Bélanger François, Gagnon Matthieu G, Steinberg Sergey V, Cunningham Philip R, Brakier-Gingras Léa
Département de Biochimie, Université de Montréal, Montréal, Qué., Canada H3T 1J4.
J Mol Biol. 2004 May 7;338(4):683-93. doi: 10.1016/j.jmb.2004.03.024.
The 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) is amongst the most conserved regions of rRNA. This tetraloop forms a GNRA motif that docks into the minor groove of three base-pairs at the bottom of helix 24 of 16S rRNA in the 30S subunit. Both the tetraloop and its receptor in helix 24 contact the 23S rRNA, forming the intersubunit bridge B2c. Here, we investigated the interaction between the 900 tetraloop and its receptor by genetic complementation. We used a specialized ribosome system in combination with an in vivo instant evolution approach to select mutations in helix 24 compensating for a mutation in the 900 tetraloop (A900G) that severely decreases ribosomal activity, impairing subunit association and translational fidelity. We selected two mutants where the G769-C810 base-pair of helix 24 was substituted with either U-A or C x A. When these mutations in helix 24 were investigated in the context of a wild-type 900 tetraloop, the C x A but not the U-A mutation severely impaired ribosome activity, interfering with subunit association and decreasing translational fidelity. In the presence of the A900G mutation, both mutations in helix 24 increased the ribosome activity to the same extent. Subunit association and translational fidelity were increased to the same level. Computer modeling was used to analyze the effect of the mutations in helix 24 on the interaction between the tetraloop and its receptor. This study demonstrates the functional importance of the interaction between the 900 tetraloop and helix 24.
封闭16S核糖体RNA（rRNA）螺旋27的900四环是rRNA中最保守的区域之一。该四环形成一个GNRA基序，该基序对接至30S亚基中16S rRNA螺旋24底部三个碱基对的小沟中。四环及其在螺旋24中的受体均与23S rRNA接触，形成亚基间桥B2c。在此，我们通过基因互补研究了900四环与其受体之间的相互作用。我们使用了一种专门的核糖体系统，并结合体内即时进化方法来选择螺旋24中的突变，以补偿900四环中的突变（A900G），该突变会严重降低核糖体活性，损害亚基缔合和翻译保真度。我们选择了两个突变体，其中螺旋24的G769 - C810碱基对被U - A或C×A取代。当在野生型900四环的背景下研究螺旋24中的这些突变时，C×A而非U - A突变严重损害了核糖体活性，干扰了亚基缔合并降低了翻译保真度。在存在A900G突变的情况下，螺旋24中的两个突变均使核糖体活性提高到相同程度。亚基缔合和翻译保真度提高到相同水平。使用计算机建模来分析螺旋24中的突变对四环与其受体之间相互作用的影响。这项研究证明了900四环与螺旋24之间相互作用的功能重要性。
