Jagath J R, Matassova N B, de Leeuw E, Warnecke J M, Lentzen G, Rodnina M V, Luirink J, Wintermeyer W
Institute of Molecular Biology, University of Witten/Herdecke, Witten, Germany.
RNA. 2001 Feb;7(2):293-301. doi: 10.1017/s1355838201002205.
Binding of Escherichia coli signal recognition particle (SRP) to its receptor, FtsY, requires the presence of 4.5S RNA, although FtsY alone does not interact with 4.5S RNA. In this study, we report that the exchange of the GGAA tetraloop sequence in domain IV of 4.5S RNA for UUCG abolishes SRP-FtsY interaction, as determined by gel retardation and membrane targeting experiments, whereas replacements with other GNRA-type tetraloops have no effect. A number of other base exchanges in the tetraloop sequence have minor or intermediate inhibitory effects. Base pair disruptions in the stem adjacent to the tetraloop or replacement of the closing C-G base pair with G-C partially restored function of the otherwise inactive UUCG mutant. Chemical probing by hydroxyl radical cleavage of 4.5S RNA variants show that replacing GGAA with UUCG in the tetraloop sequence leads to structural changes both within the tetraloop and in the adjacent stem; the latter change is reversed upon reverting the C-G closing base pair to G-C. These results show that the SRP-FtsY interaction is strongly influenced by the structure of the tetraloop region of SRP RNA, in particular the tetraloop stem, and suggest that both SRP RNA and Ffh undergo mutual structural adaptation to form SRP that is functional in the interaction with the receptor, FtsY.
大肠杆菌信号识别颗粒（SRP）与其受体FtsY的结合需要4.5S RNA的存在，尽管单独的FtsY并不与4.5S RNA相互作用。在本研究中，我们报告，通过凝胶阻滞和膜靶向实验确定，将4.5S RNA结构域IV中的GGAA四环序列替换为UUCG会消除SRP-FtsY相互作用，而用其他GNRA型四环进行替换则没有影响。四环序列中的许多其他碱基交换具有轻微或中等程度的抑制作用。四环相邻茎中的碱基对破坏或用G-C替换末端C-G碱基对可部分恢复原本无活性的UUCG突变体的功能。通过对4.5S RNA变体进行羟基自由基切割的化学探测表明，在四环序列中用UUCG替换GGAA会导致四环内部和相邻茎的结构变化；将末端C-G碱基对恢复为G-C后可逆转后者的变化。这些结果表明，SRP-FtsY相互作用受到SRP RNA四环区域结构的强烈影响，特别是四环茎，这表明SRP RNA和Ffh都经历了相互的结构适应，以形成在与受体FtsY相互作用中起作用的SRP。
