Signal Sequences Activate the Catalytic Switch of SRP RNA

Abstract

The signal recognition particle (SRP) recognizes polypeptide chains bearing a signal sequence as they emerge from the ribosome, and then binds its membrane-associated receptor (SR), thereby delivering the ribosome-nascent chain complex to the endoplasmic reticulum in eukaryotic cells and the plasma membrane in prokaryotic cells. SRP RNA catalytically accelerates the interaction of SRP and SR, which stimulates their guanosine triphosphatase (GTPase) activities, leading to dissociation of the complex. We found that although the catalytic activity of SRP RNA appeared to be constitutive, SRP RNA accelerated complex formation only when SRP was bound to a signal sequence. This crucial control step was obscured because a detergent commonly included in the reaction buffer acted as a signal peptide mimic. Thus, SRP RNA is a molecular switch that renders the SRP-SR GTPase engine responsive to signal peptide recruitment, coupling GTP hydrolysis to productive protein targeting.Secretory and transmembrane proteins are delivered to the membrane cotranslationally by the signal recognition particle (SRP) and its membrane-associated receptor (SR) (1). SRP recognizes signal sequences as they emerge from the ribosome (2) and then associates with SR at the membrane where the ribosome is transferred to the translocon. The guanosine triphosphatase (GTPase) domains of SRP and SR mediate this interaction cycle (3). Interaction of SRP with SR leads to the reciprocal stimulation of their GTPase activities, and GTP hydrolysis dissociates the complex (4,5). In Escherichia coli, SR is a single protein, FtsY, and SRP consists of 4.5S RNA and a single protein, Ffh (6). 4.5S RNA catalyzes the interaction of Ffh and FtsY, accelerating both on and off rates by a factor of more than 100 (7). If the energy of GTP hydrolysis is to be harnessed for protein targeting, recruitment of targeting substrates by SRP should be coupled to the SRP-SR interaction cycle. Both signal sequences and 4.5S RNA bind to the M domain of Ffh, which suggests that the catalytic activity of 4.5S RNA could be responsive to signal sequence binding (8). However, under typical assay conditions, 4.5S RNA is constitutively active, negating this role for the RNA (4,7,9,10). A small amount of the nonionic detergent octaethyleneglycol dodecylether (C 12 E 8 ) has been used in assays for SRP function, including kinetic characterization of the Ffh-FtsY interaction (4,

The 4.5S RNA component of the signal recognition particle is required for group A Streptococcus virulence

“…The 4.5S RNA accelerates binding between Ffh and its membrane-associated receptor FtsY, targeting Ffh-bound nascent polypeptides for secretion by the co-translational pathway (Bradshaw et al, 2009). Here, we present data Fig.…”

“…FfhFtsY complex formation also stimulates the GTPase activities of both proteins, leading to their dissociation and making them available for additional rounds of protein targeting (Powers & Walter, 1995). Thus, the 4.5S RNA functions as a molecular switch that couples GTP hydrolysis to protein secretion (Bradshaw et al, 2009).…”

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

“…It was recently shown that in cytosolic SRP pathways, the SRP RNA exerts its stimulatory effect on SRP • SR complex assembly only in the presence of cargo or a stimulatory detergent such as Nikkol that partially mimics the effect of the cargo (Bradshaw et al, 2009;Zhang et al, 2009). This led to the proposal that the SRP RNA acts as a molecular linker that turns on the GTPase cycles of SRP and SR in response to signal sequence binding in the M-domain.…”

“…Analogously cpSRP54, even though its M-and NG-domains would be prepositioned, cannot efficiently interact with the E. coli FtsY. The stimulatory effect of the SRP RNA and the M-domain of cpSRP54 is highly specific to their homologous receptors, arguing against the first two models in which the origin of the stimulatory effect would be more generic.It was recently shown that in cytosolic SRP pathways, the SRP RNA exerts its stimulatory effect on SRP • SR complex assembly only in the presence of cargo or a stimulatory detergent such as Nikkol that partially mimics the effect of the cargo (Bradshaw et al, 2009;Zhang et al, 2009). This led to the proposal that the SRP RNA acts as a molecular linker that turns on the GTPase cycles of SRP and SR in response to signal sequence binding in the M-domain.…”

“…Moreover, the SRP RNA accelerates the rate at which the SRP • SR complex hydrolyzes GTP 5-to 10-fold (Peluso et al, 2001;Siu et al, 2007). Many reports have suggested that the SRP RNA may play a regulatory role by bridging the communication between cargo binding and the GTPase cycle (Batey et al, 2000;Bradshaw and Walter, 2007;Bradshaw et al, 2009). The SRP RNA therefore plays a crucial role in the SRP pathway, explaining why it is highly conserved from bacteria to archaea to eukaryotes.…”