Abstract
SummaryThe twin-ATPase ABCE1 has a vital function in mRNA translation by recycling terminated or stalled ribosomes. As for other functionally distinct ATP-binding cassette (ABC) proteins, the mechanochemical coupling of ATP hydrolysis to conformational changes remains elusive. Here, we use an integrated biophysical approach allowing direct observation of conformational dynamics and ribosome association of ABCE1 at the single-molecule level. Our results from FRET experiments show that the current static two-state model of ABC proteins has to be expanded because the two ATP sites of ABCE1 are in dynamic equilibrium across three distinct conformational states: open, intermediate, and closed. The interaction of ABCE1 with ribosomes influences the conformational dynamics of both ATP sites asymmetrically and creates a complex network of conformational states. Our findings suggest a paradigm shift to redefine the understanding of the mechanochemical coupling in ABC proteins: from structure-based deterministic models to dynamic-based systems.
Highlights
Ribosome recycling is an integral step of mRNA translation and surveillance at the core of protein homeostasis, ribosome-based quality control, and ribosome-related diseases (Jackson et al, 2012; Mills and Green, 2017; Nu€renberg and Tampe, 2013)
SmFRET Monitors the Tweezer-like Movement at each ATP Site of ABCE1 To characterize the dynamic behavior of the two ATP sites of ABCE1 by single-molecule-based Forster resonance energy transfer (smFRET), double-cysteine variants of ABCE1 were generated (Figure 1A)
Stochastic labeling was realized by mixing of purified ABCE1 with two fluorophores, e.g., the spectrally distinct green FRET donor (D, Cy3B) and the red FRET acceptor (A, Atto647N)
Summary
Ribosome recycling is an integral step of mRNA translation and surveillance at the core of protein homeostasis, ribosome-based quality control, and ribosome-related diseases (Jackson et al, 2012; Mills and Green, 2017; Nu€renberg and Tampe , 2013). This cyclic process connects termination with initiation (Gerovac and Tampe , 2019). ABCE1 splits ribosomes either after canonical termination facilitated by release factors (e/aRF1) or after recognition of stalled and vacant ribosomes by mRNA surveillance factors (e/aPelota and Dom in yeast) (Barthelme et al, 2011; Pisarev et al, 2010; Shoemaker and Green, 2011; Strunk et al, 2012; van den Elzen et al, 2014)
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