Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability

Abstract

Terminase enzyme complexes, which facilitate ATP-driven DNA packaging in phages and in many eukaryotic viruses, constitute a wide and potentially diverse family of molecular motors about which little dynamic or mechanistic information is available. Here we report optical tweezers measurements of single DNA molecule packaging dynamics in phage T4, a large, tailed Escherichia coli virus that is an important model system in molecular biology. We show that a complex is formed between the empty prohead and the large terminase protein (gp17) that can capture and begin packaging a target DNA molecule within a few seconds, thus demonstrating a distinct viral assembly pathway. The motor generates forces >60 pN, similar to those measured with phage phi29, suggesting that high force generation is a common property of viral DNA packaging motors. However, the DNA translocation rate for T4 was strikingly higher than that for phi29, averaging approximately 700 bp/s and ranging up to approximately 2,000 bp/s, consistent with packaging by phage T4 of an enormous, 171-kb genome in <10 min during viral infection and implying high ATP turnover rates of >300 s(-1). The motor velocity decreased with applied load but averaged 320 bp/s at 45 pN, indicating very high power generation. Interestingly, the motor also exhibited large dynamic changes in velocity, suggesting that it can assume multiple active conformational states gearing different translocation rates. This capability, in addition to the reversible pausing and slipping capabilities that were observed, may allow phage T4 to coordinate DNA packaging with other ongoing processes, including viral DNA transcription, recombination, and repair.

Fig. 1.

Schematic of the experiment. T4 prohead–gp17 complexes were attached to antibody-coated microspheres and captured in one optical trap (lower left). Biotinylated DNA molecules were tethered to streptavidin-coated microspheres and captured in a second optical trap (upper left). The bottom trap was moved with respect to the top trap while measuring the force. To initiate packaging, the microspheres were brought into near contact for 1 s (middle) and then quickly separated to detect DNA binding and translocation (right).

Fig. 2.

Force and velocity measurements. (A) Force measurements on n = 33 complexes in the fixed-traps mode. As DNA packaging proceeds, the DNA tension and load opposing the motor increases. Each measurement ends with breakage of the tether (see text). (B) Analysis of the data in A yields the mean velocity vs. applied load. The error bars are standard error of the mean, calculated as SD divided by the square root of the number of measurements (n = 33). The solid line is a linear fit.

Fig. 3.

DNA translocation dynamics and static disorder. (A) DNA tether length vs. time, measured after initiation of packaging and application of a 5-pN force clamp. Each line is a recording on a different packaging complex. Thirteen of the n = 102 recorded data sets are shown. The lower dashed line indicates a packaging rate of 2,000 bp/s, and the uppermost indicates 145 bp/s, the average rate of phage φ29. (B) Distribution of average motor velocities measured for each of the recorded packaging events with n = 102 complexes in the force–clamp mode. (C) Same distribution after editing pauses and slips from the records (see text). (D) Corresponding distribution recorded with φ29 (n = 50). (E) Distribution of rates expected for simulated data sets having the same mean velocity, using the simple kinetic model (see text).

Fig. 4.

Temporal fluctuations in the velocity determined in a 2-s sampling window. (A and B) Examples of complexes exhibiting relatively small changes (A) and relatively large changes (B). (C) Histogram of SD in velocity for all complexes relative to the corresponding SDs for simulated data sets generated by a simple kinetic model (see text).