

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

Dixit¹,

Ray

Black³

2012

Proc. Natl. Acad. Sci. U.S.A.

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Viral genome packaging into capsids is powered by high-forcegenerating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient gripand-release structural change in B form DNA.DNA structure | terminase inhibitors N ucleic acid translocation into an empty procapsid is a conserved capsid assembly mechanism found among diverse DNA and RNA viruses (1). High-resolution structures of all of the conserved motor components found among tailed dsDNA bacteriophages have been determined (2-5). The small bacteriophage T4 terminase protein gp16 is required for cutting and packaging the replicative DNA concatemer in vivo but is nonessential and inhibitory for linear DNA packaging in vitro (6). Thus, T4 DNA translocation in vitro can be driven by a two-component motor consisting of a prohead portal ring channel dodecamer situated at a single packaging vertex that is docked during packaging with gp17 terminase-ATPase. A linear packaging motor mechanism proposed that a terminase to portal DNA grip-and-release driven by a motor protein conformational change drives DNA into the prohead by a DNA compression motor stroke (7-10).It has long been known that acridine dyes inhibit bacteriophage development at concentrations below those that inhibit growth of the bacterial host. Phage mutations that confer resistance to such dyes arise through different mechanisms. Among these are mutations in the phage T4 terminase gene 17, called ac and q, that confer acridine and quinacrine resistance (11). All 9-aminoacridine (9AA) treatments have been shown by electron microscopy to arrest DNA packaging in vivo; however, whether the site of action of 9AA is the DNA, the active site of the packaging enzyme, or the enzyme DNA complex-in fact, acridines are known to inactivate protease active sites or virion proteins associated with DNA (reviewed by Black et al., in r...

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“…The T4 packaging machine consists of three components ( Fig. 1): (i) an empty prohead containing a dodecameric portal protein, gp20 (25,38); (ii) a pentameric large terminase motor protein, gp17 (5,24,42); and (iii) an 11-or 12-mer small terminase regulatory protein, gp16 (2,24,35). The cone-shaped portal links the head to the motor and DNA, with its wider end inside the capsid and the narrower end protruding outside.…”

mentioning

confidence: 99%

Portal-Large Terminase Interactions of the Bacteriophage T4 DNA Packaging Machine Implicate a Molecular Lever Mechanism for Coupling ATPase to DNA Translocation

Hegde

Padilla-Sanchez

Draper

et al. 2012

J Virol

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DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11-or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.T ailed bacteriophages and herpesviruses use powerful ATPdriven machines to package their genomes into preformed capsid shells (36). They generate forces greater than 60 pN, ϳ20 times that of myosin motor, in order to compact a highly negatively charged, relatively rigid double-stranded DNA (dsDNA) to near-crystalline density (ϳ500 g/ml) (22, 39). The phage T4 employs one of the fastest and most powerful packaging machines reported to date. Packaging at a rate of up to ϳ2,000 bp/s, the T4 machine is estimated to generate twice the power (ϳ5,000 kW/ m 3 ) of an automobile engine (12, 36). The T4 packaging machine consists of three components ( Fig. 1): (i) an empty prohead containing a dodecameric portal protein, gp20 (25, 38); (ii) a pentameric large terminase motor protein, gp17 (5, 24, 42); and (iii) an 11-or 12-mer small terminase regulatory protein, gp16 (2, 24, 35). The cone-shaped portal links the head to the motor and DNA, with its wider end inside the capsid and the narrower end protruding outside. It has a central channel with a diameter of ϳ35 Å, through which DNA is threaded into the capsid. The portal works in conjunction with the motor, but its exact role is unclear. Various models such as the portal acting as a valve, DNA cruncher, or packaging sensor have been proposed (8,9,10,38,43).Five molecules of gp17 assemble on the portal into a packaging motor (Fig. 1). gp17 consists of an N-terminal ATPase domai...

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Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4

Cited by 60 publications

References 34 publications

The high‐resolution functional map of bacteriophage SPP1 portal protein

The high‐resolution functional map of bacteriophage SPP1 portal protein

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

Portal-Large Terminase Interactions of the Bacteriophage T4 DNA Packaging Machine Implicate a Molecular Lever Mechanism for Coupling ATPase to DNA Translocation

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