FIG 7.

The SecA-ribosome contact is nucleotide independent and dissociated by the presence of membranes. (A) Purified SecA (500 nM) and equimolar amounts of ribosomes bearing pBpa at position 71 of uL23 were cross-linked as described in Fig. 1 in the presence of different nucleotides (50 μM), as indicated. The cross-linking products were analyzed by SDS-PAGE and decorated with anti-SecA antibodies. One representative figure of two replicates is shown. (B) In vitro cysteine cross-linking was performed as described in Fig. 3B with SecA mutants (1 μM) containing a cysteine residue at either position 5 or 625 to ribosomes with a cysteine residue at position 71 of uL23. When indicated, SecA was preincubated with inner membrane vesicles (INVs) generated from a SecYEG-YidC-overexpressing strain. The final SecYEG concentration was 2 μM. After incubation, unbound SecA was removed by centrifugation, ribosomes were added, and cross-linking was induced by UV exposure. Cross-links were separated by SDS-PAGE and analyzed by immunoblotting. The SecA variants and the overexpressed SecY in the INVs carried a His tag and were detected with anti-His antibodies. One representative figure of at least three replicates is shown.