Activated GTPase movement on an RNA scaffold drives co-translational protein targeting

Abstract

Roughly one third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane1. 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 sequences2–4 and, via interactions with the SRP receptor5,6, delivers them to the protein translocation machinery on the target membrane7. 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.

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

“…S4C), confirming Ffh-FtsY NG domain flexibility. Our finding is consistent with the observation that targeting complexes formed with mutant FtsY(A335W), which blocks the rearrangement from the closed to the activated state, are incapable of docking to the distal site of 4.5S RNA in single-molecule FRET experiments (31). Instead, the closed complex appears to adopt intermediate states where the NG domains are found in positions ranging from the RNA tetraloop to the RNA distal end (Fig.…”

“…Instead, the closed complex appears to adopt intermediate states where the NG domains are found in positions ranging from the RNA tetraloop to the RNA distal end (Fig. 2D) (31).…”

“…Whereas the RNC stabilizes the early state (13), the transition to the activated state is triggered by binding to the SecYEG translocon (18,31). Indeed, L23 is accessible in the closed state for translocon binding (Fig.…”

Allosteric Response and Substrate Sensitivity in Peptide Binding of the Signal Recognition Particle

“…We now analyze the microsecond timescale MD trajectories in the context of a recent study by Shen et al (16), which used single molecule FRET experiments to monitor the SRP conformational dynamics. This experimental study labeled the SRP RNA distal end and NG domain and found that the SRP samples both a low efficiency and a high efficiency FRET state, with differences in FRET efficiency that correspond to distance changes of ϳ10 nm.…”

“…Both structural (12)(13)(14)(15)(16)(17)(18)(19)(20)(21) and biochemical work (22)(23)(24) suggest that the SRP exhibits multiple stable conformations that are important for protein targeting. The conserved functional core of the SRP (Fig.…”