Dynamics of the T4 Bacteriophage DNA Packasome Motor

Abstract

Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y-or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N-and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.The nucleic acid packaging machinery of double-stranded DNA and RNA bacteriophages and of herpesviruses and adenoviruses fills empty preassembled capsid precursors by broadly conserved ATP-driven motor mechanisms (1-3). In most of the large tailed double-stranded DNA bacteriophages, a prohead is filled to near liquid crystalline DNA density (ϳ500 mg/ml) from a replicative genomic concatemer in vivo by a powerful packasome motor (4). The motor consists of a multimeric terminase enzyme docked onto a unique capsid vertex containing the DNA entrance and exit channel-containing portal dodecamer. In conjunction with high ATP consumption, the terminase-portal motor translocates DNA through the prohead portal, and the terminase also cuts the genomic concatemer to form the mature viral particle DNA ends upon receiving a headful signal from the portal, hence the name terminase (5, 6).The essential components of the packasome motor appear to share many structural and mechanistic features among intensively studied double-stranded DNA phages T4, lambda, P22, SPP1, Phi29, and others (7). Accordingly, a number of general mechanisms for DNA translocation have been proposed, although some can now apparently be eliminated by more recent experiments employing a variety of genetic, biochemical, biophysical, and structural approaches. Thus, genetic and biophysical evidence suggests that the D...

DNA Scrunching in the Packaging of Viral Genomes

“…The intermediate steps are not yet known in detail, but FRET measurements reveal that conformational changes in the T4 terminase/portal complex and within the DNA substrate are both essential components of the power stroke. 21,33,69 These results support Black’s proposal that lever-like motions drive translocation by crunching DNA with a transient spring-like compression mechanism. 3,19,21 However, the specific protein–DNA contacts implied by this mechanism have not yet been identified.…”

“…It is not yet clear exactly how the events of the chemical cycle are coupled to the mechanical events that lead to DNA translocation. Do conformational changes in the ATPase crunch DNA, 3,19,21,33,69,70 or do they drive conformational changes in the connector that alternately dehydrate and rehydrate the DNA, causing DNA scrunching? 31 Or does the truth lie somewhere in between?…”

Structure, Assembly, and DNA Packaging of the Bacteriophage T4 Head

“…Consistent with this function and the torsional model is the release of DNA compression in arrested Y-DNA substrates by the addition of purified gp49 enzyme in vitro (Fig. 11A) (Dixit et al ., 2011). Active terminases labeled at the N- and C-terminal ends with a single dye molecule showed that the FRET distance between the N-terminal GFP-labeled portal protein and gp17 is 6.9 nm for the N terminus and 5.7 nm for the C terminus (Figs.…”

“…As compared to the wild type, portals containing C-terminal GFP fusions but not N-terminal GFP fusions (Dixit et al ., 2011) lock the proheads into the unexpanded conformation unless terminase packages DNA, suggesting that the portal plays a central role in controlling prohead expansion. Expansion is required to protect the packaged DNA from nuclease but not for packaging itself as measured by fluorescence correlation spectroscopy (Ray et al ., 2009).…”

“…Resolvase release of trapped Y-DNAs is also correlated with increased distance (∼0.6 nm, or 5.1 to 5.7 nm) between the terminase and the portal during packaging (B). Distances are measured from N and C termini of gp17 to the GFP–gp20 portal by FRET (C)(Dixit et al ., 2011). …”