Del Rio S, Setzer D R
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106.
Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):168-72. doi: 10.1073/pnas.90.1.168.
We have described elsewhere a number of the properties of a set of mutant forms of Xenopus transcription factor IIIA (TFIIIA) containing single amino acid substitutions that result in the structural disruption of individual zinc finger domains. These "broken finger" proteins have now been analyzed with respect to their ability to support transcription of 5S rRNA genes in vitro. Disruption of any one of the first six zinc fingers of TFIIIA has no discernible effect on the activity of the protein in supporting 5S rRNA synthesis in standard in vitro transcription assays, despite the fact that some of these mutant proteins exhibit large decreases in their binding affinity for 5S rRNA genes in binary complexes. These results indicate that the activity of TFIIIA as a transcription factor can be largely independent of its equilibrium binding constant for the 5S rRNA gene in the absence of other components of the RNA polymerase III transcriptional apparatus. In fact, this finding is consistent with the known pathway and kinetics of assembly of 5S rRNA transcription complexes. In contrast to the results obtained with finger 1-6 mutants, analogous mutations in zinc fingers 7-9 of TFIIIA result in moderate to complete loss of transcriptional activity. We interpret these results to mean that the three C-terminal zinc fingers of TFIIIA are not only involved in binding to the internal control region of 5S rRNA genes but are also required, either directly or indirectly, for higher-order interactions that are important in transcription complex assembly, stability, or activity.
我们在其他地方描述了非洲爪蟾转录因子IIIA（TFIIIA）的一组突变形式的许多特性，这些突变形式包含单个氨基酸替换，导致单个锌指结构域的结构破坏。现在已经对这些“断指”蛋白在体外支持5S rRNA基因转录的能力进行了分析。在标准的体外转录试验中，TFIIIA前六个锌指中的任何一个被破坏，对该蛋白支持5S rRNA合成的活性都没有明显影响，尽管其中一些突变蛋白在二元复合物中对5S rRNA基因的结合亲和力大幅下降。这些结果表明，在没有RNA聚合酶III转录装置的其他组分时，TFIIIA作为转录因子的活性在很大程度上可以独立于其对5S rRNA基因的平衡结合常数。事实上，这一发现与已知的5S rRNA转录复合物组装途径和动力学是一致的。与1-6号指突变体的结果相反，TFIIIA的7-9号锌指中的类似突变导致转录活性中度至完全丧失。我们将这些结果解释为意味着TFIIIA的三个C端锌指不仅参与与5S rRNA基因内部控制区的结合，而且对于转录复合物组装、稳定性或活性中重要的高阶相互作用也是直接或间接必需的。
