Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Reza Vafabakhsh, Kiran Kondabagil, Tyler Earnest, Kyung Suk Lee, Zhihong Zhang, Li Dai, Karin A. Dahmen, Venigalla B. Rao, Taekjip Ha

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)15096-15101
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number42
DOIs
StatePublished - Oct 21 2014

Keywords

  • Molecular motors
  • Single-molecule fluorescence imaging
  • Virus packaging

ASJC Scopus subject areas

  • General

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