Measurements of single DNA molecule packaging dynamics in bacteriophage lambda reveal high forces, high motor processivity, and capsid transformations.
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).
Fuller
Derek N
DN
Department of Physics, University of California, San Diego, Mail Code 0379, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Raymer
Dorian M
DM
Rickgauer
John Peter
JP
Robertson
Rae M
RM
Catalano
Carlos E
CE
Anderson
Dwight L
DL
Grimes
Shelley
S
Smith
Douglas E
DE
D000255:Adenosine Triphosphate; D010582:Bacteriophage lambda / Q000235:genetics*; D002213:Capsid; D042003:DNA Packaging; D016679:Genome, Viral; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000502:physiology
D020409:Molecular Motor Proteins; D000255:Adenosine Triphosphate