Measurements of single DNA molecule packaging dynamics in bacteriophage lambda reveal high forces, high motor processivity, and capsid transformations

Abstract

Molecular motors drive genome packaging into preformed procapsids in many double-stranded (ds)DNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage lambda. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage phi29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged approximately 600 bp/s, which is 3.5-fold higher than phi29, and the motor processivity was also threefold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging, and much greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid, which lacks an accessory protein (gpD).

Fig. 1

(A) Schematic illustration of the experiment. λ proheads were attached to antibody-coated microspheres and captured in an optical trap (bottom left). A microsphere carrying the DNA-terminase complexes was captured in a second optical trap (top left). The bottom trap was moved with respect to the top trap while monitoring the force acting on the top microsphere. To initiate DNA packaging, the microspheres were brought into near contact for ~3 s (middle) and then quickly separated to probe for DNA binding and translocation (right). (B) Force generated by individual motors measured with fixed trap positions. The recordings start at 5 pN and the force opposing the motor increases as packaging proceeds and the tension in the DNA rises. Individual recordings have been arbitrarily offset along the time axis for display purposes. Several examples of pauses of the motor, as discussed in the text, are marked by “p”. The arrow denotes the highest force measured (51 pN).

Fig. 2

(A) Dependence of pausing frequency on applied force. Frequency was calculated as the number of pauses per second that were recorded in particular force ranges. (B) Dependence of pause duration on applied force.

Fig. 3

Average motor velocity versus applied load force. The dashed line is a fit to a single decaying exponential and the solid line is a fit to a sum of two decaying exponentials, as described in the text.

Fig. 4

Packaging dynamics measured with a constant 5 pN load (force clamp). Each line is a plot of DNA tether length versus time recorded for an individual complex. Individual recordings have been arbitrarily offset along the time axis for display purposes. The plateaus seen in some records (marked “p”) indicate pauses of the motor. The section marked “s” in the far right record indicates a slip in which the motor temporarily lost grip on the DNA (see text).

Fig. 5

(A) Average packaging rate versus % of the native 48.5 kbp genome length packaged. The x-axis scale is the same as in panel B. Dashed lines indicate transition points, as discussed in the text. The average velocity was determined from N=97, 68, 21, 16, and 9 datasets that reached 40, 60, 80, 90, and 100% genome packaging, respectively. (B) Average internal force versus % of genome length packaged. Inset schematic diagrams indicate the various capsid transitions, as discussed in the text.

Fig. 6

(A) Examples of records showing a dip in the packaging rate in the vicinity of 30% packaging. (B) Examples of other records without a clearly resolved dip at that position. The two plots share the same x-axis scale.

Fig. 7

Examples of packaging measurements with a DNA construct that translocated beyond the native genome length. These datasets were recorded in force-clamp mode with a constant load of 5 pN. (A) % of genome length packaged versus time. Plots for six different complexes have been displaced arbitrarily along the time axis for clarity. (B) Velocity versus % of genome length packaged calculated in a 5 s sliding window. Plots for the six different complexes are shown in different colors.