(A) Schematic of the constructs used for production and purification of stalled RNCs. Key domains and their positions (in amino acids) are indicated, along with the mutation (3R) used to disrupt the TMD. The diagrams to the right depict RNCs with the TMD inside or outside the tunnel used for biochemical and structural analyses. (B) Anti-FLAG affinity purifications were performed on translation reactions programmed with no RNA (mock), TMD-containing transcripts, or 3R mutant transcripts. Truncation was at position 95 (tunnel) or 128 (exposed). The samples were analyzed by SDS-PAGE and visualized for total protein (top panel) or immunoblotted for SRP54, the large ribosomal protein uL6, or the small subunit protein uS9. (C) The unbound fractions of translation reactions following affinity purification of the TMD and 3R constructs (as in panel B) were analyzed relative to serial dilutions of total lysate. RNCs containing an intact TMD, whether in the tunnel or exposed, selectively deplete more than 75% of SRP from the lysate. (D) RNCs truncated at different positions relative to the TMD were affinity purified and probed for SRP and the ribosome as in panel B. Maximal recruitment is observed at all points after truncation at residue 89, when 14 TMD residues have entered the tunnel. (E) Analysis of SRP recruitment as in panel B using mutant TMDs in which 4 to 10 residues have been deleted. The truncation point was at residue 95. (F) Experiment as in panel D for the indicated truncation points using the TMD or 3R construct.