Role of the N-terminal region of ribosomal protein S7 in its interaction with 16S rRNA which binds to the concavity formed by the beta-ribbon arm andthe alpha-helix

The ribosomal protein S7, a primary 16S rRNA-binding protein, plays an esse ntial role in stabilizing the 3' major domain of 16S rRNA and also in feedb ack regulation of the str:operon, as a translational repressor. We examined amino acid residues in ribosomal protein S7 from Bacillus srearothermophil us (BstS7) that are essential for 16S rRNA binding. Truncation of the N-ter minal 10 residues of BstS7 abolished its binding to 16S rRNA, whereas remov al of the C-terminal eight residues had no effect on the binding activity. Subsequently, we used site-directed mutagenesis to identify essential basic residues in the N-terminal region for 16S rRNA binding. Mutation of Arg3 a nd Lys8 significantly weakened the binding activity, and a Smaller decrease in binding activity was observed with Arg2 and Arg9 mutations. These obser vations indicate that N-terminal basic residues, especially Arg3 and Lys8, play a crucial role as positively charged recognition groups for the negati vely charged phosphate backbone of 16S rRNA. In addition, the mutagenesis s tudy showed that Arg75, Arg78, Arg94, and Arg101, which are located in a co ncavity formed by the beta-ribbon arm and the alpha-helix (alpha 4), indivi dually make only a small contribution to 16S rRNA binding, but together pro bably form a positively charged binding site for 16S rRNA. With regard to a romatic residues, Tyr84 on the tip of the beta-ribbon arm Was found to be i nvolved in 16S rRNA binding, whereas the conserved aromatic residues Trp102 and Tyr106 in the concavity had little effect. We then probed the 16S rRNA -binding site(s) for the N-terminal region: of S7 with iron tethered to the mutant of BstS7 containing a single cysteine residue at position 4. The N- terminal region of S7 is placed in close proximity to helix 43 in the 16S r RNA, Probing also revealed additional cleavages between nucleotides 1397 an d 1438, near the P-site region in 16S rRNA, This finding is consistent with a three-dimensional model of 16S rRNA that shows close proximity of helix 43 to the P-site during three-dimensional folding.