Transient tether between the SRP RNA and SRP receptor ensures efficient cargo delivery during cotranslational protein targeting - PubMed (original) (raw)

Transient tether between the SRP RNA and SRP receptor ensures efficient cargo delivery during cotranslational protein targeting

Kuang Shen et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Kinetic control of macromolecular interactions plays key roles in biological regulation. An example of such control occurs in cotranslational protein targeting by the signal recognition particle (SRP), during which the SRP RNA and the cargo both accelerate complex assembly between the SRP and SRP receptor FtsY 10(2)-fold. The molecular mechanism underlying these rate accelerations was unclear. Here we show that a highly conserved basic residue, Lys399, on the lateral surface of FtsY provides a novel RNA tetraloop receptor to mediate the SRP RNA- and cargo-induced acceleration of SRP-FtsY complex assembly. We propose that the SRP RNA, by using its tetraloop to interact with FtsY-Lys399, provides a transient tether to stabilize the early stage and transition state of complex formation; this accelerates the assembly of a stable SRP-FtsY complex and allows the loading of cargo to be efficiently coupled to its membrane delivery. The use of a transient tether to increase the lifetime of collisional intermediates and reduce the dimension of diffusional search represents a novel and effective mechanism to accelerate macromolecular interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

FtsY–Lys399 plays a crucial role in SRP–FtsY complex assembly. (A) The basic residues on the FtsY Gα2-helix are highlighted in spacefill in the crystal structure of the Thermus aquaticus Ffh–FtsY NG-domain complex (PDB: 1RJ9). FtsY residues previously identified to be near the RNA tetraloop (21) are highlighted in magenta. (B) Rate constants of SRP–FtsY complex assembly, measured using FRET as described in Materials and Methods. Linear fits of data gave complex formation rate constants (_k_on) of 4.58 × 104 M-1 s-1 for wild-type FtsY (•) and 3.81 × 102 M-1 s-1 for mutant FtsY–K399A (▪). (C) Rate constants of SRP–FtsY complex disassembly (_k_off), determined by pulse-chase experiments as described (13, 22). Nonlinear fits of the time courses for loss of FRET from the complex (or gain in donor fluorescence) gave _k_off values of 1.46 × 10-3 s-1 for wild-type FtsY (•) and 4.05 × 10-5 s-1 for mutant FtsY–K399A (∘).

Fig. 2.

Fig. 2.

FtsY–Lys399 interacts with the SRP RNA tetraloop. (A) Effects of the FtsY–K399A and RNA(GAAU) mutations on the rate of stable complex formation, determined as described in Materials and Methods. (B) The FtsY–K399A mutation destabilizes the GTP-independent early intermediate. Nonlinear fits of the equilibrium titrations gave K d values of 8.85 μM for wild-type FtsY (•), and ≥48.4 μM for mutant FtsY–K399A (▪). (C) Effects of the FtsY–K399A and RNA(GAAU) mutations on the stability of the early complex. FRET values were measured with 10 μM FtsY.

Fig. 3.

Fig. 3.

The cpFtsY–A233K reversal mutation allows complex formation with cpFtsY to be stimulated by the SRP RNA. (A) Sequence alignment of FtsY homologues. The residue numbering is for E. coli FtsY. Bold highlights the cpFtsYs. (B) GTPase assay to measure the interaction between cpFtsY-A233K and E. coli Ffh in the absence (∘) and presence (•) of SRP RNA. Nonlinear fits of data gave _k_cat/K m values of 9.85 × 106 and 1.26 × 106 M-1 min-1 with and without the SRP RNA, respectively. For comparison, the _k_cat/K m value of the reaction of wild-type cpFtsY with Ffh is 1.77 × 106 M-1 min-1 (30).

Fig. 4.

Fig. 4.

Effect of ionic strength on the Ffh–FtsY complex assembly kinetics in the presence (A) or absence (B) of the SRP RNA, determined using the FRET assay. All the reactions also contain 50 mM K+ and 2 mM Mg2+, therefore, the ionic strength was increased from 50 to 250 mM. The complex assembly rate constants were obtained from the data in

Fig. S4

.

Fig. 5.

Fig. 5.

Mutation of FtsY–Lys399 diminishes the stimulatory effect of RNC on SRP–FtsY complex assembly. (A) Effect of FtsY–K399A on the rate constants of complex formation with cargo-loaded SRP. The inset shows the data with FtsY–K399A on an expanded scale. (B) Effect of FtsY–K399A on the equilibrium stability of the RNC–SRP–FtsY early targeting complex. The inset shows the data with FtsY–K399A on an expanded scale. Nonlinear fits of data gave K d values and FRET end points of 76.5 nM and 0.72 for wild-type FtsY (•), and approximately 2 μM and 0.35 for mutant FtsY–K399A (▪).

Fig. 6.

Fig. 6.

Model for the role of RNA tetraloop and FtsY–Lys399 on SRP–FtsY complex assembly, as described in the text. The upper panel depicts the complex assembly reaction with the assistance from the transient interaction between the RNA tetraloop and FtsY–Lys399, and the lower panel depicts the process in the absence of such a tethering interaction.

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