Abstract
Background:
The initial step of the signal recognition particle (SRP) targeting pathway requires binding to actively translating ribosomes.
Results:
SRP-ribosome binding kinetics and affinities are affected by nascent chain length.
Conclusion:
SRP targeting is likely modulated at the initial ribosome-binding step, which is influenced by translation of nascent chain.
Significance:
Understanding signal recognition particle-ribosome binding provides insights into the constraints of co-translational targeting.
The signal recognition particle (SRP) directs ribosome-nascent chain complexes (RNCs) displaying signal sequences to protein translocation channels in the plasma membrane of prokaryotes and endoplasmic reticulum of eukaryotes. It was initially proposed that SRP binds the signal sequence when it emerges from an RNC and that successful binding becomes impaired as translation extends the nascent chain, moving the signal sequence away from SRP on the ribosomal surface. Later studies drew this simple model into question, proposing that SRP binding is unaffected by nascent chain length. Here, we reinvestigate this issue using two novel and independent fluorescence resonance energy transfer assays. We show that the arrival and dissociation rates of SRP binding to RNCs vary according to nascent chain length, resulting in the highest affinity shortly after a functional signal sequence emerges from the ribosome. Moreover, we show that SRP binds RNCs in multiple and interconverting conformations, and that conversely, RNCs exist in two conformations distinguished by SRP interaction kinetics.
