“Single-molecule studies of viral DNA packaging motors in bacteriophages φ29, λ, and T4”
Douglas E. Smith – Department of Physics, University of California, San Diego, CA
key step in the assembly of many viruses is the packaging of DNA into a viral procapsid by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. Using improved techniques, we can now measure DNA binding, initiation of translocation, and DNA translocation dynamics throughout packaging. In the bacteriophage φ29 system we have studied ionic effects on portal motor function and on the forces resisting DNA packaging, confirming the importance of DNA-DNA electrostatic repulsion. We have also extended our techniques to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three viruses have different capsid sizes and shapes, different genome lengths, and structural differences in their packaging motors. We compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very high forces, as needed overcome high forces that resist the dense confinement of the stiff, highly charged DNA within the viral capsids. However, striking differences were also observed. The T4 motor translocates DNA very rapidly, at rates approaching 2000 bp/s, and exhibits large static and dynamic variability. In bacteriophage λ, the measured forces resisting packaging are equal to the measured forces driving DNA ejection. We also find evidence for an effect of λ procapsid expansion on packaging dynamics, and sudden rupture of capsids at >90% genome packaging in the absence of a putative capsid stabilizing protein, gpD.