Real-time observation of signal recognition particle binding to actively translating ribosomes.

Thomas R Noriega,Joseph D Puglisi,Peter Walter,Jin Chen

doi:10.7554/elife.04418

Thomas R Noriega, Joseph D Puglisi + Show 2 more

Open Access

https://doi.org/10.7554/elife.04418

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Abstract

The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

Highlights

The signal recognition particle (SRP) in all three kingdoms of life catalyzes the co-translational targeting of membrane and secretory proteins (Egea et al, 2005; Zhang and Shan, 2014)
To observe SRP binding at relevant concentrations, we performed single-molecule fluorescence resonance energy transfer (smFRET) experiments using zero-mode waveguides (ZMWs), in which fluorescence measurements are taken from reactions occurring within small metallic apertures (∼150 nm in diameter) that are patterned onto a glass substrate (Chen et al, 2014a)
We show that under close to physiological conditions, SRP-ribosome-nascent chain complexes (RNCs) interactions change as the nascent chain grows and a signal sequence becomes exposed

Summary

Introduction

The signal recognition particle (SRP) in all three kingdoms of life catalyzes the co-translational targeting of membrane and secretory proteins (Egea et al, 2005; Zhang and Shan, 2014). At the beginning of the targeting reaction, SRP binds to a ribosome-nascent chain complex (RNC). If the RNC displays a signal sequence, RNC-bound SRP binds the SRP receptor at the target membrane (the endoplasmic reticulum membrane in eukaryotes, or the inner membrane in prokaryotes). The membrane-localized RNC is transferred to the translocon, a protein translocation channel through which the nascent chain passes across, or into, the target membrane. The E. coli SRP, which is used in these studies, can efficiently replace mammalian SRP in in vitro targeting reactions, demonstrating that it retains the core targeting functionality (Bernstein et al, 1993; Powers and Walter, 1997)

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