Domain D2 is not involved in ribosome binding of protein S1.

<p>(<b>A</b>) Ribosome binding of proteins S1 or S1_Δ103–181 was determined 60 minutes upon induction of their synthesis in strain JE28 harbouring either plasmid pPro-S1F (lanes 1 and 2) or pPro-S1ΔD2F (lanes 3 and 4). S30 extracts (lanes 1 and 3) and purified 70 S ribosomes (lanes 2 and 4) were loaded on SDS-PAGE. The positions of proteins S1 and S1_Δ103–181 are indicated to the right. (<b>B</b>) The binding of S1 (lanes 5–7) or S1_Δ103–181 (lanes 8–10) for the ribosome was determined by <i>in vitro</i> reconstitution experiments employing 30 S(-S1) subunits. The affinity of both proteins was directly compared by a competition experiment incubating 30 S (-S1) ribosomes concomitantly with both proteins S1 and S1_Δ103–181 in equimolar amounts (lanes 12–14). Upon incubation the ribosomes were separated from unbound proteins as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032702#s4" target="_blank">Material and Methods</a>, and the proteins present in the different fractions were separated on SDS-PAGE and visualized by Coomassie staining. I, input (lanes 2, 5, 8, and 12); R, ribosome fractions (lanes 3, 6, 9, and 13); FT, flow through fractions (lanes 4, 7, 10, and 14). 30 S, 30 S ribosomes before depletion for protein S1 (lane 15); 30 S(-S1), S1 depleted ribosomes used for the study (lane 16). The positions of proteins S1 and S1_Δ103–181 are indicated to the right. Lanes 1 and 11, protein size marker.