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

Abstract

Single-molecule fluorescence microscopy techniques are used to elucidate features of the highly conserved protein-targeting machinery known as the signal recognition particle (SRP); the long SRP RNA is shown to be crucial for correct timing and precision of cargo handover to the protein-translocation machinery, a finding that could help to explain how other ribonucleosome complexes function during complex cellular processes. The signal recognition particle is a ribonucleoprotein that is essential for proper localization of proteins. It recognizes newly translated proteins and binds to the ribosome-nascent chain complex. This study uses single-molecule approaches to elucidate the mechanism by which cargo handover occurs from the signal recognition particle to the SecYEG channel that handles protein translocation into the endoplasmic reticulum. The results provide evidence for a model in which large functional RNAs act as molecular scaffolds to coordinate large-scale protein movements. Approximately 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 sequences2,3,4 and, through 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 for which precise functions have remained elusive. Here we used single-molecule fluorescence microscopy to show that the Escherichia coli SRP–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 for ensuring the productive exchange of the targeting and translocation machineries at the ribosome exit site with high spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the easy 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.