Gowda K, Chittenden K, Zwieb C
Department of Molecular Biology, The University of Texas Health Science Center at Tyler, PO Box 2003, Tyler, TX 75710, USA.
Nucleic Acids Res. 1997 Jan 15;25(2):388-94. doi: 10.1093/nar/25.2.388.
The interaction of protein SRP54M from the human signal recognition particle with SRP RNA was studied by systematic site-directed mutagenesis of the RNA molecule. Protein binding sites were identified by the analysis of mutations that removed individual SRP RNA helices or disrupted helical sections in the large SRP domain. The strongest effects on the binding activity of a purified polypeptide that corresponds to the methionine-rich domain of SRP54 (SRP54M) were caused by changes in helix 8 of the SRP RNA. Binding of protein SRP19 was diminished significantly by mutations in helix 6 and was stringently required for SRP54M to associate. Unexpectedly, mutant RNA molecules that resembled bacterial SRP RNAs were incapable of interaction with SRP54M, showing that protein SRP19 has an essential and direct role in the formation of the ternary complex with SRP54 and SRP RNA. Our findings provide an example for how, in eukaryotes, an RNA function has become protein dependent.
通过对RNA分子进行系统性定点诱变，研究了来自人类信号识别颗粒的蛋白质SRP54M与SRP RNA的相互作用。通过分析去除单个SRP RNA螺旋或破坏大SRP结构域中螺旋片段的突变，确定了蛋白质结合位点。对与SRP54富含甲硫氨酸结构域相对应的纯化多肽（SRP54M）结合活性影响最强的是SRP RNA螺旋8的变化。螺旋6中的突变显著降低了蛋白质SRP19的结合，且SRP54M缔合严格需要SRP19。出乎意料的是，类似于细菌SRP RNA的突变RNA分子无法与SRP54M相互作用，这表明蛋白质SRP19在与SRP54和SRP RNA形成三元复合物中具有重要且直接的作用。我们的研究结果为真核生物中RNA功能如何变得依赖蛋白质提供了一个例子。
