title: Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4.
abstract: Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign.
authors.forename: Reza
authors.initials: R
authors.lastname: Vafabakhsh
authors.affiliation: Department of Biology, The Catholic University of America, Washington, DC 20064.
authors.forename: Kiran
authors.initials: K
authors.lastname: Kondabagil
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign.
authors.forename: Emmy E
authors.initials: EE
authors.lastname: Earnest
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign.
authors.forename: Kyung Suk
authors.initials: KS
authors.lastname: Lee
authors.affiliation: Department of Biology, The Catholic University of America, Washington, DC 20064.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Biology, The Catholic University of America, Washington, DC 20064.
authors.forename: Li
authors.initials: L
authors.lastname: Dai
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign.
authors.forename: Karin A
authors.initials: KA
authors.lastname: Dahmen
authors.affiliation: Department of Biology, The Catholic University of America, Washington, DC 20064 rao@cua.edu tjha@illinois.edu.
authors.forename: Venigalla B
authors.initials: VB
authors.lastname: Rao
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign, Howard Hughes Medical Institute, Urbana, IL 61801; and rao@cua.edu tjha@illinois.edu.
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D017122:Bacteriophage T4 / Q000502:physiology*; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins / Q000737:chemistry; D042003:DNA Packaging; D004279:DNA, Viral / Q000737:chemistry*; D016679:Genome, Viral; D008856:Microscopy, Fluorescence; D020409:Molecular Motor Proteins / Q000737:chemistry; D038761:Photobleaching; D011487:Protein Conformation; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D020409:Molecular Motor Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: molecular motors; single-molecule fluorescence imaging; virus packaging