Dynamics of the SRP cycle: From scanning to targeting

Abstract

Inner‐membrane proteins in Escherichia coli are synthesized on ribosomes that are bound to the translocation pore (translocon) and enter the membrane co‐translationally. Ribosomes are targeted to the translocon by the signal recognition particle (SRP) and the SRP receptor, FtsY. Targeting is initiated when SRP is recruited to ribosomes that expose a signal‐anchor sequence (SAS). Here we report the kinetics of the interaction of SRP with ribosomes in different functional states and with FtsY, as followed by stopped‐flow monitoring FRET between fluorophores in SRP and the ribosome or FtsY. Rapid SRP binding to the ribosome is followed by two rearrangements the forward rate constants of which are independent of the functional state of the ribosomes. Conversely, ribosome‐SRP complex dissociation is very slow with ribosomes exposing an SAS, whereas the complex with non‐translating ribosomes dissociates rapidly. SAS‐induced stabilization of SRP on the ribosome is correlated with an acceleration of receptor binding. Thus, SRP can rapidly scan all ribosomes until it encounters a ribosome exposing an SAS, where SRP binding is strongly stabilized and membrane targeting is promoted by accelerating targeting complex formation with FtsY and, presumably, the translocon. The formation of the targeting complex is correlated with the lengthening of the nascent peptide within the exit tunnel of the ribosome.