Old, new, and widely true: The bacteriophage T4 DNA packaging mechanism

Abstract

DNA packaging into empty viral procapsids by ATP-driven motor proteins applies widely among viruses. Recent fluorescence studies of phage T4 reveal: 1) the small terminase subunit (TerS) synapses pac homologs by a twin ring mechanism to gauge DNA maturation and allow packaging by the large terminase subunit (TerL); 2) translocation of linear DNA is efficient by TerL acting alone; expansion of the procapsid is controlled by the portal-terminase assembly; 3) both ends of the packaged DNA are held at the portal, showing a loop of DNA is packaged; 4) transient spring-like compression of B form to A form-like DNA accompanies translocation; 5) the C-terminal domain of TerL is docked to the portal and moves toward it when stalled; 6) a portal bound resolvase can release stalled Y-DNA compression and allow translocation in vitro; and 7) ATP powered translocation on A form dsDNA is supported by recent hexameric helicase studies.

Keywords: A form DNA; DNA motors; DNA packaging; Helicase; Pac site; Terminase.

Fig. 1

Regulatory integration in vivo of packaging, late transcription, and replication among T4-type phages. The phage T4 terminase large subunit ATPase motor protein gp17 (TerL) and the small terminase DNA recognition protein gp16 (TerS) initiate packaging. A gp20 portal bound packaging essential gp49:Endonuclease VII resolvase removes X and Y structures blocking packaging on a branched DNA concatemer in vivo. The gp55 late sigma factor interacts with TerL and appears to be essential for initiating concatemer packaging.

Fig. 2

Evidence against a proposed prohead portal rotory motor mechanism. No rotation of six C-terminal portal-GFPs found as part of the portal dodecamer inside filled 500 mg/ml DNA active phages; no rotation of N-terminal HOC-gp20 portal protein tethered to HOC binding sites on expanded mature in vitro packaging active proheads. ESPs are immature empty small proheads, ELPs are mature and stable empty large proheads with HOC decoration protein binding sites exposed (Baumann et al., 2006).

Fig. 3

Conserved bacteriophage T4 two subunit terminase pac site prohead packaging; (A) large terminase-ATPase subunit TerL alone packages linear DNAs in vitro; (B) small terminase subunit TerS (gp16) is required in vivo for concatemer (or circular DNA) packaging; a “synapsis model” proposes DNA concatemer maturation is assessed by TerS apposing two homologous pac site DNAs. Sequence specific terminase gene amplifications requiring TerS result from apposition of two homologous pac sites (16 and 19) under genetic selection for increased TerL synthesis; normally TerS gene 16–16 pac apposition would initiate packaging; and (C) a two ring two dsDNA TerS Holliday junction strand swap signal is proposed to initiate handoff to TerL for DNA cutting and packaging.

Fig. 4

Torsional compression model for DNA packaging involving transient B to A form spring-like DNA compression as the C-terminal domain of TerL is docked on the portal of the prohead. Although short abnormal DNAs are released by the motor, such DNAs can be anchored by a leader sequence by the terminase-portal motor complex. FRET measurements show once persistence length DNA (~500 bp or more) has been encapsidated the two DNA ends are held at the portal at a fixed distance regardless of DNA length as shown, thus that a loop of DNA is packaged, and thereby preventing knotting in the capsid.

Fig. 5

Substrate and motor dimensions and dynamics by FRET: (A) Increased FRET between stalled Y-stem DNA dye pairs supports B to A form compression; the gp49:Endonuclease VII X- or Y-resolvase decreases the Y-stem DNA FRET and allows completion of packaging. In the stalled anchored Y DNA the terminase moves toward the portal as judged by FRET increase between dye labeled terminase and (B) C-terminal GFP labeled gp20 portal containing proheads, and (C) N-terminal GFP labeled gp20 portal containing proheads that allow determination of the docking orientation in the two component prohead packaging motor.