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.

Fig. 1

Detergent activates 4.5S RNA to catalyze the Ffh-FtsY interaction. (A) C₁₂E₈ stimulates the binding of Ffh and FtsY only in the presence of 4.5S RNA. Observed binding rates for formation of Ffh-FtsY complexes are plotted as a function of Ffh concentration, [Ffh], in the presence and absence of 4.5S RNA and 185 μM C₁₂E₈. Lines represent fits to the equation k_obs = k_off + k_on[Ffh]. Inset shows the slow reactions on an expanded scale. (B) C₁₂E₈ activates 4.5S RNA stimulation of Ffh-FtsY complex dissociation. Dissociation rate constants are plotted in the absence and presence of C₁₂E₈. (C) Chemical structures of C₁₂E₈, E₈, CTABr, and SDS. (D) Association rate constants for Ffh–4.5S RNA–FtsY complex formation with no detergent, 185 μM C₁₂E₈, 100 μM E₈, 70 μM CTABr, or 100 μM SDS. Error bars in (B) and (D) are SEs of the fits.

Fig. 2

ΔEspP binds SRP with micromolar affinity and stimulates 4.5S RNA catalysis of Ffh-FtsY interaction. (A) Fluorescence anisotropy of ΔEspP-FAM is plotted as a function of [Ffh]. Lines represent fits to the equation Anisotropy = Anisotropy_free + Anisotropy_bound([Ffh]/(K_d + [Ffh])). (B) C₁₂E₈ increased the K_d of ΔEspP for Ffh–4.5S RNA. K_d values for ΔEspP binding to Ffh from fluorescence anisotropy in the presence and absence of 4.5S RNA are plotted. Dark bars represent K_d in the presence of 185 μM C₁₂E₈. Error bars are SEs of the fits. (C) In the presence of 4.5S RNA, ΔEspP stimulates the association rate for Ffh-FtsY complex formation. Observed rate constants are plotted as a function of [Ffh]. Lines are fits to the equation k_obs = k_off + k_on[Ffh]. The dashed line is a reference to the binding rate in the presence of C₁₂E₈ from Fig. 1. (D) ΔEspP* activates 4.5S RNA by binding to SRP. Observed rates for 1 μM Ffh–4.5S RNA binding to 1 μM FtsY are plotted as a function of ΔEspP* concentration. The dashed line represents the equation k_obs = [(fraction bound) (maximum stimulated rate)] + [(fraction unbound)(unstimulated rate)], where the fraction bound was calculated from the K_d measured in (A); χ² = 5.4 × 10^–6.

Fig. 3

Mutations in ΔEspP that impair SRP-mediated targeting show decreased binding to SRP and decreased stimulation of 4.5S RNA. (A) ΔEspP(F12A, L15T)* stimulates SRP-FtsY complex formation less than does ΔEspP*. The dashed line represents the ΔEspP* + RNA peptide binding rate from Fig. 2C. (B) Fluorescence anisotropy of FAM-labeled ΔEspP bearing Phe¹² → Ala and Leu¹⁵ → Thr mutations [ΔEspP(F12A, L15T)] is plotted as a function of [Ffh +RNA]. Line is fit as in Fig. 2A.