Setzer D R, Menezes S R, Del Rio S, Hung V S, Subramanyan G
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
RNA. 1996 Dec;2(12):1254-69.
We have used a collection of mutant forms of Xenopus transcription factor IIIA (TFIIIA) to study its interaction with 5S rRNA. This collection includes a set of nine mutant proteins, each of which contains a structural disruption in one of the nine zinc fingers of TFIIIA (broken-finger mutants), and a pair of complementary N- and C-terminal truncation mutants. Equilibrium and kinetic binding analyses in conjunction with RNAse protection and interference assays have been used to characterize the RNA-protein interaction in each case. We find that alternative binding modes are available for specific, high-affinity recognition of 5S rRNA by TFIIIA. These binding modes are distinct kinetically and structurally, and the mode of recognition adopted by wild-type TFIIIA when binding to intact 5S rRNA is dependent on the structural integrity of zinc fingers 5 and 6 in TFIIIA and continuity of the sugar-phosphate backbone in loop A of 5S rRNA. Disruption of any of these components allows adoption of one or more alternative modes of binding. In the wild-type TFIIIA-5S rRNA complex, some portions of TFIIIA, most notably the N-terminal three zinc fingers, are prevented from interacting with 5S rRNA in an energetically optimal way, and instead adopt a mode of binding that represents a compromise with the rest of the protein.
我们利用非洲爪蟾转录因子IIIA（TFIIIA）的一系列突变体形式来研究其与5S rRNA的相互作用。该系列包括一组九个突变蛋白，每个蛋白在TFIIIA的九个锌指之一中存在结构破坏（断指突变体），以及一对互补的N端和C端截短突变体。结合RNA酶保护和干扰试验进行的平衡和动力学结合分析已用于表征每种情况下的RNA-蛋白质相互作用。我们发现TFIIIA对5S rRNA的特异性、高亲和力识别存在多种结合模式。这些结合模式在动力学和结构上是不同的，野生型TFIIIA与完整5S rRNA结合时采用的识别模式取决于TFIIIA中锌指5和6的结构完整性以及5S rRNA环A中糖-磷酸骨架的连续性。这些组分中任何一个的破坏都允许采用一种或多种替代结合模式。在野生型TFIIIA-5S rRNA复合物中，TFIIIA的某些部分，最显著的是N端的三个锌指，无法以能量最优的方式与5S rRNA相互作用，而是采用一种与蛋白质其余部分折中的结合模式。
