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

Nature volume 492, pages 271–275 (2012)Cite this article

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Abstract

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.

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Figure 1: smFRET–TIRF microscopy reveals dynamic movements of the SRP–FtsY complex on the SRP RNA.
Figure 2: The distal site of SRP RNA is crucial for GTPase activation and protein targeting.
Figure 3: Conformational rearrangements within the SRP–FtsY GTPase complex drive its movement to the RNA distal site.
Figure 4: RNC and SecYEG regulate GTPase movements on the SRP RNA.

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Acknowledgements

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. Kou and 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 Foundation Physics Frontiers Centers program (08222613) and NIH grant GM065367.

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Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Kuang Shen, David Akopian & Shu-ou Shan

  2. Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Sinan Arslan & Taekjip Ha

  3. Howard Hughes Medical Institute, Urbana, 61801, Illinois, USA

    Taekjip Ha

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  1. Kuang Shen
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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.

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Correspondence to Shu-ou Shan.

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Shen, K., Arslan, S., Akopian, D. et al. Activated GTPase movement on an RNA scaffold drives co-translational protein targeting. Nature 492, 271–275 (2012). https://doi.org/10.1038/nature11726

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