Molecular Mechanism of GTPase Activation at the Signal Recognition Particle (SRP) RNA Distal End

Kuang Shen,Yaqiang Wang,Yu-Hsien Hwang Fu,Qi Zhang,Juli Feigon,Shu-Ou Shan

doi:10.1074/jbc.m113.513614

Abstract

The signal recognition particle (SRP) RNA is a universally conserved and essential component of the SRP that mediates the co-translational targeting of proteins to the correct cellular membrane. During the targeting reaction, two functional ends in the SRP RNA mediate distinct functions. Whereas the RNA tetraloop facilitates initial assembly of two GTPases between the SRP and SRP receptor, this GTPase complex subsequently relocalizes ∼100 Å to the 5',3'-distal end of the RNA, a conformation crucial for GTPase activation and cargo handover. Here we combined biochemical, single molecule, and NMR studies to investigate the molecular mechanism of this large scale conformational change. We show that two independent sites contribute to the interaction of the GTPase complex with the SRP RNA distal end. Loop E plays a crucial role in the precise positioning of the GTPase complex on these two sites by inducing a defined bend in the RNA helix and thus generating a preorganized recognition surface. GTPase docking can be uncoupled from its subsequent activation, which is mediated by conserved bases in the next internal loop. These results, combined with recent structural work, elucidate how the SRP RNA induces GTPase relocalization and activation at the end of the protein targeting reaction.

Highlights

A large GTPase movement on the signal recognition particle (SRP) RNA activates GTP hydrolysis to complete protein targeting
An Intact Primary Docking Site Is Required for GTPase Activation at the RNA Distal End—To provide mechanistic details on how the SRP RNA distal end triggers GTP hydrolysis in the Ffh-FtsY GTPase complex, we revisited the 92-mer SRP RNA, the minimal RNA construct that can stimulate GTP hydrolysis [27]
Deletion of every nucleotide beyond C101 reduced the kcat value of the SRP-FtsY complex to levels in the absence of the SRP RNA (Fig. 2, A and B), even though single mutations of most of these nucleotides exhibited no significant defect (Fig. 3). These observations suggest that an intact docking site at the distal end of SRP RNA is required to stimulate GTPase activation

Summary

Introduction

A large GTPase movement on the signal recognition particle (SRP) RNA activates GTP hydrolysis to complete protein targeting. Results: GTPase activation requires distinct primary and secondary docking sites and catalytic bases, optimally positioned on the SRP RNA. Loop E plays a crucial role in the precise positioning of the GTPase complex on these two sites by inducing a defined bend in the RNA helix and generating a preorganized recognition surface. GTPase docking can be uncoupled from its subsequent activation, which is mediated by conserved bases in the internal loop. These results, combined with recent structural work, elucidate how the SRP RNA induces GTPase relocalization and activation at the end of the protein targeting reaction

Results

Discussion

Conclusion