Dynamic switch of the signal recognition particle from scanning to targeting

Abstract

Ribosomes synthesizing inner membrane proteins in Escherichia coli are targeted to the membrane by the signal recognition particle (SRP) pathway. By rapid kinetic analysis we show that after initial binding to the ribosome, SRP undergoes dynamic fluctuations in search of additional interactions. Non-translating ribosomes, or ribosomes synthesizing non-membrane proteins, do not provide these contacts, allowing SRPs to dissociate rapidly. A nascent peptide in the exit tunnel stabilizes SRPs in a standby state. Binding to the emerging signal-anchor sequence (SAS) of a nascent membrane protein halts the fluctuations of SRP, resulting in complex stabilization and recruitment of the SRP receptor. We propose a kinetic model where SRP rapidly scans all ribosomes until it encounters a ribosome exposing an SAS. Binding to the SAS switches SRP into the targeting mode, in which dissociation is slow and docking of the SRP receptor is accelerated.

Interplay between trigger factor and other protein biogenesis factors on the ribosome

“…Previous kinetic analyses revealed that, similar to what we report here for TF, SRP scans ribosomes rapidly until binding is stabilized on translating ribosomes that have their peptide exit tunnel filled ('stand-by') or expose a signal-anchor sequence ('targeting') 13 . Assuming an SRP concentration in vivo of 0.1 mM and diffusion-controlled binding, as observed in vitro 13 , the effective rate of SRP binding to ribosomes or RNCs is about 10 s À 1 .…”

“…Assuming an SRP concentration in vivo of 0.1 mM and diffusion-controlled binding, as observed in vitro 13 , the effective rate of SRP binding to ribosomes or RNCs is about 10 s À 1 . This rate matches the rate of TF dissociation from non-TF-substrate RNCs, Lep75-RNC in our experiments, which may optimize the access of SRP to its substrate RNCs.…”

“…13) and TF labelled with Bodipy-FL (Bpy; acceptor) at position 99. To examine the kinetics of ribosome-TF complex formation, we rapidly mixed MDCC-labelled ribosomes with TF(Bpy) and monitored the increase of the acceptor signal (Fig.…”

“…In the 'scanning' mode, SRP binds to non-translating ribosomes or ribosomes that do not synthesize membrane proteins in a readily reversible fashion 13 , with effective dissociation rates in the range of 10 s À 1 , allowing other RPBs to bind in a stochastic fashion. However, the extent to which RPBs can enter when SRP is stabilized on translating ribosomes with short nascent chains ('stand-by'; effective off-rate about 1 s À 1 ) or on ribosomes synthesizing membrane proteins ('targeting'; effective off-rate about 0.1 s À 1 ) 12,13 is not clear.…”

“…SRP scans translating ribosomes for emerging hydrophobic signal-anchor sequences (SAS). Binding to an SAS switches SRP to the targeting mode and initiates cotranslational membrane targeting by binding to the SRP receptor and transfer to the translocon 12,13 . SRP and TF together can bind to the ribosome, although both contact protein L23 at the peptide exit ( Fig.…”