Activated GTPase movement on an RNA scaffold drives cotranslational protein targeting

Abstract

Roughly one third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane 1 . The proper localization of these proteins is mediated by a universally conserved protein targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences 2-4 and, via interactions with the SRP receptor 5,6 , delivers them to the protein translocation machinery on the target membrane 7 . The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA whose precise functions have remained elusive. Here, we used single molecule fluorescence microscopy to demonstrate that the SRP-receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism to ensure the productive exchange of the targeting and translocation machineries at the ribosome exit site with exquisite spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the facile exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes.Cotranslational protein targeting face fundamental challenges in both spatial and temporal coordination. Spatially, both the SRP 2-4 and SecYEG (or Sec61p) translocon 7 contact the L23 ribosomal protein and the signal sequence, raising puzzling questions about how the translating ribosome is transferred from the targeting to translocation machinery. Temporally, guanosine-5′-triphosphate (GTP) hydrolysis by the SRP-SRP receptor complex, Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

Single-Molecule Studies of Bacterial Protein Translocation

“…57 The dynamics of this process has recently been investigated using the single-molecule fluorescence assay based on Forster's resonance energy transfer (FRET) between fluorophores conjugated within the E. coli SRP. 58 Individual SRP molecules bearing fluorophores at the Ffh protein and at the distal end of the 4.5S RNA were specifically immobilized on a surface and imaged by a total internal reflection fluorescence (TIRF) microscope. The background fluorescence is strongly reduced in the TIRF microscope, as the formed evanescence excitation wave illuminates the volume only within 100 nm of the surface, so detection of single surface-bound molecules can be achieved.…”