Progression of nascent Lep through the ribosome. (A) Schematic representation of the 9, 24, 40, and 50Lep constructs with a cross-linking probe at position 3. The transmembrane regions (H1) and methionine tags are presented as thick gray lines and white bars, respectively. (B) In vitro translation of nascent 9–50LepTAG3 constructs was performed in the presence of (Tmd)Phe-tRNA^sup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated 9, 15, 24, 44, and 50LepTAG3 were immunoprecipitated with antisera as indicated. (C) Schematic representation of the cross-links observed in B. Numbers indicate the distance in amino acids of the cross-linking probe to the PTC. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, L4 and L22 cross-link to a truncated translation product.

Interactions of nascent LepKO, pre-PhoE, and RpoB with ribosomal proteins and cytosolic chaperones. (A) Schematic representation of LepTAG3 and LepKOTAG3. The sequences of wild-type and mutated H1 are depicted. (B and C) 15–44LepKOTAG3 (B) and 15/44pre-PhoETAG3 and 15/44RpoBTAG3 (C) were produced, cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; >, TF cross-link.

Tunnel view of the large ribosomal subunit from Deinococcus radiodurans. The 40–amino acid nascent Lep (blue) and TM (red) is stretched from P-site tRNA (salmon pink) to the ribosomal exit tunnel and makes contacts with ribosomal proteins L22 (yellow), L4 (green), and L23 (orange).

The initial targeting and insertion steps of Lep. (A) In vitro translation of nascent 32–50LepTAG3 was performed in the presence of IMVs and (Tmd)Phe-tRNA^sup. After translations, samples were kept in the dark or UV irradiated and were subsequently extracted with sodium carbonate and spun down. UV-irradiated pellet fractions of 44LepTAG3 were immunoprecipitated as indicated. (B) Quantifications of SecY and YidC cross-linking to 38–50LepTAG3 (A, lanes 13–18) relative to the amount of nonirradiated carbonate-resistant nascent chains (A, lanes 4–9). Highest values for cross-linking efficiency were taken as 100%. Images in different panels represent different parts of the gel or different exposure times. *, SecY cross-link; o, YidC cross-link.

SRP is not oriented toward the ribosomal exit before it is able to interact with H1. (A) Schematic representation of 30 and 44Lep with a cross-linking probe at position 3 and 48Pf3Lep and 48Pf3LepKO with a cross-linking probe at position seven. H1 and the Pf3 extension are depicted as a thick gray line and a thin white bar, respectively. The four mutations in H1 to obtain the Pf3LepKO construct are the same as in Fig. 3 and are indicated here with four asterisks. (B) The constructs shown in A were translated in vitro with and without the addition of 350 nM of purified SRP. After translation, samples were cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, TF cross-link.