Interaction of the sarcin/ricin domain of 23 S ribosomal RNA with proteins L3 and L6.
We investigated interaction of an RNA domain covering the target site of alpha-sarcin and ricin (sarcin/ricin domain) of Escherichia coli 23 S rRNA with ribosomal proteins. RNA fragments comprising residues 2630-2788 (Tox-1) and residues 2640-2774 (Tox-2) of 23 S rRNA were transcribed in vitro and used to analyze the binding proteins by gel shift and filter binding. Protein L6 bound to both Tox-1 (Kd: 0.31 microM) and Tox-2 (Kd: 0.18 microM), and L3 bound only to Tox-1 (Kd: 0.069 microM) in a solution containing 10 mM MgCl2 and 175 mM KCl at 0 degreesC. Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA. Binding of L6 protected a single base, A-2757, and strongly enhanced reactivity of C-2752. 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 L6 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 together 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 cooperative interaction of L3 and L6 with the RNA. The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the protein binding.
Uchiumi
T
T
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan. uchiumi@giptc.shinshu-u.ac.jp
Sato
N
N
Wada
A
A
Hachimori
A
A
D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism*; D005656:Fungal Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism*; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000378:metabolism*; D012276:Ricin / Q000378:metabolism*
D005656:Fungal Proteins; D012338:RNA, Ribosomal, 23S; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; C031249:ribosomal protein L6; C014801:alpha-sarcin; D012276:Ricin; D004722:Endoribonucleases