Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

Abstract

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.

Figure 1

Chemical crosslinking of TF to arrested nascent ICDH polypeptides of varying length. SecM-arrested ³⁵S-labelled nascent polypeptides were synthesized in vitro exposing different lengths of ICDH polypeptides outside the ribosomal tunnel (4–146 aa, indicated by a filled circle). The interaction of TF with these nascent chains was probed by chemical crosslinking with DSS. Crosslinking products (indicated by an asterisk) were visualized by SDS–PAGE and autoradiography.

Figure 2

Site-specific crosslinking of TF-Bpa variants to arrested nascent ICDH. (A) Structural model of ribosome-associated TF (Ferbitz et al, 2004). The ribosome (grey) is cut in half to visualize the ribosomal tunnel with a modelled nascent chain (magenta). TF (blue) is shown in ribbon representation docked to the ribosomal protein L23 (green) and in surface representation rotated by 90° to allow one to view the interior. Positions of UV-activatable Bpa crosslinker are indicated in green; red numbers label Bpa positions on the interior of TF; exterior positions are labelled with white numbers. (B) SecM-arrested nascent ICDH polypeptides of varying length were synthesized in vitro in the presence of TF variants. After translation, UV light was applied to crosslink TF-Bpa variants to nascent chains. Crosslinks were detected by SDS–PAGE and autoradiography. Only crosslinking products are depicted. ICDH-16-SecM (with a total length of 47 aa including the SecM linker, marked by a star) represents the minimal length of a nascent chain required for site-specific TF crosslinking.

Figure 3

Interactions of non-arrested ICDH with TF during ongoing translation. TF-Bpa variants were analysed for crosslinking to non-arrested ICDH during ongoing translation by applying UV light during synthesis. The released full-length ICDH (A, supernatant) was separated from ribosome-associated incompletely synthesized ICDH nascent polypeptides (B, pellet) by ultracentrifugation. TF–ICDH crosslinking products are indicated by asterisks. As control, UV irradiation was applied after synthesis to the supernatant fraction (C).

Figure 4

Localization of nascent chains with different folding states within TF. The folding-competent SH3-wt domain and the unfolded mutant SH3-m10 (see text) were synthesized in vitro and analysed for their interactions with TF-Bpa variants. (A, B) The SH3 variants were stalled by SecM arrest (SH3-wt-SecM, SH3-m10-SecM) and UV irradiation was applied (A) after synthesis or (B) during ongoing synthesis. (C) Non-arrested SH3 and SH3-m10 were generated, UV light was applied during ongoing synthesis and the ribosome-dissociated crosslinking products of TF-Bpa variants and released full-length SH3-wt or SH3-m10 were analysed.

Figure 5

Generation of stabilized TF–RNCs. TF–RNCs were generated and stabilized by disulphide bond formation. (A) Schematic description of the model nascent chain, Strep-SH3-2Cys-SecM, composed of a triple Strep tag (3xStrep), a folded SH3-wt domain (SH3-wt), a peptide-linker of 13 aa (Pep44-linker) harbouring a single cysteine (Cys2, asterisk) and the SecM arrest sequence (SecM). (B) Analysis of disulphide bond formation of TF-wt (cysteine-free) and TF-S61C variant with model nascent chains harbouring the single cysteine at different positions in the Pep44-linker (Cys2, Cys7, Cys12). Formation of disulphide bridges (asterisk) was induced by the addition of diamide after in vitro synthesis. (C) TF-S61C and nascent Strep-SH3-2Cys-SecM (filled circle) efficiently build disulphide bonds (asterisk) upon diamide addition, which can be resolved by the reducing agent dithiothreitol. (D) Site-specific crosslinking of purified RNCs of Strep-SH3-2Cys-SecM and TF-Bpa variants. Crosslinking products were analysed by western blot analysis using antibodies specific for TF or the Strep tag of the model nascent chain.

Figure 6

Cryo-EM analysis of TF bound to an RNC. (A) Structure of the E. coli RNC (50S subunit, blue; 30S subunit, yellow; P-site tRNA, green) in complex with TF interacting with the nascent chain (red). (B) Same perspective as in (A) but with the ribosome sliced along the tunnel. The tunnel exit is marked with a star. (C) Schematic drawing of the TF interacting with the nascent chain. The 50S subunit is shown in blue and the 30S subunit is in yellow. The nascent chain (orange) connected to the peptidyl P-tRNA (green) is shielded by TF (red). (D) Crystal structure of the E. coli TF (red ribbon, crosslinked residue 61 is shown as orange spheres) fitted into the EM density (grey mesh). The density next to the tunnel exit (black star) attributed to the nascent chain is shown in orange. The ribosome is visualized as a grey surface. (E) Orientation of the TF (red ribbon) on the ribosome (rRNA, grey ribbon). The proteins in the vicinity of the tunnel exit (black star) are highlighted in different colours (L23, magenta; L22, light brown; L29, cyan; L24, green). (F) Same as (E) but tilted by 90° towards the viewer and including the TF density shown as a grey mesh.

Figure 7

Model of the passage of nascent polypeptides through TF. TF directs the nascent chains through its interior in a sequential and length-dependent manner. (A) Initially (with a length of 40–60 aa), the N terminus of the nascent chain slides along the N domain, where it might be accessible by means of the lateral openings from both sides for processing factors such as PDF, MAP or SRP. (B) Up to a length of 90 aa, the nascent chain traverses through the C-terminal arms towards the PPIase domain and engages the entire interior. (C) Upon further elongation, the nascent chain might leave TF or, alternatively, may accumulate and perhaps fold in the interior of the TF chaperone. On demand, the folding of a subset of newly synthesized proteins is further assisted by cytosolic chaperones.