Moazed D, Van Stolk B J, Douthwaite S, Noller H F
J Mol Biol. 1986 Oct 5;191(3):483-93. doi: 10.1016/0022-2836(86)90143-9.
Zamir, Elson and their co-workers have shown that 30 S ribosomal subunits are reversibly inactivated by depletion of monovalent or divalent cations. We have re-investigated the conformation of 16 S rRNA in the active and inactive forms of the 30 S subunit, using a strategy that is designed to eliminate reversible ion-dependent conformational effects that are unrelated to the heat-dependent Zamir-Elson transition. A combination of structure-specific chemical probes enables us to monitor the accessibility of pyrimidines at N-3 and purines at N-1 and N-7. Chemically modified bases are identified by end-labeling followed by analine-induced strand scission (in some cases preceded by hybrid selection), or by primer extension using synthetic DNA oligomers. These studies show the following: The transition from the active to the inactive state cannot be described as a simple loosening or unfolding of native structure, such as that which is observed under conditions of more severe ion depletion. Instead, it has the appearance of a reciprocal interconversion between two differently structured states; some bases become more reactive toward the probes, whilst others become less reactive as a result of inactivation. Changes in reactivity are almost exclusively confined to the "decoding site" centered at positions 1400 and 1500, but significant differences are also detected at U723 and G791 in the central domain. This may reflect possible structural and functional interactions between the central and 3' regions of 16 S rRNA. The inactive form also shows significantly decreased reactivity at positions 1533 to 1538 (the Shine-Dalgarno region), in agreement with earlier findings. The principal changes in reactivity involve the universally conserved nucleotides G926, C1395, A1398 and G1401. The three purines show reciprocal behavior at their N-1 versus N-7 positions. G926 loses its reactivity at N-1, but becomes highly reactive at N-7 as a result of the transition of the inactive state. In contrast, A1398 and G1401 become reactive at N-1, but lose their hyper-reactivity at N-7. The possible structural and functional implications of these findings are discussed.
扎米尔、埃尔森及其同事已表明，单价或二价阳离子的耗尽会使30 S核糖体亚基可逆失活。我们重新研究了30 S亚基活性形式和非活性形式中16 S rRNA的构象，采用了一种策略，旨在消除与热依赖性扎米尔 - 埃尔森转变无关的可逆离子依赖性构象效应。结构特异性化学探针的组合使我们能够监测嘧啶在N - 3位和嘌呤在N - 1位和N - 7位的可及性。化学修饰的碱基通过末端标记随后进行苯胺诱导的链断裂（在某些情况下先进行杂交选择），或使用合成DNA寡聚物进行引物延伸来鉴定。这些研究表明：从活性状态到非活性状态的转变不能被描述为天然结构的简单松弛或展开，比如在更严重的离子耗尽条件下所观察到的那样。相反，它表现为两种不同结构状态之间的相互转换；一些碱基对探针的反应性增强，而另一些碱基由于失活反应性降低。反应性的变化几乎完全局限于以1400和1500位为中心的“解码位点”，但在中央结构域的U723和G791处也检测到显著差异。这可能反映了16 S rRNA中央区域和3'区域之间可能的结构和功能相互作用。非活性形式在1533至1538位（夏因 - 达尔加诺区域）的反应性也显著降低，这与早期研究结果一致。反应性的主要变化涉及普遍保守的核苷酸G926、C1395、A1398和G1401。这三种嘌呤在其N - 1位与N - 7位表现出相反的行为。G926在N - 1位失去反应性，但由于转变为非活性状态在N - 7位变得高度反应性。相反，A1398和G1401在N - 1位变得有反应性，但在N - 7位失去其高反应性。讨论了这些发现可能的结构和功能意义。
