Single-molecule FRET supports the two-state model of argonaute action

  • Zander A
  • Holzmeister P
  • Klose D
 et al. 
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Argonaute can be found in all three domains of life and is the functional core of the eukaryotic RNA-silencing machinery. In order to shed light on the conformational changes that drive Argonaute action, we performed single-molecule FRET measurements employing a so far uncharacterized member of the Argonaute family, namely Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo). We show that MjAgo is a catalytically active Argonaute variant hydrolyzing exclusively DNA target strands out of a DNA/DNA hybrid. We studied the interplay between Argonaute and nucleic acids using fluorescent dyes covalently attached at different positions of the DNA guide as steric reporters. This allowed us to determine structurally confined parts of the protein scaffold and flexible regions of the DNA guide. Single-molecule FRET measurements demonstrate that the 3'end of the DNA guide is released from the PAZ domain upon target strand loading. This conformational change does not necessitate target strand cleavage but a fully complementary target strand. Thus, our data support the two state model for Argonaute action.

Author-supplied keywords

  • Amber suppressor strategy
  • Archaea
  • Argonaute
  • Biophysics
  • Dynamics
  • FRET
  • Fluorescence
  • Single-molecule fluorescence spectroscopy

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