UCSF

Secretory and transmembrane proteins are delivered to the ER membrane or eukaryotic cells or the plasma membrane of prokaryotic cells cotranslationally by the interaction of the signal recognition particle (SRP) with its membrane associated receptor (SR). SRP recognizes hydrophobic signal sequences in proteins as they are translated and delivers the ribosome nascent chain to the membrane by associating with SR. The ribosome is then transferred to the translocation channel, and synthesis of the protein continues through the membrane. Homologous GTPase domains of SRP and SR mediate their interaction cycle: SRP-SR association reciprocally stimulates their GTPase activities, and GTP hydrolysis disassembles the complex. How this cycle of GTP hydrolysis is coupled to productive targeting was unknown. Additionally, SRP requires an RNA subunit that catalyzes the SRP-SR interaction, accelerating both on and off rates by over 100 fold. The mechanism of SRP RNA catalysis and its role in protein targeting were also mysteries.

The first chapter describes the identification of mutations in the SRP protein that abrogate the activity of SRP RNA. This demonstrated a link between conformational changes in the SPR protein and the activity of SRP RNA. Furthermore, these studies suggested that SRP RNA activity might be controlled by signal sequence binding to SRP. The second chapter describes the discovery that the activity of SRP RNA to accelerate SRP-SR association requires that SRP be bound to a signal sequence. This effect was previously not observed because it was masked by a small amount of detergent included in the reaction buffer that acted as a signal sequence mimic. This couples the SRP-SR interaction with cargo recruitment by SRP and ensures that GTP hydrolysis is productive. The third chapter describes the discovery that the structurally and evolutionarily related N-terminal helices of SRP and SR are autoinhibitory to complex formation in the absence of SRP RNA and that SRP RNA relieves this autoinhibition. Using NMR spectroscopy and enzymatic assays, we found that truncation of the N-terminal helix of SR allows it to adopt its SRP bound conformation. These studies demonstrate that SRP RNA controls a conformational switch in the SRP and SR to coordinate SRP-SR interaction with cargo recruitment by SRP.