Signal sequences activate the catalytic switch of SRP RNA - PubMed (original) (raw)

Signal sequences activate the catalytic switch of SRP RNA

Niels Bradshaw et al. Science. 2009.

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.

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Figures

Fig. 1

Fig. 1

Detergent activates 4.5_S_ RNA to catalyze the Ffh-FtsY interaction. (A) C12E8 stimulates the binding of Ffh and FtsY only in the presence of 4.5_S_ 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 4.5_S_ 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 4.5_S 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–4.5_S 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

Fig. 2

ΔEspP binds SRP with micromolar affinity and stimulates 4.5_S_ 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 = Anisotropyfree + Anisotropybound([Ffh]/(_K_d + [Ffh])). (B) C12E8 increased the K_d of ΔEspP for Ffh–4.5_S RNA. K_d values for ΔEspP binding to Ffh from fluorescence anisotropy in the presence and absence of 4.5_S 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 4.5_S 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 4.5_S RNA by binding to SRP. Observed rates for 1 μM Ffh–4.5_S 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

Fig. 3

Mutations in ΔEspP that impair SRP-mediated targeting show decreased binding to SRP and decreased stimulation of 4.5_S_ 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.

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