A Multistep Mechanism for Assembly of the SRP–SR Complex
Two GTPases in the signal recognition particle (SRP) and its receptor (SR) control the delivery of newly synthesized proteins to the ER or plasma membrane. During the protein targeting reaction, the 4.5S SRP RNA accelerates the association between the two GTPases by 400-fold. Using fluorescence resonance energy transfer (FRET), we demonstrate here that formation of a stable SRP–SR complex involves two distinct steps: a fast initial association between SRP and SR to form an early, GTP-independent complex, followed by a GTP-dependent conformational rearrangement to form the stable, final complex. We also found that the 4.5S SRP RNA significantly stabilizes the early, GTP-independent intermediate. Further, mutational analyses show that there is a strong correlation between the ability of the mutant SRP RNAs to stabilize the early intermediate and their ability to accelerate SRP–SR complex formation. We propose that the SRP RNA, by stabilizing the transient early intermediate, can give this intermediate a longer dwell time and therefore a higher probability to rearrange to the final, stable complex. This provides a coherent model that explains how the 4.5S RNA exerts its catalytic role in SRP–SR complex assembly.
KeywordsHydrolysis Filtration Hydroxyl EDTA Titration
We thank members of the Shan Laboratory for comments on the manuscript. This work was supported by NIH grant GM078024 to S.S. S.S. was supported by the Burroughs Wellcome Fund career award, the Henry and Camille Dreyfus foundation, the Beckman Young Investigator award, and the Packard and Lucile award in science and engineering. X.Z. was supported by a fellowship from the Ulric B. and Evelyn L. Bray Endowment Fund.
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