Genome Packaging in the Complex Double-Stranded DNA Viruses. Terminase enzymes are responsible for processive excision of an individual genome from a concatemeric packaging substrate (genome maturation) and for translocation of the duplex into a pre-formed procapsid shell (genome packaging). Two basic strategies for genome packaging, unit-length and headful, are summarized at bottom, right. Details are provided in the text.

Lambda packaging complexes stalled with ATP-γS are attached by the DNA end to a microsphere (orange) and trapped with optical tweezers. This microsphere is brought near a second trapped microsphere coated with antibodies that bind the capsid (green). When exposed to ATP the motor packages the DNA. After ∼1.5-6 kbp of DNA (blue) is packaged, the complex is moved (dashed arrow) into a region containing either no nucleotide or 0.5 mM ATP-γS, AMP-PNP, or ADP. The length of the DNA outside the capsid versus time is measured.

(A) Representative measurements of DNA length released vs. time in a solution with either no nucleotide (“apo” state, red lines), ADP (0.5 mM, blue), ATP-γS (0.5 mM, black), or AMP-PNP (0.5 mM, magenta). Y-axis indicates the length of DNA released from the nucleocapsid bound to terminase. Gripping is evidenced by horizontal portions of each curve, while slipping is evident during the sloped regions. (B) Zoomed plot showing examples of the pauses with no nucleotide.

Average metrics characterizing the gripping/slipping dynamics, comparing lambda with T4 results measured previously47. Due to the low slipping velocity with the ATP analogs, we could not reliably score individual gripping events, so frequencies and durations are not reported. In the T4 study periods where slipping occurred were determined using the 2σ criterion (see methods) so the same criteria was used to analyze the lambda data presented in this table. All nucleotide conditions were 0.5 mM.

Transient slipping velocities measured in 1 s time intervals with 5 pN applied force. For these plots, and the subsequent figures, periods of slipping vs. gripping were determined using the 3σ criterion (see SI methods).

(A) Average transient slipping velocity vs. applied force. (B) Gripping frequency vs. force, calculated as the number of pauses per amount of time spent slipping. Blue points indicate measurements with no nucleotide and orange points those with 0.5 mM ADP. Error bars indicate standard errors of the means. (C) Mean grip and slip durations vs. force. Blue: slipping with no nucleotide, orange: gripping with no nucleotide, yellow: slipping with 0.5 mM ADP, and purple: gripping with 0.5 mM ADP. (D) Fraction of time spent slipping vs. force, calculated as fraction of 1 s time bins in which slipping occurs, with no nucleotide (blue) or 0.5 mM ADP (orange). Error bars indicate standard errors of the means.

(A) Example of repeated slip/clamp/re-package measurements with 10 pN force. The DNA is packaged with ATP (blue) and when the solution is changed from ATP to ADP (upward arrows) the DNA slips out but is end clamped (red). Each time the complex is moved back into ATP (downward arrows) the DNA is re-packaged. (B) Examples of length of DNA released vs. time measured during slipping with no nucleotide and 5 pN force. Slipping usually arrests when the measured length is around 10 kb, consistent with the full length of the DNA substrate. The observed variation in the final lengths is fully attributable to limits in the accuracy of absolute DNA length measurements with the optical tweezers due variations in the sizes of microspheres used (see SI). (C) Histogram of differences in the DNA length measured in repeated end clamp events with single complexes.