Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

Derek N. Fuller, Dorian M. Raymer, John Peter Rickgauer, Rae M. Robertson, Carlos E. Catalano, Dwight L. Anderson, Shelley N Grimes, Douglas E. Smith

Research output: Contribution to journalArticlepeer-review

151 Scopus citations

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 λ. 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 φ{symbol}29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged ∼600 bp/s, which is 3.5-fold higher than φ{symbol}29, 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).

Original languageEnglish (US)
Pages (from-to)1113-1122
Number of pages10
JournalJournal of Molecular Biology
Volume373
Issue number5
DOIs
StatePublished - Nov 9 2007

Bibliographical note

Funding Information:
We thank Brandy Pappas, James Tsay, Jiao Xie, Mladen Nisavic, and Daniel Cohen for technical assistance and Dr Paul Jardine for thoughtful comments and discussion. Our research was supported by NIH grant GM-071552, The Kinship Foundation, and the Arnold and Mabel Beckman Foundation. D.F. was also supported by NIH grant P01-DK54441 through the Molecular Biophysics Training Program at UCSD. C.E.C was supported by NIH grant GM-063943.

Keywords

  • bacteriophage lambda
  • molecular motor
  • optical tweezers
  • single-molecule
  • viral DNA packaging

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