FIG. 4.

Cross-linking of ribosome-nascent-chain complexes to SRP components. Coupled transcription-translation reactions were programmed with pTM1-MIA2 linearized with either StuI (lanes 1 to 4), EcoRI (lanes 5 to 8), or NgoMI (lanes 9 to 12) to produce short mRNAs lacking a stop codon. Reactions were performed for 30 min in the absence or presence of microsomal membranes as indicated. Subsequently, ribosome-nascent-chain complexes were centrifuged at 75,000 rpm in a Beckman Optima TLX ultracentrifuge for 30 min at 2°C. Aliquots from the resuspended pellets were subjected to a cross-linking reaction (2 h, 0°C) with the homobifunctional cross-linking reagent DSS as indicated. Molecular mass markers are indicated on the right. The open arrowhead marks the cross-linked product (58aa × unknown protein) of about 60 kDa in lane 2; a solid arrowhead and an asterisk identify the cross-linked products in lanes 6 and 10, respectively. Note that no specific cross-linking products were detected in lanes 4, 8, and 12, where the corresponding reactions translated in the presence of microsomal membranes were applied.