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Published December 13, 2012 | Supplemental Material + Accepted Version
Journal Article Open

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


Approximately one-third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane. 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 and, through interactions with the SRP receptor delivers them to the protein-translocation machinery on the target membrane. 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.

Additional Information

© 2012 Macmillan Publishers Limited. Received 31 July; accepted 26 October 2012. Published online 12 December 2012. We thank N. Ban and members of the Shan group for comments on the manuscript, C. Richards, L. Cai, T. Zhiyentayev, K. Lee and R. Zhou for help with RNC labelling and the instrument and software setup, and C. L. Guo, S. Kouand H. Lester for discussions. This work is supported by National Institutes of Health (NIH) grant GM078024 to S.-o.S., an NIH instrument supplement to grant GM45162 to D.C. Rees, and Caltech matching fund 350270 for the single-molecule instruments. S.-o.S. was supported by the Beckman Young Investigator award, the David and Lucile Packard Fellowship in Science and Engineering, and the Henry Dreyfus Teacher-Scholar award. T. H. was supported by National Science FoundationPhysics Frontiers Centers program (08222613) and NIH grant GM065367. Author Contributions: K.S., S.A., T.H. and S.-o.S. conceived the experiments. K.S. purified and labelled Ffh, FtsY, DNA, RNA and RNC. D.A. purified SecYEG and performed the GTPase assay in Fig. 4a. K.S. and S.A. carried out smFRET measurements under the direction of T.H. K.S. and S.A. analysed the data. K.S. and S.-o.S. wrote the paper with inputs from all other authors.

Attached Files

Accepted Version - nihms418620.pdf

Supplemental Material - nature11726-s1.pdf


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