FIG. 4.

TMEA cross-linking of Aer and Tar. Cells expressing various combinations of Tar and Aer cross-linking reporters were grown in tryptone broth at 30°C, treated with TMEA, and analyzed as described in Materials and Methods. About 50% of Aer and Tar subunits become cross-linked under these conditions (not shown; see reference 23). The gel regions containing two-subunit (e.g., Tar∼Tar) and three-subunit (e.g., Tar∼Tar∼Tar) cross-linking products are shown; uncrosslinked Aer and Tar molecules are not. The gel on the left (first three lanes) was blotted with a combination of anti-Aer and anti-Tsr; the gel on the right was blotted with anti-Aer. Lane 1, pNP1 (Aer vector control)/UU1604 (Tar-S364C); lane 2, pKG158 (Aer-S356C)/UU1604; lane 3, pKG193 (Aer-S356C/I367P)/UU1604; lane 4, pKG193 (Aer-S356C/I367P)/UU1250 (no transducers); lane 5, pKG158 (Aer-S356C)/UU1250. Strains containing pKG158 were induced at 15 μM IPTG; strains containing pKG193 were induced with 60 μM IPTG to compensate for lower expression of Aer proteins with the I367P lesion. The faint bands at the Tar∼Tar position, most apparent in lanes 4 and 5, are unidentified cellular proteins that cross-react with the anti-Aer serum.