Horie K, Wada A, Fukutome H
J Biochem. 1981 Aug;90(2):449-61. doi: 10.1093/oxfordjournals.jbchem.a133492.
The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.
为了获取核糖体中核糖体RNA（rRNA）的构象信息，研究了大肠杆菌空核糖体与吖啶橙（AO）的相互作用。吖啶橙以两种不同模式与RNA结合：通过结合的AO分子堆叠进行协同外部结合以及在核苷酸碱基之间插入。游离的16S和23S rRNA对AO的亲和力几乎相同。在1 mM Mg2+条件下，AO可以在约40%的rRNA磷酸基团上实现堆叠结合。与游离的16S rRNA相比，30S亚基中堆叠结合位点的数量降至约1/3。在50S亚基中，与游离的23S rRNA相比，堆叠结合位点的数量仅为1/5。Mg2+离子对AO与核糖体结合的抑制作用比对游离rRNA的抑制作用更强。与rRNA相比，Mg2+在活性核糖体中对堆叠结合强度的降低似乎更明显。“紧密偶联”的70S颗粒比游离亚基更难进行堆叠结合。在低Mg2+条件下不可逆地失去形成70S能力的30S亚基对AO的亲和力与活性30S亚基的亲和力非常不同，但与游离rRNA的亲和力相似，尽管失活后堆叠结合位点的数量变化不大。结合了堆叠AO的70S和30S核糖体具有正常的沉降常数，但50S亚基可逆地形成聚集体。
