Conformation of the signal recognition particle in ribosomal targeting complexes

Abstract

The bacterial signal recognition particle (SRP) binds to ribosomes synthesizing inner membrane proteins and, by interaction with the SRP receptor, FtsY, targets them to the translocon at the membrane. Here we probe the conformation of SRP and SRP protein, Ffh, at different stages of targeting by measuring fluorescence resonance energy transfer (FRET) between fluorophores placed at various positions within SRP. Distances derived from FRET indicate that SRP binding to nontranslating ribosomes triggers a global conformational change of SRP that facilitates binding of the SRP receptor, FtsY. Binding of SRP to a signal-anchor sequence exposed on a ribosome-nascent chain complex (RNC) causes a further change of the SRP conformation, involving the flexible part of the Ffh(M) domain, which increases the affinity for FtsY of ribosome-bound SRP up to the affinity exhibited by the isolated NG domain of Ffh. This indicates that in the RNC-SRP complex the Ffh(NG) domain is fully exposed for binding FtsY to form the targeting complex. Binding of FtsY to the RNC-SRP complex results in a limited conformational change of SRP, which may initiate subsequent targeting steps.

FIGURE 1.

FRET measurements. (A) Positions of engineered cysteine residues in Ffh. N, G, and M domains are arranged as described previously (Buskiewicz et al. 2005a,b). Positions of cysteine residues at which fluorescent dyes were introduced are indicated by spheres and numbers. (B) 4.5S RNA constructs: full-length 4.5S RNA and the truncated constructs 4.5S RNA_21–81 and 4.5S RNA_30–78. (C–E) Fluorescence decay of OG attached to Ffh at positions 17 (C), 201 (D), or 344 (E) with Alx555 at the 3′ end of 4.5S RNA_21–81. (1) RNC–SRP, donor alone, (2) SRP, donor+acceptor, (3) RNC–SRP, donor+acceptor, (4) excitation pulse.

FIGURE 2.

FRET distances in free SRP and the SRP–RNC complex. (A) Distances between position 344 in the M domain of Ffh and the indicated positions in the N domain (17, 25) and G domain (84, 152, 165, 201, 203). (B) Distances between the 3′ end of 4.5S RNA_21–81 and the indicated positions in Ffh. (White bars) Free SRP; (black bars) SRP–RNC complex.

FIGURE 3.

Model of SRP bound to RNC. The position of Ffh domains N, G, and M derived from FRET is in red, the arrangement of the RNA and Ffh domains in the cryo-EM model (Halic et al. 2006a) are in green and blue, respectively. Red spheres label positions used for FRET and placed by pairwise alignment using FRETsg (Supplementary Information); blue spheres label positions according to the cryo-EM model; green sphere indicates position 81 of 4.5S RNA in the cryo-EM model. The 50S subunit is indicated in gray, proteins L23 and L29 in orange and purple, respectively. (np) Nascent peptide from the cryo-EM model.

FIGURE 4.

FRET distances in SRP at different stages of targeting. FRET distances were measured in free SRP (white bars), SRP-70S (light gray), SRP-RNC (dark gray), or SRP-RNC-FtsY (black) complexes. (A) Distances between the indicated positions and position 344 in the M domain. (B) Distances from the indicated positions to the 3′ end of 4.5S RNA_21–81. (C) Affinity of FtsY binding to SRP in different functional stages. Fluorescence titrations were carried out with SRP formed from Ffh(OG84) and full-length 4.5S RNA. Free SRP (▪); SRP bound to vacant 70S ribosomes (▼); SRP bound to Lep50-RNC (●); isolated Ffh(NG)(OG84) domain (○). Nonlinear fitting (continuous lines; see Materials and Methods) yielded the following K _d values: FtsY-SRP, (70 ± 9) nM; FtsY-SRP-70S, (20 ± 3) nM; FtsY-SRP-RNC, (3 ± 0.1) nM; FtsY-Ffh(NG), (3 ± 0.2) nM.

FIGURE 5.

Influence of TF on the conformation of ribosome-bound SRP. Changes of distances upon binding of TF to the complex of SRP with RNC (A) or vacant ribosomes (B) were measured by FRET. Distances between the indicated positions in M and NG domains, labeled with OG, and the 3′ end of 4.5S RNA_21–81, labeled with Alx 647, were measured in free SRP (white bars), SRP-RNC or SRP-ribosome (gray bars), and SRP-RNC-TF or SRP-ribosome-TF (black) complexes.

FIGURE 6.

Effect of 4.5S RNA. Distances from position 344 in the Ffh(M) domain to the indicated positions in the Ffh(NG) domain were measured in the absence of ribosomes (white bars) and with RNCs (black bars). (A) Ffh, (B) Ffh–4.5S RNA_30–78, (C) Ffh–4.5S RNA_21–81, (D) Ffh–4.5S RNA.