Dynamics of co-translational protein targeting

Abstract

Most membrane and secretory proteins are delivered co-translationally to protein translocation channels in their destination membrane by the signal recognition particle (SRP) and its receptor. This co-translational molecular machinery is conserved across all kingdoms of life, though it varies in composition and function. Here we report recent progress towards understanding the mechanism of SRP function, focusing on findings about Escherichia coli SRP's conformational dynamics throughout the targeting process. These insights shed light on a key checkpoint in the targeting cycle: how SRP regulates engagement of an actively translating ribosome with the translocation machinery at the membrane.

Figure 1. SRP binds to actively translating RNCs after emergence of a signal sequence-containing nascent chain

Representative single-molecule fluorescence vs. time trajectory of Cy3B-labeled 50S ribosomal subunits, Cy5-labeled SRP, and unlabeled translation mix delivered at time = 0 to pre-initiation complexes assembled on a truncated LepB mRNA (encoding the first 115 amino acids), immobilized in Zero Mode Waveguides, and imaged by TIRF microscopy. The top panel shows a cartoon representation of the molecular events throughout the time series. The bottom panel shows the fluorescence intensity of the Cy3B (green), and Cy5 (red) signals upon 532 nm excitation. “AU” indicates “arbitrary units”. * denotes 50S ribosomal subunit joining. Figure from (Noriega et al. 2014 eLife).

Figure 2. SRP-FtsY GTPase complex relocalization to the SRP RNA distal end

A. Crystal structure of SRP in complex with FtsY trapped with GMPPCP. Full-length 4.5S SRP RNA in tan, with the tetraloop and distal end indicated. Ffh in violet with M and NG domains indicated, and FtsY(NG) in green. PDB: 2XXA. The figure was prepared using PyMOL. B. E. coli 4.5S SRP RNA secondary structure. The tetraloop and distal end GTPase complex binding site are indicated in tan boxes.

Figure 3. Conformational rearrangements within the SRP-FtsY GTPase complex drive its movement to the RNA distal site

A-D. smFRET histograms of (A) free SRP in the open state and of the Ffh-FtsY complex in the (B) early, (C) closed, and (D) activated states. (E) Transition density plot depicting the range of E_FRET values sampled by the Ffh-FtsY (GMPPNP) GTPase complex movements. The plot depicts idealized E_FRET before a transition versus E_FRET after the transition as 2-D population histograms. At least two distinct intermediate states, M1 and M2, are sampled by the Ffh-FtsY GTPase complex along the path between the proximal and distal sites of the SRP RNA. (F) SRP-FtsY complex bound to RNC_FtsQ, and (G) SRP-FtsY complex bound to RNC_FtsQ and SecYEG. Figure modified from (Shen et al. 2012 Nature).

Figure 4. Mechanistic model of co-translational protein targeting

E. coli SRP is composed of 4.5S RNA (violet) and Ffh (blue). SRP binds a ribosome-nascent chain complex (RNC) (gray) and exposed signal sequence (magenta) in its GTP-bound state (“T”). Ffh associates with FtsY (green), driving membrane localization. SecYEG promotes a conformational rearrangement of the Ffh-FtsY GTPase complex from the proximal to the distal site on 4.5S RNA, allowing transfer of the RNC onto SecYEG. GTP hydrolysis triggers SRP-FtsY disassembly and recycling (GDP = “D”).