Interaction of the sarcin/ricin domain of 23 S ribosomal RNA with proteinsL3 and L6

We investigated interaction of an RNA domain covering the target;site of al pha-sarcin and ricin (sarcin/ricin domain) of Escherichia coil 23 S rRNA wi th ribosomal proteins. RNA fragments comprising residues 2630-2788 (Tox-1) and residues 2840-2774 (Tox-2) of 23 S rRNA were transcribed in vitro and u sed to analyze the binding proteins by gel shift and filter binding. Protei n L6 ;bound to both Tox-1 (K-d: 0.31 mu M) and Tox-2 (K-d: 0.18 mu M), and L3 bound only to Tox-1 (K-d: 0.069 mu M) in a solution containing 10 mM MgC l2 and 175 mM KCl at 0 degrees C. Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA, Binding of L 6 protected a single base, A-2757, and strongly enhanced reactivity of C-27 52, A direct role of A-2757 in the L6 binding was verified by site-directed mutagenesis; replacements of A-2757 with G and C impaired the 1.6 binding. On the other hand, binding of L3 protected A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750. Interestingly, binding of L6 and L3 to gether protected additional bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the other stem-loop. This appears to be due to cooper ative interaction of L3 and L6 with the RNA. The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the pro tein binding.