Uchiumi T, Sato N, Wada A, Hachimori A
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.
J Biol Chem. 1999 Jan 8;274(2):681-6. doi: 10.1074/jbc.274.2.681.
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.
我们研究了大肠杆菌23S rRNA中覆盖α-肌动蛋白和蓖麻毒素靶位点的RNA结构域（肌动蛋白/蓖麻毒素结构域）与核糖体蛋白的相互作用。体外转录包含23S rRNA第2630 - 2788位残基（Tox - 1）和第2640 - 2774位残基（Tox - 2）的RNA片段，并通过凝胶迁移和滤膜结合分析与之结合的蛋白。在0℃、含有10 mM MgCl₂和175 mM KCl的溶液中，蛋白L6与Tox - 1（解离常数Kd：0.31 μM）和Tox - 2（Kd：0.18 μM）均结合，而L3仅与Tox - 1结合（Kd：0.069 μM）。使用化学探针硫酸二甲酯对全长23S rRNA进行足迹分析。L6的结合保护了单个碱基A - 2757，并强烈增强了C - 2752的反应性。通过定点诱变验证了A - 2757在L6结合中的直接作用；用G和C取代A - 2757会损害L6的结合。另一方面，L3的结合保护了A - 2632、A - 2634、A - 2635、A - 2675、A - 2726、A - 2733、A - 2749和A - 2750。有趣的是，L6和L3一起结合除了保护另一个茎环中的A - 2740、A - 2741、A - 2748、A - 2753、A - 2764、A - 2765和A - 2766外，还保护了肌动蛋白/蓖麻毒素环中的额外碱基A - 2657、A - 2662、C - 2666和C - 2667。这似乎是由于L3和L6与RNA的协同相互作用。结合蛋白对肌动蛋白/蓖麻毒素结构域构象调节的结果进行了讨论。
